Seleção de antígenos de Leishmania infantum por imunoproteômica para emprego no diagnóstico da leishmaniose visceral humana / Selection of Leishmania infantum antigens by immunoproteomics for use in the diagnosis of human visceral leishmaniasis

O controle da leishmaniose visceral (LV) requer um diagnóstico e tratamento adequados, uma vez que o diagnóstico preciso é fundamental para um regime medicamentoso eficaz para os pacientes. Nesse contexto, as ferramentas biotecnológicas devem ser aprimoradas para o manejo clínico e a avaliação epidemiológica da doença. No entanto, existem limitações relacionadas com a sensibilidade e/ou especificidade dos antígenos usados atualmente, mostrando a necessidade de identificação de novas moléculas para serem testadas em um diagnóstico sorológico mais sensível e específico. Neste sentido, no presente estudo, uma abordagem imunoproteômica foi usada para identificar proteínas antigênicas das formas promastigotas e amastigotas da espécie Leishmania infantum, causadora de LV em nosso país, por meio de seu reconhecimento por anticorpos em soros de pacientes com a doença. Amostras de indivíduos saudáveis residentes em região endêmica da doença e de pacientes com Doença de Chagas foram utilizadas com a função de se obter proteínas mais específicas ao parasito Leishmania para serem avaliadas no diagnóstico da LV. Como resultados obtidos, um total de 29 e 21 proteínas foram identificadas nos extratos de formas promastigotas e amastigotas dos parasitos, respectivamente. Para a validação da capacidade diagnóstica, duas proteínas, endonuclease III e GTP-binding protein, foram selecionadas, clonadas, expressas e purificadas para serem testadas em experimentos de ELISA. Os resultados dos testes mostraram valores de sensibilidade e especificidade superiores a 99,0% para a identificação da LV. Os antígenos ainda exibiram um diferencial ao apresentarem baixa reatividade sorológica em pacientes curados e tratados, sugerindo a possibilidade de que as mesmas possam ser aplicadas como marcadores prognósticos da doença. Em conclusão, o estudo imunoproteômico se mostrou eficaz na seleção de proteínas antigênicas de L. infantum e duas delas, endonuclease III e GTP-binding protein, foram bem avaliadas para o diagnóstico da LV frente a um painel sorológico, além de demonstrarem um potencial para monitoramento de pacientes com LV após o tratamento.

The control of visceral leishmaniasis (VL) requires an adequate diagnosis and treatment, since an accurate diagnosis is essential for an effective medication regimen for patients. In this context, biotechnological tools must be improved for the clinical management and epidemiological assessment of the disease. However, there are limitations related to the sensitivity and / or specificity of the antigens currently used, showing the necessity to identify new molecules to be tested in a more sensitive and specific serological diagnosis. In this sense, in the present study, an immunoproteomics approach was used to identify antigenic proteins of the Leishmania infantum promastigote and amastigote forms, which causes VL in our country, through its recognition by antibodies in sera of patients with the disease. Samples from healthy individuals living in an endemic region of the disease and from patients with Chagas disease were used to obtain more specific proteins for the Leishmania parasite, aiming their future application in the VL diagnosis. As results obtained, a total of 29 and 21 proteins were identified in the extracts of parasitic promastigotes and amastigotes, respectively. For validation of the diagnostic capacity, two proteins, endonuclease III and GTP-binding protein, were selected, cloned, expressed and purified to be tested in ELISA experiments. The test results showed sensitivity and specificity values greater than 99.0% for the identification of VL. The antigens also exhibited a differential when presenting low serological reactivity in cured and treated patients, suggesting the possibility that they can be applied as prognostic markers of the disease. In conclusion, the immunoproteomic study proved to be effective in the selection of L. infantum antigenic proteins and two of them, endonuclease III and GTP-binding protein, were well evaluated for the diagnosis of VL against a serological panel, in addition, demonstrating a potential for monitoring patients with VL after treatment.

Expression, purification and micelle reconstruction of the transmembrane domain of the human amyloid precursor protein for NMR studies / 生物工程学报

The multiple-step cleavage of amyloid precursor protein (APP) generates amyloid-β peptides (Aβ), highly toxic molecules causing Alzheimer's disease (AD). The nonspecific cleavage between the transmembrane region of APP (APPTM) and γ-secretase is the key step of Aβ generation. Reconstituting APPTM under physiologically-relevant conditions is crucial to investigate how it interacts with γ-secretase and for future AD drug discovery. Although producing recombinant APPTM was reported before, the large scale purification was hindered by the use of biological protease in the presence of membrane protein. Here, we expressed recombinant APPTM in Escherichia coli using the pMM-LR6 vector and recovered the fusion protein from inclusion bodies. By combining Ni-NTA chromatography, cyanogen bromide cleavage, and reverse phase high performance liquid chromatography (RP-HPLC), isotopically-labeled APPTM was obtained in high yield and high purity. The reconstitution of APPTM into dodecylphosphocholine (DPC) micelle generated mono dispersed 2D 15N-1H HSQC spectra in high quality. We successfully established an efficient and reliable method for the expression, purification and reconstruction of APPTM, which may facilitate future investigation of APPTM and its complex in more native like membrane mimetics such as bicelle and nanodiscs.

Clinical observation of Qiliqiangxin capsule combined with recombinant human brain natriuretic peptide in patients with acute heart failure / 中华内科杂志

Objective: To observe the clinical effect of Qiliqiangxin capsule combined with recombinant human brain natriuretic peptide in acute left heart failure patients 7 days after onset as well as the effects of plasma MDA and ET-1. Methods: In total, 240 hospitalized patients with acute left heart failure from October 2017 to May 2021 were selected from the Department of Emergency and Critical Care Center of Beijing Anzhen Hospital, Capital Medical University and the Department of Cardiology of the Jilin Provincial People's Hospital. They were randomly divided into routine treatment group and combined treatment group, with 120 cases in each group. The routine treatment group was treated with vasodilation, diuresis, cardiotonic and recombinant human brain natriuretic peptide. The combined treatment group was treated with Qiliqiangxin capsules based on the routine treatment group. One week later, the changes in clinical efficacy, ejection fraction, left ventricular commoid diameter, and plasma BNP, MDA, and ET-1 were compared between the two groups before and after treatment. SPSS 11.5 statistical software was used. The measurement data was expressed in x¯±s, the independent sample t-test was used for comparison between groups, and the paired t-test was used for comparison before and after treatment within groups. Counting data was expressed as case (%), and the rank sum test was used for inter-group comparison. Result: In terms of clinical efficacy, the total effective rate of the combined treatment group was significantly higher than that of the conventional treatment group, and the difference was statistically significant (P<0.05). Compared with the routine treatment group, the left ventricular ejection fraction in the combined treatment group was significantly increased (P<0.05). The levels of plasma BNP, MDA and ET-1 were significantly decreased (P<0.05). Conclusion: Qiliqiangxin capsule combined with rhBNP treatment can effectively improve the clinical symptoms of acute heart failure, as well as reduce the lipid peroxidation product MDA content and endothetin ET-1 level in blood. The clinical application value of the Qiliqiangxin capsule needs to be further confirmed by further trials.

Effect of Recombinant Human Thrombopoietin (rhTPO) on Hematopoietic Reconstitution in Allogeneic Hematopoietic Stem Cell Transplantation Model / 中国实验血液学杂志

OBJECTIVE@#To explore the effect of recombinant human thrombopoietin (rhTPO) on hematopoietic reconstruction in allogeneic hematopoietic stem cell transplantation (allo-HSCT) model.@*METHODS@#The C57BL/6 mice were employed as the donors, and BALB/c mice as recipients. The bone marrow mononuclear cells of the donor mice were extracted and pretreated, which then were injected with 5×106 per mouse through the tail vein of the recipient to establish an allo-HSCT model. The implantation of hematopoietic stem cells in the recipient mice was detected by flow cytometry on the 28th day after transplantation. Next, the successfully modeled recipient mice were randomly divided into experimental group and control group. The rhTPO was injected into mice in the experimental group on the first day after transplantation, while the saline was injected into mice in the control group. Both groups were injected for 14 consecutive days. The peripheral blood and bone marrow hematopoiesis of the two groups were observed on day 1, 3, 7, 14, and 21 after transplantation.@*RESULTS@#The expression rate of H-2Kb in the bone marrow of recipient mice was 43.85% (>20%) on the 28th day after transplantation, which indicated that the recipient mice were successfully chimerized. Meanwhile, counts of PLTs on the day 3, 7, 14, and 21 after transplantation in the experimental group were higher than those in the control group with statistical significances (P<0.05). In addition, hematopoietic function of bone marrow was suppressed in both groups on day 1, 3 and 7 after transplantation, but hematopoietic bone marrow hyperplasia was better in the experimental group than in the control group. On day 14 and 21 after transplantation, the hematopoietic function of bone marrow in the two groups was recovered, and the experimental group showed more obvious than the control group.@*CONCLUSION@#rhTPO can effectively stimulate the production of PLTs and facilitate the recovery of white blood cells and hemoglobin after allo-HSCT, and promote hematopoietic recovery and reconstitution of bone marrow.

Cloning, expression and purification of fructose-2, 6-bisphosphatase gene CpF2KP in papaya / 生物工程学报

Papaya, which is mainly cultivated in the southeastern region of China, is one of the four famous fruits in Lingnan. It is favored by people because of its edible and medicinal value. Fructose-6-phosphate, 2-kinase/fructose-2, 6-bisphosphatase (F2KP) is a unique bifunctional enzyme with a kinase domain and an esterase domain that catalyzes the synthesis and degradation of fructose-2, 6-bisphosphate (Fru-2, 6-P2), an important regulator of glucose metabolism in organisms. In order to study the function of the gene CpF2KP encoding the enzyme in papaya, it is particularly important to obtain the target protein. In this study, the coding sequence (CDS) of CpF2KP, with a full-length of 2 274 bp, was got from the papaya genome. The amplified sequence of full-length CDS was cloned into the vector PGEX-4T-1 which was double digested with EcoR I and BamH I. The amplified sequence was constructed into a prokaryotic expression vector by genetic recombination. After exploring the induction conditions, the results of SDS-PAGE showed that the size of the recombinant GST-CpF2KP protein was about 110 kDa. The optimum IPTG concentration and temperature for CpF2KP induction were 0.5 mmol/L and 28 ℃, respectively. The purified sin[A1] gle target protein was obtained after purifying the induced CpF2KP protein. In addition, the expression level of this gene was detected in different tissues, and showed that the gene was expressed at the highest level in seeds and the lowest in pulp. This study provides an important basis for further revealing the function of CpF2KP protein and studying the involved biological processes of this gene in papaya.

Advances in epigenetic regulation of Chinese hamster ovary cells / 生物工程学报

Chinese hamster ovary (CHO) cells play an irreplaceable role in biopharmaceuticals because the cells can be adapted to grow in suspension cultures and are capable of producing high quality biologics exhibiting human-like post-translational modifications. However, gene expression regulation such as transgene silencing and epigenetic modifications may reduce the recombinant protein production due to the decrease of expression stability of CHO cells. This paper summarized the role of epigenetic modifications in CHO cells, including DNA methylation, histone modification and miRNA, as well as their effects on gene expression regulation.

Effect of Recombinant Human Thrombopoietin (rhTPO) on Long-term Hematopoietic Recovery in Mice with Acute Radiation Sickness and Relative Mechanism / 中国实验血液学杂志

OBJECTIVE@#To investigate the effect and relative mechanism of Recombinant Human Thrombopoietin (rhTPO) on long-term hematopoietic recovery in mice with acute radiation sickness.@*METHODS@#Mice were intramuscularly injected with rhTPO (100 μg/kg) 2 hours after total body irradiation with 60Co γ-rays (6.5 Gy). Moreover, six months after irradiation, peripheral blood, hematopoietic stem cells (HSC) ratio, competitive transplantation survival rate and chimerization rate, senescence rate of c-kit+ HSC, and p16 and p38 mRNA expression of c-kit+ HSC were detected.@*RESULTS@#Six months after 6.5 Gy γ-ray irradiation, there were no differences in peripheral blood white blood cells, red blood cells, platelets, neutrophils and bone marrow nucleated cells in normal group, irradiated group and rhTPO group (P>0.05). The proportion of hematopoietic stem cells and multipotent progenitor cells in mice of irradiated group was significantly decreased after irradiation (P<0.05), but there was no significant changes in rhTPO group (P>0.05). The counts of CFU-MK and BFU-E in irradiated group were significantly lower than that in normal group, and rhTPO group was higher than that of the irradiated group(P<0.05). The 70 day survival rate of recipient mice in normal group and rhTPO group was 100%, and all mice died in irradiation group. The senescence positive rates of c-kit+ HSC in normal group, irradiation group and rhTPO group were 6.11%, 9.54% and 6.01%, respectively (P<0.01). Compared with the normal group, the p16 and p38 mRNA expression of c-kit+ HSC in the irradiated mice were significantly increased (P<0.01), and it was markedly decreased after rhTPO administration (P<0.01).@*CONCLUSION@#The hematopoietic function of mice is still decreased 6 months after 6.5 Gy γ-ray irradiation, suggesting that there may be long-term damage. High-dose administration of rhTPO in the treatment of acute radiation sickness can reduce the senescence of HSC through p38-p16 pathway and improve the long-term damage of hematopoietic function in mice with acute radiation sickness.

Protein expression, purification and mouse antiserum preparation of monkeypox virus A23R / 细胞与分子免疫学杂志

Objective To express the monkeypox virus (MPXV) A23R protein in Escherichia coli and purify by Ni-NTA affinity column, and to prepare mouse antiserum against MPXV A23R. Methods The recombinant plasmid pET-28a-MPXV-A23R was constructed and transformed into Escherichia coli BL21 to induce the expression of A23R protein. After optimizing the conditions of expression, A23R protein was highly expressed. Recombinant A23R protein was purified by Ni-NTA affinity column and identified by Western blot analysis. The purified protein was used to immunize mice for preparing the A23R polyclonal antibody, and the antibody titer was detected by ELISA. Results The expression of A23R recombinant protein reached the peak under the induced conditions of 0.6 mmol/L isopropyl-β-D-thiogalactoside (IPTG), 37 DegreesCelsius and 20 hours. The purity of the protein was about 96.07% and was identified by Western blot analysis. The mice were immunized with recombinant protein, and the titer of antibody reached 1:102 400 at the 6th week after immunization. Conclusion MPXV A23R is expressed highly and purified with a high purity and its antiserum from mouse is obtained with a high titre.

Preparation and identification of rabbit anti-mouse coiled-coil domain containing 189(Ccdc189)polyclonal antibody / 细胞与分子免疫学杂志

Objective To prepare a rabbit anti-mouse coiled-coil domain containing 189 (Ccdc189) polyclonal antibody. Methods The pET-28a-Ccdc189 prokaryotic expression plasmid was constructed and transformed into E.coli BL21. IPTG was used to induce the expression of Ccdc189 prokaryotic protein. Adult male New Zealand rabbits were immunized with purified recombinant protein to obtain rabbit anti-mouse Ccdc189 polyclonal antibody. The specificity of the polyclonal antibody was identified by Western blot analysis, indirect ELISA and immunofluorescence histochemical staining. Results The pET-28a-Ccdc189 recombinant plasmid was successfully constructed and the expression of the Ccdc189 recombinant protein was induced. ELISA revealed that the titer of the polyclonal antibody was 1:1 000 000. Western blot and immunofluorescence staining demonstrated that the Ccdc189 polyclonal antibody could specifically identify the Ccdc189 prokaryotic protein and the Ccdc189 protein in adult wild-type mouse testis. Conclusion A polyclonal antibody with high specificity against mouse Ccdc189 was successfully created.

Clonamiento, expresión y seroreactividad de la proteína recombinante de ensamblaje de lipopolisacáridos - D (LptD) de Bartonella bacilliformis / Cloning, expression and seroreactivity of the recombinant lipopolysaccharide assembly protein - D (LptD) from Bartonella bacilliformis

RESUMEN Objetivo. Evaluar in silico y a nivel serológico el potencial antigénico del dominio extracelular recombinante de la proteína de ensamblaje de lipopolisacáridos - D (LptD) de Bartonella bacilliformis (dexr_LptD). Materiales y métodos. Mediante el análisis in silico se realizó la selección de una proteína de B. bacilliformis con potencial antigénico e inmunogénico. El gen de la proteína seleccionada se clonó en Escherichia coli TOP10 y se expresó en Escherichia coli BL21 (DE3) pLysS. La proteína recombinante fue expresada usando isopropil-β-D-1-tiogalactopiranósido (IPTG) y se optimizaron las condiciones de inducción. Por último, se purificó con resina Ni-IDA (His60 Ni Superflow) y se realizó un ensayo de Western Blot. Resultados. In silico, la proteína seleccionada fue LptD por estar localizada en la membrana externa y ser antigénica e inmunogénica. Las condiciones optimizadas para la inducción del dexr_LptD fueron 0,5 mM IPTG, 16 h, medio TB (Terrific Broth), etanol al 3% (v/v), 28 ºC, OD600: 1-1,5 y 200 r.p.m. La purificación se realizó en condiciones denaturantes a pequeña escala y se obtuvo 2,6 µg/mL de dexr_LptD parcialmente purificada. El ensayo de Western Blot mostró una reacción positiva entre los sueros provenientes de pacientes con la enfermedad de Carrión y dexr_LptD, ello evidencia la antigenicidad del dexr_LptD. Conclusiones. El dexr_LptD muestra antigenicidad in silico y a nivel serológico, estos resultados son base para posteriores estudios sobre candidatos vacunales contra la enfermedad de Carrión.

ABSTRACT Objective. To evaluate in silico and at the serological level the antigenic potential of the recombinant extracellular domain of the lipopolysaccharide assembly protein - D (LptD) of Bartonella bacilliformis (dexr_LptD). Materials and Methods. Through in silico analysis, we selected a B. bacilliformis protein with antigenic and immunogenic potential. The selected protein gene was cloned into Escherichia coli TOP10 and expressed in Escherichia coli BL21 (DE3) pLysS. Recombinant protein was expressed using isopropyl-β-D-1-thiogalactopyranoside (IPTG) and induction conditions were optimized. Finally, it was purified with Ni-IDA resin (His60 Ni Superflow) and a Western Blot assay was conducted. Results. In silico, the selected protein was LptD because it is located in the outer membrane and is antigenic and immunogenic. Optimized conditions for dexr_LptD induction were 0.5 mM IPTG, 16 hours, TB (Terrific Broth) medium, 3% (v/v) ethanol, 28 ºC, OD600: 1-1.5 and 200 rpm. Purification was carried out under denaturating conditions on a small scale and we obtained 2.6 μg/mL of partially purified dexr_LptD. The Western Blot assay showed a positive reaction between the sera from patients with Carrión's Disease and dexr_LptD, which shows the antigenicity of dexr_LptD. Conclusions. The dexr_LptD shows antigenicity both in silico and at the serological level, these results are the basis for further studies on vaccine candidates against Carrion's Disease.

Knockout of caspase-7 gene improves the expression of recombinant protein in CHO cell line through the cell cycle arrest in G2/M phase

BACKGROUND: Chinese hamster ovary cell line has been used routinely as a bioproduction factory of numerous biopharmaceuticals. So far, various engineering strategies have been recruited to improve the production efficiency of this cell line such as apoptosis engineering. Previously, it is reported that the caspase-7 deficiency in CHO cells reduces the cell proliferation rate. But the effect of this reduction on the CHO cell productivity remained unclear. Hence, in the study at hand the effect of caspase-7 deficiency was assessed on the cell growth, viability and protein expression. In addition, the enzymatic activity of caspase-3 was investigated in the absence of caspase-7. RESULTS: Findings showed that in the absence of caspase-7, both cell growth and cell viability were decreased. Cell cycle analysis illustrated that the CHO knockout (CHO-KO) cells experienced a cell cycle arrest in G2/M phase. This cell cycle arrest resulted in a 1.7-fold increase in the expression of luciferase in CHO-KO cells compared to parenteral cells. Furthermore, in the apoptotic situation the enzymatic activity of caspase-3 in CHO-KO cells was approximately 3 times more than CHO-K1 cells. CONCLUSIONS: These findings represented that; however, caspase-7 deficiency reduces the cell proliferation rate but the resulted cell cycle arrest leads to the enhancement of recombinant protein expression. Moreover, increasing in the caspase-3 enzymatic activity compensates the absence of caspase-7 in the caspase cascade of apoptosis.

Studies on the recombinant production and anticancer activity of thermostable L- asparaginase I from Pyrococcus abyssi / Estudos sobre a produção recombinante e atividade anticancerígena da L-asparaginase I termoestável de Pyrococcus abyssi

L-Asparaginase catalysing the breakdown of L-Asparagine to L-Aspartate and ammonia is an enzyme of therapeutic importance in the treatment of cancer, especially the lymphomas and leukaemia. The present study describes the recombinant production, properties and anticancer potential of enzyme from a hyperthermophilic archaeon Pyrococcus abyssi. There are two genes coding for asparaginase in the genome of this organism. A 918 bp gene encoding 305 amino acids was PCR amplified and cloned in BL21 (DE3) strain of E. coli using pET28a (+) plasmid. The production of recombinant enzyme was induced under 0.5mM IPTG, purified by selective heat denaturation and ion exchange chromatography. Purified enzyme was analyzed for kinetics, in silico structure and anticancer properties. The recombinant enzyme has shown a molecular weight of 33 kDa, specific activity of 1175 U/mg, KM value 2.05mM, optimum temperature and pH 80°C and 8 respectively. No detectable enzyme activity found when L-Glutamine was used as the substrate. In silico studies have shown that the enzyme exists as a homodimer having Arg11, Ala87, Thr110, His112, Gln142, Leu172, and Lys232 being the putative active site residues. The free energy change calculated by molecular docking studies of enzyme and substrate was found as ∆G ­ 4.5 kJ/mole indicating the affinity of enzyme with the substrate. IC50 values of 5U/mL to 7.5U/mL were determined for FB, caco2 cells and HepG2 cells. A calculated amount of enzyme (5U/mL) exhibited 78% to 55% growth inhibition of caco2 and HepG2 cells. In conclusion, the recombinant enzyme produced and characterized in the present study offers a good candidate for the treatment of cancer. The procedures adopted in the present study can be prolonged for in vivo studies.

A L-asparaginase, que catalisa a degradação da L-asparagina em L-aspartato e amônia, é uma enzima de importância terapêutica no tratamento do câncer, especialmente dos linfomas e da leucemia. O presente estudo descreve a produção recombinante, propriedades e potencial anticancerígeno da enzima de Pyrococcus abyssi, um archaeon hipertermofílico. Existem dois genes que codificam para a asparaginase no genoma desse organismo. Um gene de 918 bp, que codifica 305 aminoácidos, foi amplificado por PCR e clonado na cepa BL21 (DE3) de E. coli usando o plasmídeo pET28a (+). A produção da enzima recombinante foi induzida sob 0,5mM de IPTG, purificada por desnaturação seletiva por calor e cromatografia de troca iônica. A enzima purificada foi analisada quanto à cinética, estrutura in silico e propriedades anticancerígenas. A enzima recombinante apresentou peso molecular de 33 kDa, atividade específica de 1.175 U / mg, valor de KM 2,05 mM, temperatura ótima de 80º C e pH 8. Nenhuma atividade enzimática detectável foi encontrada quando a L-glutamina foi usada como substrato. Estudos in silico mostraram que a enzima existe como um homodímero, com Arg11, Ala87, Thr110, His112, Gln142, Leu172 e Lys232 sendo os resíduos do local ativo putativo. A mudança de energia livre calculada por estudos de docking molecular da enzima e do substrato foi encontrada como ∆G ­ 4,5 kJ / mol, indicando a afinidade da enzima com o substrato. Valores de IC50 de 5U / mL a 7,5U / mL foram determinados para células FB, células caco2 e células HepG2. Uma quantidade de enzima (5U / mL) apresentou inibição de crescimento de 78% a 55% das células caco2 e HepG2, respectivamente. Em conclusão, a enzima recombinante produzida e caracterizada no presente estudo é uma boa possibilidade para o tratamento do câncer. Os procedimentos adotados na presente pesquisa podem ser aplicados para estudos in vivo.

Construction of recombinant adenovirus expressing capsid protein of serotype O foot-and-mouth disease virus and analysis of its immunogenicity / 生物工程学报

In order to construct a recombinant replication deficient human type 5 adenovirus (Ad5) expressing a foot-and-mouth disease virus (FMDV) capsid protein, specific primers for P12A and 3B3C genes of FMDV-OZK93 were synthesized. The P12A and 3B3C genes were then amplified and connected by fusion PCR, and a recombinant shuttle plasmid pDC316-mCMV-EGFP-P12A3B3C expressing the FMDV-OZK93 capsid protein precursor P12A and 3B3C protease were obtained by inserting the P12A3B3C gene into the pDC316-mCMV-EGFP plasmid. The recombinant adenovirus rAdv-P12A3B3C-OZK93 was subsequently packaged, characterized and amplified using AdMaxTM adenovirus packaging system, and the expression was verified by infecting human embryonic kidney cell HEK-293. The humoral and cellular immunity levels of well-expressed and purified recombinant adenovirus immunized mice were evaluated. The results showed that rAdv-P12A3B3C-OZK93 could be stably passaged and the maximum virus titer reached 1×109.1 TCID50/mL. Western blotting and indirect immunofluorescence showed that rAdv-P12A3B3C-OZK93 expressed the FMDV-specific proteins P12A and VP1 in HEK-293 cells. In addition, the PK cell infection experiment confirmed that rAdv-P12A3B3C-OZK93 could infect porcine cells, which is essential for vaccination in pigs. Comparing with the inactivated vaccine group, the recombinant adenovirus could induce higher FMDV-specific IgG antibodies, γ-IFN and IL-10. This indicates that the recombinant adenovirus has good immunity for animal, which is very important for the subsequent development of foot-and-mouth disease vaccine.

Preparation of a recombinant tumor-targeting ribosome inactivating protein luffin-α-NGR and evaluation of its antitumor activity / 生物工程学报

Loofah seeds ribosome inactivating protein luffin-α was fused with a tumor-targeting peptide NGR to create a recombinant protein, and its inhibitory activity on tumor cells and angiogenesis were assessed. luffin-α-NGR fusion gene was obtained by PCR amplification. The fusion gene was ligated with pGEX-6p-1 vector to create a recombinant plasmid pGEX-6p-1/luffin-α-NGR. The plasmid was transformed into E. coli BL21, and the target protein was isolated and purified by GST affinity chromatography. The luffin-α-NGR fusion gene with a full length of 849 bp was successfully obtained, and the optimal soluble expression of the target protein was achieved under the conditions of 16 ℃, 0.5 mmol/L IPTG after 16 h induction. SDS-PAGE and Western blotting confirmed the recombinant protein has an expected molecular weight of 56.6 kDa. Subsequently, the recombinant protein was de-tagged by precision protease digestion. The inhibitory effects of the recombinant protein on liver tumor cells HepG2 and breast cancer cells MDA-MB-231 were significantly stronger than that of luffin-α. The Transwell and CAM experiment proved that the recombinant protein luffin-α-NGR also had a significant inhibitory effect on tumor cells migration and neovascularization. The inhibitory activity on tumor cells and angiogenesis of the recombinant luffin-α-NGR protein lays a foundation for the development of subsequent recombinant tumor-targeting drugs.

Preparation and characterization of Ad-ERα-36-Fc-GFP / 生物工程学报

ERα-36 is a novel subtype of estrogen receptor α which promotes tumor cell proliferation, invasion and drug resistance, and it serves as a therapeutic target. However, only small-molecule compounds targeting ERα-36 are under development as anticancer drugs at present. Gene therapy approach targeting ERα-36 can be explored using recombinant adenovirus armed with decoy receptor. The recombinant shuttle plasmid pDC316-Ig κ-ERα-36-Fc-GFP was constructed via genetic engineering to express an Ig κ-signaling peptide-leading secretory recombinant fusion protein ERα-36-Fc. The recombinant adenovirus Ad-ERα-36-Fc-GFP was subsequently packaged, characterized and amplified using AdMaxTM adenovirus packaging system. The expression of fusion protein and functional outcome of Ad-ERα-36-Fc-GFP transduction were further analyzed with triple-negative breast cancer MDA-MB-231 cells. Results showed that the recombinant adenovirus Ad-ERα-36-Fc-GFP was successfully generated. The virus effectively infected MDA-MB-231 cells which resulted in expression and secretion of the recombinant fusion protein ERα-36-Fc, leading to significant inhibition of EGFR/ERK signaling pathway. Preparation of the recombinant adenovirus Ad-ERα-36-Fc-GFP provides a basis for further investigation on cancer gene therapy targeting ERα-36.

Desenvolvimento de teste diagnóstico não invasivo capaz de detectar anticorpos anti-SARS-CoV-2 em urina / Development of a non-invasive diagnostic test capable of detecting anti-SARS-CoV-2 antibodies in urine

A plataforma de ELISA (ensaio de imunoabsorção por ligação enzimática) tem sido amplamente utilizada para detectar anticorpos anti-SARS-CoV-2 gerados após a exposição ao vírus ou à vacinação. A amostra comumente utilizada para a realização do teste é o soro. Até o momento, nenhum estudo havia investigado a urina do paciente como amostra para detectar anticorpos específicos para o vírus SARS-CoV-2. A urina é um espécime biológico que traz vantagens significativas inerentes ao tipo de amostra, que compreende coleta não invasiva, de fácil manuseio e armazenamento. Neste trabalho, propomos um ELISA indireto in house baseado no uso de urina e proteínas recombinantes do Nucleocapsídeo (N) ou da Spike (S) do vírus SARS-CoV-2. As proteínas recombinantes (r) de SARS-CoV-2, N e as subunidades da proteína S (S-Glic, S1-NGlic e RBD-NGlic), foram avaliadas usando um painel composto por aproximadamente 200 amostras de urina e de soro. A presença de anticorpos anti-SARS-CoV-2 na urina foi detectada com sensibilidade e especificidade similares ou superiores ao soro, nas quais foram obtidos valores de sensibilidade de 94,0%, 75,0%, 81,38% e 89,66%, e especificidade de 100%, 96,0%, 96,77% e 96,77%, frente às proteínas rSARS-CoV-2 N, S-Glic, S1-NGlic e RBDNGlic, respectivamente. Dessa forma, os dados apresentados sugerem que a urina poderia ser considerada como uma potencial amostra biológica para aplicação em plataformas de imunodiagnóstico para a infecção por SARS-CoV-2, trazendo benefícios tanto no contexto individual quanto populacional.

The Enzyme-linked immunosorbent assay (ELISA) method has been widely used to detect anti-SARS-CoV-2 antibodies generated after exposure to the virus or vaccination. The sample usually used to perform the test is the serum. Thus far, no study has investigated the urine of patients as biological sample to detect specific SARS-CoV-2 antibodies. Urine is a biological specimen with significant advantages inherent to the type of sample, which comprises non-invasive collection, easy handling and storage. In this work, we propose an in house urine-based indirect ELISA using recombinant proteins from Nucleocapsid (N) and Spike (S) of the SARSCoV-2 virus. SARS-CoV-2 recombinant N and S protein subunits (Gly-S, NonGly-S1 and NonGly-RBD) were evaluated in an ELISA platform with a panel composed about 200 urine and serum samples. The presence of anti-SARS-CoV-2 antibodies in urine was detected with similar or superior sensitivity and specificity to serum, in which sensitivity values of 94.0%, 75.0%, 81.38% and 89.66% were obtained, while specificity values were of 100.0%, 96.0%, 96.77% and 96.77%, respectively, against rSARS-CoV-2 N, S-Glic, S1-NGlic and RBD-NGlic proteins. In conclusion, the data presented suggest that urine could be considered as a potential biological sample for application in immunodiagnostic platforms for SARS-CoV-2 infection, with benefits to the individual and population context.

Prokaryotic expression of a recombinant protein of adeno-associated virus capsid conserved regions and preparation of its polyclonal antibody / 南方医科大学学报

OBJECTIVE@#To express and purify the antigenic peptide of adeno-associated virus (AAV) capsid conserved regions in prokaryotic cells and prepare its rabbit polyclonal antibody.@*METHODS@#The DNA sequence encoding the conserved regions of AAV capsid protein was synthesized and cloned into the vector pET30a to obtain the plasmid pET30a-AAV-CR for prokaryotic expression and purification of the conserved peptides. Coomassie blue staining and Western blotting were used to identify the AAV conserved peptides. Japanese big ear white rabbits were immunized with AAV conserved region protein to prepare polyclonal antibody, with the rabbits injected with PBS as the control group. The antibody titer was determined with ELISA, and the performance of the antibody for recognizing capsid protein sequences of AAV1-AAV10 was assessed with Western blotting and immunofluorescence assay.@*RESULTS@#The plasmid pET30a-AAV-CR was successfully constructed, and a recombinant protein with a relative molecular mass of 17000 was obtained. The purified protein induced the production of antibodies against the conserved regions of AAV capsid in rabbits, and the titer of the purified antibodies reached 1:320 000. The antibodies were capable of recognizing a wide range of capsid protein sequences of AAV1-AAV10.@*CONCLUSION@#We successfully obtained the polyclonal antibodies against AAV capsid conserved region protein from rabbits, which facilitate future studies of AAV vector development and the biological functions of AAV.

Diagnostic value of serum tumor markers CEA, CYFRA21-1, SCCAg, NSE and ProGRP for lung cancers of different pathological types / 南方医科大学学报

OBJECTIVE@#To evaluate the diagnostic value of the serum tumor markers carcinoembryonic antigen (CEA), cytokeratin-19-fragment (CYFRA21-1), squamous cell carcinoma associated antigen (SCCAg), neuron-specificenolase (NSE) and pro-gastrin-releasing peptide (ProGRP) for lung cancers of different pathological types.@*METHODS@#This study was conducted among patients with established diagnoses of lung adenocarcinoma (LADC, n=137), lung squamous cell carcinoma (LSCC, n=82), small cell lung carcinoma (SCLC, n=59), and benign chest disease (BCD, n=102). The serum tumor markers were detected for all the patients for comparison of the positivity rates and their serum levels. ROC curve was used for analysis of the diagnostic efficacy of these tumor markers either alone or in different combinations.@*RESULTS@#In patients with LADC, the positivity rate and serum level of CEA were significantly higher than those in the other groups (P < 0.05); the patients with LSCC had the highest positivity rate and serum level of SCCAg among the 4 groups (P < 0.05). The positivity rates and serum levels of ProGRP and NSE were significantly higher in SCLC group than in the other groups (P < 0.05). CYFRA21-1 showed the highest positivity rate and serum level in LADC group and LSCC group. With the patients with BCD as control, CEA showed a diagnostic sensitivity of 62.8% and a specificity of 93.1% for LADC, and the sensitivity and specificity of SCCAg for diagnosing LSCC were 64.6% and 91.2%, respectively. CYFRA21-1 had the highest diagnostic sensitivity for LADC and LSCC. The diagnostic sensitivity and specificity of ProGRP for SCLC were 83.1% and 98.0%, respectively. When combined, CYFRA21-1 and CEA showed a high sensitivity (78.8%) and specificity (86.3%) for diagnosing LADC with an AUC of 0.891; CYFRA21-1 and SCCAg had a high sensitivity (84.1%) and specificity (87.3%) for diagnosing LSCC with an AUC of 0.912. NSE combined with ProGRP was highly sensitive (88.1%) and specific (98.0%) for diagnosis of SCLC, with an AUC of 0.952. For lung cancers of different pathological types, the combination of all the 5 tumor markers showed no significant differences in the diagnostic power from a combined detection with any two of the markers (P>0.05).@*CONCLUSION@#CEA, CYFRA21-1, SCCAg, NSE and ProGRP are all related to the pathological type of lung cancers and can be used in different combinations as useful diagnostic indicators for lung cancers.

Effect of artificial liver with double plasma molecular absorb system model on patients' platelets and corresponding treatment strategy / 北京大学学报(医学版)

OBJECTIVE@#To compare the effects of artificial liver treatment with double plasma molecular adsorption system(DPMAS) mode and traditional plasma exchange (PE) mode on platelets in patients, and to evaluate the clinical efficacy of recombinent human thrombopoietin (rhTPO) in the treatment of thrombocytopenia.@*METHODS@#A total of fifteen patients undergoing artificial liver with DPMAS model admitted to the Fifth Affiliated Hospital of Guangzhou Medical University from January 2018 to November 2020 were selected and included in the DPMAS group, and another 15 patients receiving PE were selected and included in the PE group. The improvement of clinical symptoms, such as fatigue, jaundice, oliguria, edema, etc. before and after artificial liver treatment was compared between the two groups, and the trend of blood routine (especially platelet), coagulation function and other indexes before and after treatment were compared between the two groups. The use of rhTPO and the number of platelets were recorded during treatment.@*RESULTS@#The improvement rate of clinical symptoms in DPMAS group was 86.67%, which was higher than that in PE group, but the difference was not statistically significant (P>0.05). There was no statistical significance in the outcome of the two groups within 90 days (P>0.05). There was no significant difference in white blood cell (WBC) and hemoglobin (HB) between the two groups after treatment (P>0.05). However, the level of platelet(PLT) in DPMAS group was significantly lower than that before treatment (P < 0.05), and was significantly lower than that in PE group (P < 0.05). After treatment, the international normalized ratio (INR) level in PE group was significantly improved (P < 0.05), but there was no significant difference in the INR level in DPMAS group (P>0.05). The patients in the DPMAS group received an average of (8.2±3.1) doses of rhTPO and (1.5±0.3) IU of platelet transfusions during hospitalization. In DMPAS group, platelets increased significantly after infusion of terbium.@*CONCLUSION@#Compared with PE mode, the artificial liver with DPMAS mode can reduce platelet levels in patients, but the application of rhTPO can stimulate platelet regeneration and increase platelet levels in the patients, thereby reducing the risk of bleeding due to platelet hypoplasia.

Effects of methacrylic anhydride gelatin hydrogel loaded with silver and recombinant human basic fibroblast growth factor on deep partial-thickness burn wounds in rabbits / 中华烧伤杂志

Objective: To investigate the effects of methacrylic anhydride gelatin (GelMA) hydrogel loaded with silver and recombinant human basic fibroblast growth factor (rh-bFGF) on deep partial-thickness burn wounds in rabbits. Methods: The experimental research method was adopted. Low-concentration GelMA materials, medium-concentration GelMA materials and high-concentration GelMA materials containing different concentrations of methacrylic anhydride (MA) were prepared, after adding photoinitiator, low-concentration GelMA hydrogels, medium-concentration GelMA hydrogels, and high-concentration GelMA hydrogels were obtained, respectively. The nuclear magnetic resonance spectroscopy was performed to detect the hydrogen nuclear magnetic resonance spectra of the above-mentioned three concentrations of GelMA materials, and to calculate the degree of substitution according to the spectrum diagram. The three-dimensional microstructure and pore size of 3 types of above-mentioned GelMA hydrogels were detected by field emission scanning electron microscopy (FESEM), with 9 samples measured. According to the selected concentration of MA, ten kinds of solutions of GelMA with different concentration of silver (silver-containing GelMA) were synthesized, and the silver-containing GelMA solution of each concentration was divided into three parts, and then exposed to ultraviolet light lasting for 20, 25, and 35 s, respectively. After adding photoinitiator,the corresponding silver-containing GelMA hydrogels were obtained. The residual degradation rate of silver-containing GelMA hydrogel with different photocrosslinking times was detected by collagenase degradation method at degradation of 12, 24, 36, and 48 h; and the time required for complete degradation was detected, and the sample number was 5. The inhibition zone diameter of GelMA hydrogel under above screened photocrosslinking times containing 10 concentrations of silver against Staphylococcus aureus was measured to reflect its antibacterial ability, and the sample numbers were all 5. The silver-containing GelMA hydrogel with statistical significance compared with the antibacterial circle diameter of the silver-containing GelMA hydrogel containing the lowest concentration (no silver) was considered as having antibacterial activity. The three-dimensional microstructure and pore size of the silver-containing GelMA hydrogels with antibacterial activity and the lowest drug concentration selected were detected by FESEM, and the sample numbers were all 9. The freeze-dried alone GelMA hydrogel and the freeze-dried silver-containing GelMA hydrogel were soaked in phosphate buffer solution for 24 h, respectively, then the swelling rate of the two GelMA hydrogel were calculated and compared by weighing method, and the sample number was 5. GelMA hydrogel containing silver and rh-bFGF, namely compound hydrogel for short, was prepared according to the preliminary experiment and the above experimental results. The appearance of the composite hydrogel was observed in general, and its three-dimensional microstructure and pore size were detected by FESEM. The deep partial-thickness burn wound was made on the back of 30 rabbits (aged 4-6 months, female half and half). Meanwhile, with the rabbit head as the benchmark, the wounds on the left side of the spine were treated as composite hydrogel treatment group, and the wounds on the right side were treated as gauze control group, and which were treated accordingly. On post injury day (PID) 3, 7, 14, 21, and 28, the healing of wounds in the two groups was observed. On PID 7, 14, 21, and 28, the wound healing area was recorded and the healing rate was calculated, with a sample number of 30. Data were statistically analyzed with analysis of variance for repeated measurement, one-way analysis of variance, and independent sample t test. Results: The substitution degree among low-concentration GelMA materials, medium-concentration GelMA materials, and high-concentration GelMA materials was significantly different (F=1 628.00, P<0.01). The low-concentration GelMA hydrogel had a loose and irregular three-dimensional spatial network structure with a pore size of (60±17) μm; the medium-concentration GelMA hydrogel had a relatively uniform three-dimensional spatial network and pore size with a pore size of (45±13) μm; the high-concentration GelMA hydrogel had the dense and disordered three-dimensional spatial network with a pore size of (25±15) μm, the pore sizes of 3 types of GelMA hydrogels were significantly differences (F=12.20, P<0.01), and medium concentration of MA was selected for the concentration of subsequent materials. The degradability of silver-containing GelMA hydrogels with different concentrations of the same photocrosslinking time was basically same. The degradation residual rates of silver-containing GelMA hydrogels with 20, 25, and 35 s crosslinking time at 12 h were (74.2±1.7)%, (85.3±0.9)%, and (93.2±1.2)%, respectively; the residual rates of degradation at 24 h were (58.3±2.1)%, (65.2±1.8)%, and (81.4±2.6)%, respectively; the residual rates of degradation at 36 h were (22.4±1.9)%, (45.2±1.7)%, and (68.1±1.4)%, respectively; the residual rates of degradation at 48 h were (8.2±1.7)%, (32.4±1.3)%, and (54.3±2.2)%, respectively, and 20, 25, and 30 s photocrosslinking time required for complete degradation of silver-containing GelMA hydrogels were (50.2±2.4), (62.4±1.4), and (72.2±3.2) h, and the difference was statistically significant (F=182.40, P<0.01), 25 s were selected as the subsequent photocrosslinking time. The antibacterial diameters of 10 types of silver-containing GelMA hydrogels against Staphylococcus aureus from low to high concentrations were (2.6±0.4), (2.5±0.4), (3.2±0.4), (12.1±0.7), (14.8±0.7), (15.1±0.5), (16.2±0.6), (16.7±0.5), (16.7±0.4), and (16.7±0.6) mm, respectively, and which basically showed a concentration-dependent increasing trend, and the overall difference was statistically significant (F=428.70, P<0.01). Compared with the silver-containing GelMA hydrogel with the lowest concentration, the antibacterial circle diameters of other silver-containing GelMA hydrogels with antibacterial ability from low to high concentration were significantly increased (with t values of 26.35, 33.84, 43.65, 42.17, 49.24, 55.74, and 43.72, respectively, P<0.01). The silver-containing GelMA hydrogel with the antibacterial diameter of (12.1±0.7) mm had the lowest antibacterial activity against Staphylococcus aureus and the lowest drug loading concentration, and the concentration of silver was selected for the concentration of subsequent materials. The microscopic morphology of the silver-containing GelMA hydrogel containing silver element with a pore size of (45±13) μm had a regular and linear strip-like structure. After soaking for 24 h, the swelling ratio of silver-containing GelMA hydrogel was similar to that of alone GelMA hydrogel. The composite hydrogel was colorless, clear and transparent, and its three-dimensional microstructure was a regular and uniform grid, with a filament network structure inside, and the pore size of (40±21) μm. On PID 3, a large amount of necrotic tissue and exudate of rabbit wound in composite hydrogel group were observed, and scattered scabs, a small amount of necrotic tissue and exudate of rabbit wound in gauze control group were observed. On PID 7, the area of rabbit wound in composite hydrogel group was significantly reduced, and adhesion of rabbit wound and gauze in gauze control group was observed. On PID 14, In composite hydrogel group, the rabbit wound surface was ruddy, and the growth of granulation tissue was observed, and in gauze control group, the rabbit wound base was pale, and the blood supply was poor. On PID 21, the rabbit wounds in composite hydrogel group healed completely, and rabbit wound in gauze control group had healing trend. On PID 28, new hair could be seen on rabbit wound surface in composite hydrogel group; oval wound of rabbit in gauze control group still remained. On PID 7, 14, 21, and 28, the wound healing areas of rabbit in composite hydrogel group were significantly larger than those in gauze control group (with t values of 2.24, 4.43, 7.67, and 7.69, respectively, P<0.05 or P<0.01). Conclusions: The medium-concentration GelMA hydrogel has good physical and chemical properties in terms of swelling and degradability. The screened silver-containing GelMA hydrogels had the lowest antibacterial activity and the lowest drug loading concentration. Composite hydrogel can significantly shorten the healing time of deep partial-thickness burn wounds in rabbits.