Diseño y evaluación de una proteína multiepitópica como candidata para vacuna contra la enfermedad de carrión / Design and evaluation of a multiepitopic protein as a candidate for a carrion disease vaccine

RESUMEN Objetivos. Diseñar y evaluar una proteína multiepítope como candidato a vacuna contra la enfermedad de Carrión. Materiales y métodos. Mediante herramientas bioinformáticas se seleccionó epítopes de proteínas de membrana externa y se diseñó una proteína multiepítope. El gen de la proteína multiepítope fue subclonado en el plásmido de expresión pET28b y transformado en E. coli BL21 pLys. La proteína multiepítope fue expresada usando isopropil-β-D-1-tiogalactopiranósido y purificada usando resina. Esta proteína purificada fue utilizada para inmunizar ratones BALB/c y se obtuvo anticuerpos policlonales. Se realizaron ensayos de invasión in vitro usando una cepa de Bartonella bacilliformis (B. bacilliformis) a eritrocitos humanos. Resultados. La proteína multiepítope M1 presenta epítopes conservados entre aislamientos de B. bacilliformis, no tóxicos, no homólogos a proteínas humanas y superficiales. Los ratones inmunizados presentaron niveles de anticuerpos IgG capaces de reducir in vitro la tasa de invasión de B. bacilliformis a eritrocitos humanos. Conclusiones. La proteína multiepítope M1 podría servir como candidato a vacuna contra la enfermedad de Carrión; sin embargo, se requiere de más estudios para caracterizar el uso de este antígeno como vacuna.

ABSTRACT Objectives. To design and assess a multiepitopic protein as a candidate for a vaccine against Carrion disease. Materials and Methods. Using bioinformatics tools, epitopes of external membrane proteins were selected and a multiepitopic protein was designed. The multiepitopic protein gene was subcloned into the expression plasmid pET28b and transformed into E. coli BL21 pLys. The multiepitopic protein was expressed using isopropyl-β-D-1-thiogalactopyranoside and purified using resin. This purified protein was used to immunize BALB/c mice obtaining polyclonal antibodies. In vitro invasion assays were conducted using a strain of Bartonella bacilliformis (B. bacilliformis) in human red blood cells. Results. The multiepitopic protein M1 presents preserved epitopes between isolates of B. bacilliformis with are non-toxic, and not homologous to human and surface proteins. Immunized mice presented IgG antibody levels capable of reducing in vitro the rate of invasion of B. bacilliformis into human red blood cells. Conclusions. Multiepitopic protein M1 may serve as a candidate for a Carrion disease vaccine; however, more studies are needed to characterize the use of this antigen as a vaccine.

Construction of eukaryotic expression vector of hypoxic inducible VEGF and its in vitro delivery application and functional identification / 解放军医学杂志

Objective To construct the eukaryotic expression vector of hypoxia inducible vascular endothelial growth factor (VEGF), and establish its in vitro delivery method. Methods Erythropoietin (EPO) enhancer was inserted into eukaryotic expression vector pGL4.73 [hRluc/SV40] (pSV) promoter by gene recombination technique to construct hypoxia inducible expression system (pEPO-SV). Renilla luciferase (Rluc) was used as downstream reporter gene. Then the VEGF165 gene was inserted into the pEPO-SV plasmid instead of Rluc, and the pEPO-SV-VEGF and pSV-VEGF expression vectors were obtained by inserting the pEPO-SV-VEGF gene into pSV as control. The pSV plasmid expressing Rluc or VEGF165 and pEPO-SV plasmid were transfected in vitro into human embryonic kidney 293T cells. The expression of Rluc or VEGF165 was used to identify the hypoxia induction function of the constructed vector after being treated under normal and hypoxic conditions for 24h and 48h. The intracellular delivery method of plasmids was then established based on poly (lactic acid-glycolic acid) copolymer (PLGA) nanoparticles as carrier, and the efficiency of the eukaryotic expression plasmids induced by hypoxia was evaluated under the in vitro hypoxia model. Results In the construction of plasmid, the successful insertion and correctness of EPO enhancer and VEGF165 gene were confirmed by restriction endonuclease digestion, PCR amplification and DNA sequencing. The plasmid expressing Rluc or VEGF165 was transfected into 293T cells respectively. There was no significant difference in the expression of reporter gene Rluc (one, plasmid pSV and pEPO-SV fluorescence expression values were 2448.24±158.51 and 3173.97±379.92, the second, plasmid pSV and pEPO-SV fluorescence expression values were 55 500.00±3237.05 and 51 193.18±866.32, respectively) or target gene VEGF165 in normal culture (P>0.05). But the expression of Rluc (In the cobalt chloride of hypoxia, the fluorescence expression values of pSV and pEPO-SV were 4857.70±1223.28 and 16 432.64±1618.73, respectively. In the hypoxia incubator, the fluorescence expression values of pSV and pEPO-SV were 2504.45±213.20 and 17 274.35±685.60, respectively) or VEGF165 in hypoxia was significantly higher than that in control group (P<0.01). The results showed that the constructed pEPO-SV and pEPO-SV-VEGF plasmids had typical hypoxia inducible expression activity. PLGA nanoparticles were used to in vitro deliver pEPO-SV and pSV in 293T cells. The results of detecting the reporter gene Rluc in normal culture and hypoxic conditions were consistent with those mentioned above, that is, under normal conditions, the 24h and 48h fluorescence expression values of plasmids pSV and pEPO-SV were 149.44±4.01 and 127.09±15.05, 1074.91±114.78 and 1064.56±137.48, respectively; under hypoxic conditions, the 24h and 48h fluorescence expression values of pSV and pEPO-SV were 3265.34±440.00 and 8828.87±637.03, 3202.06±33.43 and 9114.75±292.06, respectively. Conclusion A typical hypoxia inducible VEGF eukaryotic expression system has been successfully established, and an in vitro effective delivery method is also established, which may have an important application prospect in ischemia, hypoxia and other tissue injury diseases.

Construction and identification of the recombinant M13-IN5 phage and its effect on Chlamydia trachomatis / 中华皮肤科杂志

Objective To construct active phages against Chlamydia trachomatis,and to evaluate its effect on Chlamydia trachomatis.Methods The M13 phage was recombined with the IN5 sequences encoding the capsid protein VP1 of chlamydiophage phiCPG1,and then the recombinant M13-IN5 phage was obtained.PCR amplification,enzyme digestion and sequencing were performed to verify whether the target fragment was inserted into the phage successfully.The viability of the phage was evaluated by plaque formation assay.Cell counting kit-8 (CCK8) assay was conducted to evaluate the effect of M13 phage and recombinant M13-IN5 phage at the titer of 1011 plaque-forming units (PFU)/ml on the proliferation of Hela cells,and Hela cells uninfected with chlamydia served as the blank control group.Western blot analysis was performed to determine the expression of the IN5 loop protein in the recombinant M13-IN5 phage,M13 phage and Escherichia coli ER2738 at exponential growth phase.Cultured standard Chlamydia trachomatis serovar E strain was treated with M13 phage and recombinant M13-IN5 phage at the titer of 1011 PFU/ml separately,and chlamydia control group without the treatment with phages was set up.After 36-hour infection,confocal microscopy was performed to detect the location of the M13 phage and the recombinant M13-IN5 phage.Moreover,iodine staining was conducted to count inclusion bodies at 36,48,60 and 72 hours separately after infection.Statistical analysis was carried out by a two-sample t-test for comparisons between two groups,one-way analysis of variance (ANOVA) for intergroup comparison,and Bonferroni test for multiple comparisons.Results The bioactive recombinant M13 phage containing the IN5 loop gene was constructed successfully,and Western blot analysis confirmed that the recombinant phage expressed IN5 loop/p Ⅲ fusion protein with a high titer of 3.05 × 1011 PFU/ml.As CCK8 assay showed,there was no significant difference in proliferation of Hela cells among the blank control group,M 13 phage group and recombinant M13-IN5 phage group (A450 values:3.63 ± 0.01,3.55 ± 0.02,3.70 ± 0.01,respectively,F =12.0,P ＞ 0.05).Confocal microscopy showed overlap between the phage fluorescence and chlamydial inclusion body fluorescence.The M13-IN5 phage group and M13 phage group both showed significantly decreased number of inclusion bodies compared with the control group (both P ＜ 0.05) at 36 and 72 hours after chlamydial infection,and the number of inclusion bodies was significantly lower in the M 13-IN5 phage group than in the M13 phage group (P ＞ 0.05).After 48,and 60 hours of chlamydial infection,the number of inclusion bodies did not differ among the M13 phage group,M13-IN5 phage group and control group (both P ＞ 0.05).Conclusions The recombinant M13-IN5 phage was bioactive and could successfully express the IN5 loop protein.In the in vitro experiments,the recombinant phage could enter into chlamydia inclusion bodies,and markedly inhibited the infection of Chlamydia trachomatis.

Construction and identification of TSOL18 recombinant Lactococcus lactis secretory and non-secretory vaccines of Taenia solium / 中华地方病学杂志

Objective To construct recombinant non-secretory pMG36e-TSOL18/L.lactis and secretory pMG36e-SP-TSOL18/L.lactis vaccines of Taenia solium. Methods Taking sequence of Taenia solium TSOL18 gene as template, genetic optimization was carried out using lactic acid bacteria as a host system, TSOL18 gene and SP-TSOL18 gene were synthesized using PCR-based accurate synthesis (PAS), through the design of full-length primers, the addition of restriction enzyme cutting sites Sac Ⅰ and Hind Ⅲ and protective bases, and the SPUSP45 secretory signal peptide sequence in the N terminal. TSOL18 gene and SP-TSOL18 gene were cloned into Escherichia coli-L.lactis shuttle expression plasmid pMG36e to construct intracellular expression vector pMG36e-TSOL18 and secreted expression vector pMG36e-SP-TSOL18. The two recombinant plasmids were identified by enzyme digestion and sequencing, and electroporated into L.lactis MG1363 to construct the recombinant pMG36e-TSOL18/L.lactis and pMG36e-SP-TSOL18/L.lactis vaccines of Taenia solium, and they were identified by PCR. Results TSOL18 gene fragment and pMG36e vector fragment were obtained by double digestion with Sac Ⅰ and Hind Ⅲ, which were consistent with the expected results; TSOL18 gene standard sequence was aligned and the matching degree was 100％, and both were inserted into Sac Ⅰ and Hind Ⅲ of pMG36e vector. Our PCR results showed that both recombinant pMG36e-TSOL18/L.lactis and pMG36e-SP-TSOL18/L.lactis were 393 bp gene fragment products. Conclusion The recombinant pMG36e-TSOL18/L.lactis and pMG36e-SP-TSOL18/L.lactis vaccines of Taenia solium are successfully constructed.

Aplicación de la nanobiotecnología con el sistema CRISPR-cas / Application of the nanobiotechnology with the system CRISP-Cas

Resumen Introducción: La nanobiotecnología y la biología sintética son ciencias que impactan en la actualidad con el lanzamiento de aplicaciones innovadoras y beneficiosas para el ser humano, estas ciencias se han fusionado para fabricar nuevos componentes para la construcción de células totalmente artificiales y la creación de biomoléculas sintéticas. Objetivo: Conocer las aplicaciones de la nanobiotecnología relacionadas con el uso del sistema CRISPR/Cas en el almacenamiento de información en el ADN bacteriano y alternativas terapéuticas. Materiales y métodos: Se realizó una revisión bibliográfica sobre las principales aplicaciones de la nanobiotecnología, en las bases de datos ScienceDirect, SciELO, PubMed y en revistas como: Nature biotechnology, Biochemistry, Science y Journal Microbiology. Resultados: La revisión de literatura describe y analiza las nuevas aplicaciones nanobiotecnológicas utilizadas para escribir información en el código genético de las células bacterianas, en el que se emplean el sistema basado en repeticiones palindrómicas cortas agrupadas y regularmente interespaciadas (CRISPR/Cas) y la producción de ADN sintético, así como las alternativas terapéuticas relacionadas con la terapia génica. Conclusión: Entre las aplicaciones nanobiotecnológicas se han demostrado dos métodos para grabar información en el ADN de células bacterianas, de Escherichia coli y Sulfolobus tokodai vinculados con el empleo del sistema CRISPR/Cas y la producción de ADN sintético, así como el uso del CRISPR/Cas en la terapia génica y celular.

Abstract Introduction: Nanobiotechnology and synthetic biology are sciences that impact today with the launching of innovative and beneficial applications for the human being. These sciences have been amalgamated to manufacture new components for the construction of totally artificial cells and the creation of synthetic biomolecules. Objective: To know the applications of nanobiotechnology related to the use of the system CRISPR/Cas in the storage of bacterial DNA and therapeutic alternatives. Materials and methods: A bibliographical review on the main applications of nanobiotechnology was carried out in ScienceDirect, SciELO, PubMed databases and in magazines such as: Nature Biotechnology, Biochemistry, Science and Journal Microbiology. Results: The literature review describes and analyzes the new nanobiotechnology applications used to write information in the genetic code of bacterial cells, in which the system is used based on short grouped and regularly interspaced palindromic repetitions (CRISPR/Cas) and the production of synthetic DNA, as well as therapeutic alternatives related to gene therapy. Conclusion: Among the nanobiotechnology applications, two methods to record information in the DNA of bacterial cells Escherichia coli and Sulfolobus Tokodai have been shown, which are linked to the use of the system CRISPR/Cas and the production of synthetic DNA, as well as the use of CRISPR/Cas in gene and cellular therapy.

A new target for gene therapy-splicing factor PUF60 / 国际肿瘤学杂志

Poly(U)-binding-splicing factor 60 KDa (PUF60), a U2-related splicing factor that facilitates 3′splice-site recognition at the early stage of spliceosome assembly.High expression of PUF60 is related to the occurrence of multiple tumors, such as colorectal cancer, ovarian cancer, gastric cancer, liver cancer, et al.PUF60 can regulate the expression of c-myc through the core-human transcription factor Ⅱ basal transcription factor.Anti-PUF60 antibodies are detected in the sera of patients with early-stage and recurrent tumor, and the levels are significantly decreased after the operation.PUF60 can be used as a combined or independent test index for the diagnosis of cancer, which can be a potential new target for gene therapy.

Seroconversión frente a primovacunación reforzada contra hepatitis B en niños con cáncer / Seroconversion in response to a reinforced primary hepatitis B vaccination in children with cancer

Introducción: La respuesta inmune a los antígenos de las vacunas está disminuida en los niños con cáncer. El objetivo de este estudio fue evaluar la seroconversión frente a vacuna ADN recombinante contra hepatitis B al momento del inicio de la quimioterapia y/o remisión en niños con cáncer. Pacientes y método: Estudio prospectivo, bicéntrico, controlado, no aleatorizado de niños con diagnóstico reciente de cáncer pareados con niños sanos. Los casos fueron vacunados a tiempo 0, 1 y 6 meses, a dosis de 20 y 40 μg si eran < ó > 10 años, respectivamente, con vacuna ADN recombinante contra hepatitis B, en el momento del diagnóstico en el caso de los tumores sólidos y luego de la remisión en el caso de los tumores hematológicos. El grupo control recibió el mismo esquema, con dosis de 10 o 20 μg respectivamente. Se midieron anticuerpos séricos anti-HBs a los 2, 8 y 12 meses posvacunación. Seroconversión se definió como títulos anti-HBs > 10 mUI/ml al octavo mes. Resultados: Un total de 78 niños con cáncer y 25 controles fueron evaluados con títulos anti-HBs al octavo mes. La tasa de seroconversión fue de 26,9%, en niños con cáncer, sin diferencia por edad, género ni tipo de tumor (p = 0,13; 0,29; y 0,44, respectivamente), y de 100% en el grupo control (p < 0,0001, comparado con los niños con cáncer). En el seguimiento a los 12 meses solo el 31,9% de los niños con cáncer presentaba títulos anti-HBs > 10 mUI/ml. Conclusiones: La vacunación contra hepatitis B con vacuna ADN recombinante, con esquema reforzado de 3 dosis, en el momento del inicio de la quimioterapia y/o remisión provee una respuesta inmune insuficiente en la mayoría de los niños con cáncer. En esta población debieran evaluarse vacunas de tercera generación, con adyuvantes más inmunogénicos, esquemas reforzados a los 0, 1, 2 y 6 meses, medición de títulos de anticuerpos al octavo y duodécimo mes, eventual uso de refuerzos y reevaluación de inmunogenicidad si correspondiese.

Introduction: Immune response against vaccine antigens may be impaired in children with cancer. The aim of this study was to evaluate the seroconversion response against hepatitis B vaccination (HBV) at the time of chemotherapy onset and/or remission in children with cancer. Patients and method: Prospective, two-centre, controlled, non-randomised study conducted on children recently diagnosed with cancer, paired with healthy subjects. Cases received HBV at time 0, 1 and 6 months with DNA recombinant HBV at a dose of 20 and 40 μg if < or > than 10 years of age, respectively, at the time of diagnosis for solids tumours and after the remission in case of haematological tumours. Controls received the same schedule, but at of 10 and 20 μg doses, respectively. HBs antibodies were measured in serum samples obtained at 2, 8 and 12 months post-vaccination. Protective titres were defined as > 10 mIU/ml at 8th month of follow up. Results: A total of 78 children with cancer and 25 healthy controls were analysed at month 8th of follow up. Seroconversion rates in the cancer group reached 26.9%, with no differences by age, gender or type of tumour (P = .13, .29, and .44, respectively). Control group seroconversion was 100% at the 8th month, with P < .0001 compared with the cancer group. At month 12 of follow up, just 31.9% of children with cancer achieved anti-HBs antibodies > 10 mIU/ml. Conclusions: Vaccination against hepatitis B with three doses of DNA recombinant vaccine at an increased concentration, administrated at the time of onset of chemotherapy and/or remission provided an insufficient immune response in a majority of children with cancer. More immunogenic vaccines should be evaluated in this special population, such as a third generation, with more immunogenic adjuvants, enhanced schedules at 0, 1, 2, 6 month, evaluation of antibody titres at month 8 and 12 h to evaluate the need for further booster doses.

Construction of recombinant HIF-1α and NIS lentiviral expression plasmid and its functional identification / 中华核医学与分子影像杂志

Objective To construct a recombinant lentivirus vector containing the human NIS gene and HIF-1α with the myosin light chain-2v(MLC-2v) as a promoter and to investigate the specific expression and feasibility of NIS as a reporter gene in cardiomyocytes.Methods The target gene HIF-1α and NIS were subcloned into the lentivirus (Lv)-elongation factor (EF)1-HIF-1α-internal ribosome entry site (IRES)-NIS and Lv-MLC-HIF-1α-IRES-NIS lentivirus vectors.The recombinated vectors were transfected into Hela cells by lipofectamine 2000.The expression of HIF-1α and NIS in the transfected Hela cells was detected by indirect immunofluorescence and Western blot.The H9C2 cells were exposed to different multiplicities of infection (MOI; 5,10,20,40) with packaged virus particles.The infection efficiency was detected by Western blot.MOI 20 was used for H9C2,NIH-3T3 and L6 cell lines and the specificity of the MLC-2v promoter was detected by the count of NIS protein in the 3 different cell lines with Western blot.The function and features of NIS protein were evaluated by dynamic iodine uptake and NaClO4 iodine uptake inhibition tests in vitro.Two-sample t test was used to analyze the data.Results The two recombinant lentivirus vectors were constructed successfully.The HIF-1α protein was expressed in the cytoplasm and the NIS protein was expressed on the cell membrane in Hela cells.The grey levels of NIS and HIF-1α proteins in the positive control were 69.8 and 71.9,respectively,which were 109.4 and 92.7 after being prompted by EF1,and 141.9 and 132.4 by MLC-2v.The expression of these proteins was much higher by EF1 promoter than that by MLC-2v promoter.The optimal MOI for the Lv-MLC-HIF-1α-IRES-NIS virus to infect H9C2 cells was 20.With the MOI of 20,the grey levels of NIS protein promoted by EF1 were 23.4,29.8 and 28.6 for H9C2,NIH-3T3 and L6 cells infected with Lv-EF1-HIF-1α-IRES-NIS virus,respectively.The expression of NIS protein promoted by MLC-2v was much higher in H9C2 cells than the other two cell lines.The grey level of NIS protein was 157.9 in H9C2 cells,178.8 in L6 cells and 217.3 in NIH-3T3 cells.The NIS protein expressed in infected H9C2 cells showed high radioiodine uptake.The peak of iodine uptake was 4 287.2 counts · min-1 at 40 min which was 16.85 times of the control group (254.4 counts · min-1) (t=5.34,P＜ 0.01).The inhibition rate of iodine uptake was up to 85.5％ (3 666.4/4 287.2,t=21.3,P＜0.01) by NaClO4.Conclusions MLC-2v promoter allows specific expression of the external gene HIF-1α and NIS in myocardium.The cardiomyocytes transfected with NIS gene acquires the function of iodine uptake.Therefore,NIS may have a potential to be the reporter gene to monitor the external gene therapy in ischemic cardiomyopathy.

Bioterrorismo e a facilidade de acesso à biotecnologia e seus insumos / Bioterrorism and easy access to biotechnology and its inputs

Os fundamentalismos surgiram no Ocidente a partir de questões religiosas e posteriormente difundiram-se para outras partes do mundo tomando outras conotações, principalmente políticas. As técnicas de manipulação genética difundiram-se pelas universidades, que formam mestres e doutores com os conhecimentos básicos sobre clonagem gênica, que se tornou de domínio público. Todos os insumos para clonagem gênica podem ser adquiridos por meio de catálogos via internet. Podem-se recrutar profissionais fanáticos e com a competência para a manipulação genética de organismos patogênicos, lado perverso da biotecnologia. Os conflitos étnicos, culturais e religiosos estão associados a um cenário de contrastes entre os países ricos e carentes de matéria-prima e aqueles pobres, mas detentores de insumos básicos e energia, e atingem a sua forma mais aguda nos fundamentalismos. Grupos de fanáticos têm pleno acesso a essa biotecnologia. Estariam assim as populações civis vulneráveis aos ataques do bioterrorismo com armas biológicas geneticamente modificadas?.

Fundamentalism arose in the West based in religious matters and afterward diffused to other parts of theworld with other connotations, especially political. Genetic manipulation techniques spread to universities,which has given masters and doctors the basic knowledge on gene cloning, which has become public domain.All inputs for gene cloning may be obtained through online catalogs. Fanatic professionals may be recruited,with qualification for genetic manipulation of pathogenic organisms, the negative side of biotechnology. Eth-nic, cultural and religious conflicts are linked to a series of contrasts between countries that are rich but witha lack of raw materials and the poor countries that possess basic input and energy sources, when it reachesthe highest fundamentalist form. Fanatic groups have complete access to this biotechnology. Are civilian po-pulations in vulnerable to bioterrorist attacks involving genetically modified biological weapons?

Construction and titration of rat CGRP gene recombinant lentivirus / 重庆医学

Objective To construct lentiviral vector carrying rat′s calcitonin gene-related peptide(CGRP) gene for the following-up study on the function of CGRP .Methods CGRP gene segment was subcloned into shuttle plasmid ,become Puc57-CGRP .The pLenO-DCE-CGRP expression vector was be constructed by double digests .The pLenO-DCE-CGRP and 4 auxiliary packaging plas-mids were co-transfected into 293T cells .Cells were cultured for 48 hours .The supernant was collected and concentrated ,and then the viral titers were tested by multiple proportions dilution method and flow cytometer .The expression levels of CGRP were detec-ted in CGRP-modified 293T cells by Real-time PCR .Results The results of digestion and sequencing show that the pLenO-DCE-CGRP vector was constructed successfully .The titer of the lentiviral particles was 5 .1 × 108 TU /mL .Conclusion The high-titer lentvirus vector containing CGRP gene is constructed successfully ,which lay a foundation for transfecting mesenchymal stem cell (MSC) and studying the function of CGRP .

The effects of miR-181a on proliferation, migration and invasion abilities of esophageal carcinoma cell line TE11 / 肿瘤

Objective: To investigate the effect of up-regulation of miR-181a expression mediated by constructing miR-181a recombinant eukaryotic expression vector pcDNA3.1(+)-miR-181a on cell proliferation, migration and invasion abilities of esophageal carcinoma cell line TE11. Methods: PCR primers were designed and miR-181a precursor sequence was amplified from 95C cell genomic DNA. The product fragments were cloned into pcDNA3.1(+) to construct the recombinant vector pcDNA3.1(+)-miR-181a. The recombinant eukaryotic expression vector pcDNA3.1(+)-miR-181a was transfected into TE11 cells. The expression of miR-181a mRNA was detected by real-time fluorogenic quantitative-PCR (RFQ-PCR). The effects of pcDNA3.1(+)-miR-181a on cell proliferation, migration and invasion abilities of TE11 cells were detected by MTT, wound healing and Boyden chamber methods, respectively. Results: The miR-181a recombinant eukaryotic expression vector pcDNA3.1(+)-miR-181a was successfully established. RFQ-PCR revealed that the mature miR-181a was able to effectively express in TE11 cells transfected with recombinant vector pcDNA3.1(+)-miR-181a (P<0.05). The overexpression of miR-181a could significantly increase the proliferation, migration and invasion abilities of TE11 cells. Conclusion: Overexpression of miR-181a can increase cell proliferation, migration and invasion abilities of esophageal carcinoma TE11 cells. These results may provide experiment references for further research of the role of miR-181a in cancer development and progression. Copyright© 2011 by Tumor.

Genetic optimization, recombinant expression and functional study of human augmenter of liver regeneration / 第二军医大学学报

Objective: To construct recombinant expression vector of human augmenter of liver regeneration (ALR) and study its protective effect on liver function. Methods: ALR cDNA was synthesized and inserted into expression vector pET28a+. The recombinant plasmid was tranformed into BL21 and the expression of ALR was induced by isopropyl-β-D-thiogalactoside(IPTG). MTT method was used for cell proliferation assay; the protective effect of recombinant product on liver function was observed in CCl4-induced acute toxic mouse model. Results: Recombinant expression plasmid of ALR was confirmed correct by restriction enzyme digestion and sequencing. The purified expression product had strong stimulatory effect on hepatocyte proliferation. Low and medium dosages of expression product decreased aminotransferase level in acute chemical injury mouse model. Conclusion: The recombinant expression vector of ALR has been correctly constructed and the expressed rALR can simulate hepatocyte regeneration.

Construction and verification of replication-deficient recombinant adenovirus to silence CTGF gene by AdEasy system / 肿瘤

Objective:To design and construct the replication-deficient recombinant adenovirus Ad-siCTGF which can silence the expression of connective tissue growth factor (CTGF) by RNA interference and verified its function. Methods:A specific sequence, which was verified to be able to silence CTGF gene with high efficiency, was cloned into pSES-HUS vector to produce the shuttle plasmid pSES-siCTGF. The plasmid after Pme Ⅰ linearization was cotransduced with pAdEasy into BJ5183 E.coli strains to construct recombinant vector Ad-siCTGF. After linearization treatment with Pac Ⅰ enzyme digestion Ad-siCTGF was transfected into HEK293 cells via liposome mediation. The recombinant adenovirus was packaged. The titer of the Ad-siCTGF was increased after three times of cross-infection. 4T1 cells were infected with the adenovirus. The silencing efficiency was tested by real-time fluorescence quantitative (RFQ)-PCR and Western blotting.Results:Pac Ⅰ enzyme digestion electrophoresis indentified that recombinant adenovirus was successfully constructed. The titer of the recombinant adenovirus Ad-siCTGF was 2.6×10~(10) pfu/mL after amplification and purification. The CTGF mRNA and protein expression in 4T1 cells were decreased by 36.27% and 31.56%, respectively, compared with the control groups.Conclusion:The recombinant adenovirus which can silence the expression of CTGF was successfully constructed. It laid a good foundation for further investigation of the action mechanism of CTGF in tumor cells.

Construction and identification of a recombinant adenovirus vector with indoleamine 2,3-deoxygenase and chimeric albumin promoter / 中华普通外科杂志

Objective To construct a recombinant adenovirus vector encoding for indoleamine 2,3-dioxygenase(IDO)and chimetric albumin promoter,evaluate the mRNA and protein expression levels in Hepa 1-6cell.Methods Full-length mouse derived IDO cDNA was subeloned into pAdTraek-ALB shuttle Plasmid.The product was linearized to homologous recombination with AdEasy-l vector in BJ5183 bacteria.The positive clone was identified by restriction endonuclease digestion and further confirmed by sequencing.The recombined adenoviruses DNA were transfected into AD-293 cells for packaging and amplification of Ad-ALB/IDO.The expression of IDO was monitored by RT-PCR and EGFP fluorescence in infected cells.The recombinant viruses with Hepa 1-6 cells were cultured and the mRNA and protein expression levels monitored bv RT-PCR and Western blot, respectively. Results Construction of recombinant andenoviruses containing IDO and albumin promoter was confirmed by restriction endonuclease digestion and sequencing.The expression of IDO was identified by RT-PCR in transfected AD-293 cell.The virus titer was 2.9×10~6 pfu/ml.The IDO mRNA and protein expression levels were detectable after transfected Hepa 1-6 cells by RT-PCR and Western blot. Conclusion A recombinant adenovirus Ad-ALB/IDO was susceessfullyconstructed.

Construction and identification of recombinant Bb-Eg95 vaccine of Echinococcus granulosus / 中国地方病学杂志

Objective To construct and identify recombinant Bifutobacteria (rBb)-Eg95 vaccine of Echinococcus granulosus (Eg). Methods The total RNA was extracted from hydatid cyst protoscoleces shattered by ultrasound, Eg95 antigen encoding gene was obtained by reverse transcription-polymerase chain reaction(RT-PCR) from the template of total RNA using the primer designed according to the DNA sequence of Eg95, the gene was cloned into Escherichia coli-Bifutobacteria(E.coli-Bb) shuttle plasmid pGEX-1λT and transformed into E.coli BL2 (DE3) competent cell to construct recombinant plasmid pGEX-Eg95 using BamH Ⅰ and EcoR Ⅰ, the recombinant plasmid was identified by restriction endonuclease digestion, then was electroporated into Bb to construct rBb-Eg95 vaccine, the vaccine was identified by PCR. Results Four hundred and seventy-one bp Eg95 gene was amplified by RT-PCR, the products of restriction endonuclease digestion were the same as expected(471 bp Eg95 gene and 4947 bp pGEX-1λT), 471 bp Eg95 gene fragment was amplified by PCR from the template of pGEX-Eg95 extracted from rBb vaccine. Conclusion rBb-Eg95 vaccine of Eg is successfully constructed, which lays the theoretical foundation for exploitation and utilization of this vaccine.

Transcatheter arterial chemoembolization combined with tumor necrosis factor for primary hepatic cancer / 肿瘤

Objective: To observe and compare short-term therapeutic effect and adverse effect of recombinant mutant human tumor necrosis factor (rmhTNF) via transcatheter arterial chemoembolization in the treatment of primary hepatic cancer. Methods: Sixty patients with confirmed primary hepatic cancer in clinic were randomly divided into two groups (n = 30). Group A (therapeutic group) received conventional TACE first and then the catheter was inserted into the hepatic artery and rmhTNF, chemotherapeutic drugs, and hyper liquid iodide oil were emulsified and infused to induce intraarterial embobilization. Group B (control group) received one cycle of conventional TACE and the second cycle of conventional TACE 1 month later. After two cycles of interventional treatment the short-term therapeutic effect and adverse reaction of the two groups were evaluated. Results: Comparison of two groups, the tumor volume were obviously reduced in the therapeutic group. There was significant difference in the short-term therapeutic efficacy (P 0.05). Some patients experienced shiver after infusion of rmhTNF. Conclusion: TACE combined with rmhTNF is an effective method for primary hepatic cancer with good short-term outcome and no obvious adverse reaction. It is worthy of popularization in clinic.

Genetic diagnosis of two haemophilia families with recombination / 中华检验医学杂志

objective To make genetic diagnosis in two haemophilia families with recombination. Methods For hemophilia A(HA)family,screening of the F Ⅷ intron 22 and intron 1 inversion mutations was employed to identify the mutation. Linkage analysis with 8 polymorphic markers was adopted in the pedigree. For hemophilia B(HB)family,DNA sequencing of all coding regions of FⅨ gene Was used to detect the mutation directly. The muhifluorescent PCR method employing six FⅨ related STR was adopted in linkage analysis.Results In the HA family,the proband was positive in inversion 1 detection and the relative female was inversion 1 carrier. But linkage analysis with polymorphic markers showed contrary resuhs. Some markers certified that the female inherited the disease chromosome of the family while the others showed contrary results.In the HB family,it was unsuccessful in sequencing the exon 7 of the F Ⅸ gene in the proband and there was no mutation found in the other parts. The relative female and her amniocyte DNA were successful in sequencing the whole F Ⅸ gene and no mutation was detected.The linkage analysis of the family showed contrary results. Recombination occured in these two families. Conclusions Although the linkage analysis iS convenient and effective in carrier and prenatal diagnosis of hemophilia families. The recombination risk shouldn't be neglected especially when the polymorphic markers give inconsistent information for linkage analysis. It is necessary to find some high inforrnative markers intragenic or on the telomeric side to the gene in order to prevent the risk of recombination.

Inhibitory effects of recombinant human parvovirus H-1 vector expressing p21 (rhH1 Δ/p21) on the growth of human gastric cancer cell line HGC27 / 肿瘤

Objective: To investigate the effects of recombinant parvovirus H-1 vector expressing p21(rhH1 Δ p21) on human gastric cancer cell line HGC27, and to further reveal the biological function of p21wif1 to provide the basis for cancer gene therapy. Methods: The recombinant parvovirus H-1 vector expressing p21 (rhH1 Δ/p21) was constructed by reverse transcriptase-polymerase chain reaction (RT-PCR), and was transfected into HGC27 cell line. The morphological changes of HGC27 cell were observed. The transgene protein expressions in the gastric cancer cells were detected by Western blot. The inhibitory effects of rhH1 Δ/p21 on the growth of HGC27 cells were measured by MTT assay. The cell cycle distribution was determined by flowcytometry. Results: The rhH1 Δ/p21 was successfully constructed, with a titer of 3.5 × 107 PFU/mL. The transgene protein p21 was over-expressed in the HGC27 cells. The cell cycle distribution was changed. The proportion of cells in G1 phase significantly increased. The cell growth was significantly inhibited. Conclusion: rhH1 Δ/p21 induces G1 arrest and inhibits the proliferation of gastric cancer HGC27 cells. It indicates that rhH1 Δ/p21 gene therapy can effectively inhibit the growth of gastric cancer cells in vitro.

High-level Expression of Foreign Genes In vivo and In vitro by Improved DNA-Based Replicon Vector Derived From Semliki Forest Virus / 生物化学与生物物理进展

The design of DNA-based alphavirus vectors significantly improves the utility of these replicon vectors. The DNA-based replicon vectors can be used in expressing foreign genes and preparing RVP in virto efficiently, also in developing replicon vaccines and gene therapy vectors in vivo. The approach involved the conversion a RNA-based replicon vector into a layered DNA-based replicon vector by the RNA polymerase Ⅱ promoter and transcription termination/polyadenylation signal transcribed replicon RNA from DNA. When DNA-based alphavirus vector tranfected into cells, the first layer includes a eukaryotic RNA polymerase Ⅱ expression cassette that initiates transcription of RNA in nucleus. Following transport of this RNA from the nucleus to the cytoplasm, the second layer, autocatalytic amplification of the RNA vector corresponds to virus RNA replication cycle and results in high level expression of foreign gene. DNA and RNA-based bifunctional replicon expression vector pSCTA and helper vector pSHCTA were successfully constructed by replacing the SP6 promoter used in the original system pSFV1 and pSFV-helper2 derived from Semliki Forest virus (SFV) with CMV promoter and T7 promoter, and inserting BGH transcription termination and polyadenylation signal downstream 3′-untranslated region (UTR). In order to obtain DNA-based highly efficient replicon vectors, they were further modified to construct additional three DNA-based SFV replicon expression vectors and corresponding helper vectors. To investigate the efficiency of foreign gene expression level by the four different DNA-based SFV expression vectors and recombinant virus particle (RVP) prepared by cotranfecting with corresponding helper vectors, improved DNA-based replicon vectors pSCAR and pSHCAR derived from SFV were developed. high level protein could be generated using the new vector system by transfecting DNA into BHK21 cells and High titer of RVP produced by cotranfecting with helper vector. Antigen genes were also expressed in cells by the replicon expression vector. Additionally, reporter gene expression was observed in mice muscle following injection with SFV DNA vector. Anti-?-Gal antibody response and cell-mediated immune response were induced after intramuscular inoculation of the ?-Gal-encoding SFV replicon DNA. The results suggested that highly efficient DNA-based replicon vectors pSCAR and pSHCAR were constructed by modifying the SFV vectors. The improved DNA-based replicon vectors enhance the utility of them, and can be developed as potentially replicon vaccines and gene therapy vectors.

The changes of GABA and Glu in rat inferior colliculus after unilateral cochlear ablation / 中国耳鼻咽喉头颈外科

OBJECTIVE To observe the changes of GABA and Glu in rat inferior colliculus following unilateral cochlear ablation and explore the function and significance of GABA and Glu in reorganization of auditory center after deafferentation. METHODS Twenty-five Sprangue Dawley (SD) rats were divided randomly into 5 groups. The content of GABA and Glu were measured by 835-50 type Amino Acid Automatism Analyzer and compared at 1 week, 2 week and 1 month after unilateral cochlear ablation respectively. RESULTS Compared with sham operated groups, the content levels of GABA decreased (from 78.00?7.50 to 51.65?10.36, about decreasing 33.6 %)1 week after unilateral cochlear ablation and there was a significant difference in GABA levels between 2 groups(P0.05). CONCLUSION The dynamic change of GABA and Glu in rat inferior colliculus reflected the neuronal activity, which implied both GABA and Glu may play an important role in reorganization of auditory center after unilateral cochlea ablation.