Expression optimization and preliminary identification of recombinant monoclonal antibody IgA against SARS-CoV-2 / 中国生物制品学杂志

@#Objective To prepare monoclonal antibody IgA against severe acute respiratory syndrome coronavirus 2(SARSCoV-2),optimize relevant expression conditions to increase the expression level,and preliminarily explore the effect of IgA antibody on anti-SARS-CoV-2.Methods The plasmid encoding IgAl-F61 antibody sequence was mixed with polyethyleneimine(PEI) transfection reagent and then transfected into EXPi293F~(TM) cells to transiently express antibody protein;The optimal culture conditions and expression levels of monomeric IgAl(mIgAl)-F61 and dimeric IgAl(dIgAl)-F61 in EXPi293F~(TM) cells were determined by optimizing the ratio of heavy chain(Hc),light chain(Lc) and joining chain(Jc),the proportion of plasmid and PEI,and the harvest time after transfection.The supernatant after transfection was purified by affinity chromatography,and then determined for the concentration by BCA,analyzed for the expression integrity and purity of antibody by SDS-PAGE and size exclusion chromatography-high performance liquid chromatography(SEC-HPLC),and detected for the neutralizing activity of antibody by pseudovirus neutralization assay.Results The optimal expression level of mIgAl-F61 was 123.45 μg/mL and the purity of purified antibody was over 95% when the ratio of Hc to Lc was 1:2,the ratio of plasmid to PEI was 1:3,and the supernatant was harvested 5 d after transfection;The highest purity of dIgAlF61 was more than 90% when the ratio of Hc:Lc:Jc was 1:2:1.The results of pseudovirus neutrali-zation assay against Omicron BA.4/5 showed that dIgAl-F61 exhibited better neutralizing activity than IgG-F61,and the value of half maximum inhibitory concentration(IC_(50)) was reduced by about 4 times.Conclusion Recombinant monoclonal antibodies mIgAl-F61 and dIgAl-F61 against SARS-CoV-2 were successfully expressed with high purity and dIgAl showed better neutralizing activity than IgG in vitro.

Expression optimization and preliminary identification of recombinant monoclonal antibody IgA against SARS-CoV-2 / 中国生物制品学杂志

@#Objective To prepare monoclonal antibody IgA against severe acute respiratory syndrome coronavirus 2(SARSCoV-2),optimize relevant expression conditions to increase the expression level,and preliminarily explore the effect of IgA antibody on anti-SARS-CoV-2.Methods The plasmid encoding IgAl-F61 antibody sequence was mixed with polyethyleneimine(PEI) transfection reagent and then transfected into EXPi293F~(TM) cells to transiently express antibody protein;The optimal culture conditions and expression levels of monomeric IgAl(mIgAl)-F61 and dimeric IgAl(dIgAl)-F61 in EXPi293F~(TM) cells were determined by optimizing the ratio of heavy chain(Hc),light chain(Lc) and joining chain(Jc),the proportion of plasmid and PEI,and the harvest time after transfection.The supernatant after transfection was purified by affinity chromatography,and then determined for the concentration by BCA,analyzed for the expression integrity and purity of antibody by SDS-PAGE and size exclusion chromatography-high performance liquid chromatography(SEC-HPLC),and detected for the neutralizing activity of antibody by pseudovirus neutralization assay.Results The optimal expression level of mIgAl-F61 was 123.45 μg/mL and the purity of purified antibody was over 95% when the ratio of Hc to Lc was 1:2,the ratio of plasmid to PEI was 1:3,and the supernatant was harvested 5 d after transfection;The highest purity of dIgAlF61 was more than 90% when the ratio of Hc:Lc:Jc was 1:2:1.The results of pseudovirus neutrali-zation assay against Omicron BA.4/5 showed that dIgAl-F61 exhibited better neutralizing activity than IgG-F61,and the value of half maximum inhibitory concentration(IC_(50)) was reduced by about 4 times.Conclusion Recombinant monoclonal antibodies mIgAl-F61 and dIgAl-F61 against SARS-CoV-2 were successfully expressed with high purity and dIgAl showed better neutralizing activity than IgG in vitro.

Impact of metabolic enzymes overexpression on transient expression of anti-hLAG3 by CHO cells / 生物工程学报

To enhance recombinant protein production by CHO cells, We compared the impact of overexpression of metabolic enzymes, namely pyruvate carboxylase 2 (PYC2), malate dehydrogenase Ⅱ (MDH2), alanine aminotransferase Ⅰ (ALT1), ornithine transcarbamylase (OTC), carbamoyl phosphate synthetase Ⅰ (CPSⅠ), and metabolism related proteins, namely taurine transporter (TAUT) and Vitreoscilla hemoglobin (VHb), on transient expression of anti-hLAG3 by ExpiCHO-S. Overexpression of these 7 proteins could differentially enhance antibody production. OTC, CPSI, MDH2, and PYC2 overexpression could improve antibody titer by 29.2%, 27.6%, 24.1%, and 20.3%, respectively. Specifically, OTC and MDH2 could obviously improve early-stage antibody production rate and the culture period was shortened by 4 days compared with that of the control. In addition, OTC and MDH2 had little impact on the affinity of anti-hLAG3. In most cases, overexpression of these proteins had little impact on the cell growth of ExpiCHO-S. MDH2 and ALT1 overexpression in H293T cells could also improve antibody production. Overall, overexpression of enzymes involved in cellular metabolism is an effective tool to improve antibody production in transient expression system.

Transient expression of a green fluorescent protein in tobacco and maize chloroplast

BACKGROUND: Maize is one of the most important crops worldwide and has been a target of nuclear-based transformation biotechnology to improve it and satisfy the food demand of the ever-growing global population. However, the maize plastid transformation has not been accomplished due to the recalcitrant condition of the crop. RESULTS: In this study, we constructed two different vectors with homologous recombination sequences from maize (Zea mays var. LPC13) and grass (Bouteloua gracilis var. ex Steud) (pZmcpGFP and pBgcpGFP, respectively). Both vectors were designed to integrate into rrn23S/rrn16S from an inverted repeat region in the chloroplast genome. Moreover, the vector had the mgfp5 gene driven by Prrn, a leader sequence of the atpB gene and a terminator sequence from the rbcL gene. Also, constructs have an hph gene as a selection marker gene driven by Prrn, a leader sequence from rbcL gene and a terminator sequence from the rbcL gene. Explants of maize, tobacco and Escherichia coli cells were transformed with both vectors to evaluate the transitory expression­an exhibition of green and red fluorescent light under epifluorescence microscopy. These results showed that both vectors were expressed; the reporter gene in all three organisms confirmed the capacity of the vectors to express genes in the cell compartments. CONCLUSIONS: This paper is the first report of transient expression of GFP in maize embryos and offers new information for genetically improving recalcitrant crops; it also opens new possibilities for the improvement in maize chloroplast transformation with these vectors.

Evaluación del potencial biotecnológico de un promotor derivado del virus de la distorsión de la hoja de maracuyá (PLDV), un begomovirus que infecta maracuyá / Evaluation of the biotechnological potential of a promoter derived from passion fruit leaf distortion virus (PLDV), a begomovirus that infects passionfruit

RESUMEN Los avances biotecnológicos en plantas requieren la bioprospección de nuevos promotores para la expresión de genes de interés agronómico, en particular, es necesario caracterizar nuevos promotores con expresión tejido específica. El objetivo de esta investigación fue evaluar la actividad de expresión del promotor del gen AV1 que codifica para la proteína de la cápside (CP) del virus de la distorsión de la hoja de maracuyá (Passion fruit leaf distortion virus, PLDV) mediante ensayos transitorios de biobalística de baja presión. Se realizó un análisis de la región promotora del gen AV1 empleando herramientas bioinformáticas. Se construyó una fusión traduccional (CP-PLDV-GUS), que porta la región promotora del gen AV1 de PLDV fusionada al gen reportero uidA (GUS). CP-PLDV-GUS fue bombardeado sobre hojas de plántulas de tabaco cultivadas in vitro empleando una pistola de genes. Como control positivo se utilizó el plásmido pBI121 que porta el gen GUS bajo el control del promotor 35S de CaMV. Se llevaron a cabo 11 repeticiones, donde la unidad experimental fue la hoja y la variable de respuesta, la expresión transitoria del gen GUS representado por el número de puntos azules observados en las hojas bombardeadas. Como resultado, el análisis estadístico no paramétrico demostró que existe evidencia muestral suficiente para confirmar que, tanto el promotor AV1 del PLDV y 35S de CaMV presentan una actividad de expresión semejante. Finalmente, el promotor del gen AV1 de PLDV mostró una fuerte actividad de expresión del gen reportero en las células del mesófilo de las hojas, el cual podría ser usado para conferir expresión tejido específica en plantas transgénicas.

ABSTRACT Biotechnological advances in plants require the bioprospecting of new promoters for the gene´s expression of agronomic interest, in particular, it is necessary to characterize new promoters with tissue-specific expression The objective of this research was to evaluate the expression activity of the AV1 gene promoter that codes for the capsid protein (CP) of the Passion fruit leaf distortion virus (PLDV) by means of transient tests of low pressure biobalistics. An analysis of the promoter region was carried out using bioinformatics tools. A CP-PLDV-GUS translational fusion was constructed, which carries the promoter region of the AV1 gene of PLDV fused to the uidA reporter gene (GUS). CP-PLDV-GUS was bombarded on leaves of tobacco seedlings grown in vitro using a gene gun. As a positive control pBI121 carrying the GUS gene under the control of the 35S promoter of CaMV was used. It was carried out 11 repetitions where the experimental unit was the leaf and the response variable the transient expression of the GUS gene represented by number of blue dots observed in the bombarded leaves. As a result, the non-parametric statistical analysis showed that there is sufficient sample evidence to confirm that both the AV1 promoter of PLDV and 35S of CaMV exhibit similar expression activity. Finally, the promoter of the AV1 gene of PLDV showed a strong activity of expression of the reporter gene in the leaf mesophyll cells, which could be used to confer tissue-specific expression in transgenic plants.

Cloning and activity analysis of AvTPS1 promoter of terpene synthase from Amomum villousm / 中草药

Objective To clone the unknown sequence of terpene synthase (TPS) AvTPS1 promoter from Amomum villousm and analyze its activity. Methods In this research, AvTPS1 DNA sequence was amplified and cloned from genomic DNA (gDNA) of A. villousm leaves. Furthermore, the promoter of AvTPS1 was cloned by FPNI-PCR and the sequence was analyzed. The recombinant vector pCAM-AvTPS1p with AvTPS1 promoter for the expression of GUS gene was constructed. The activity of AvTPS1 promoter was verified by transient expression of Agrobacterium-mediated infiltration using the leaves of Nicotiana benthamiana. Results The gene sequence of AvTPS1 was 2 444 bp including seven exons and six introns. The 568 bp AvTPS1 promoter was successfully cloned using FPNI-PCR. Furthermore the sequence had ten kinds of cis-elements including conserved elements TATA-box, CAAT-box, MYC2 related element G-box and other elements. Finally, the GUS staining showed the tobacco leaves infiltrated by the pCAM-AvTPS1p were blue. Conclusion The AvTPS1 promoter can drive the transcription of GUS gene and then it was verified to have the promoter activity. These results give foundation for future research on the function of AvTPS1 involved in the terpenoid biosynthesis and its relationship with the transcription factors.

Prokaryotic expression of Aquilaria sinensis AsMAPK3 gene and its localization in transient expression system in onion epidermis / 中草药

Objective To construct a prokaryotic vector of AsMAPK3 gene from Aquilaria sinensis, the original plant of agarwood, and induce the recombinant proteins expression so as to study the subcellular localization of AsMAPK3. This work will prepare materials for antibody preparation and lay a foundation for screening the interaction proteins and further studying their functions. Methods Partial cDNA sequence was amplified by PCR and recombined to pET-28a vector to construct a prokaryotic expression vector pET-28a-AsMAPK3, and induced the expression of the fusion protein. The full-length cDNA of AsMAPK3 was amplified and subcloned to pAN580 vector to construct a pAN580-AsMAPK3 transient expression vector. The recombinant plasmid of pAN580-AsMAPK3 was introduced into the onion epidermis by gold particle bombardment, and GFP fluorescence was observed by luorescence microscope. Results The Escherichia coli BL21 (DE3) containing the recombinant plasmid was induced with 0.5 mmol/L isopropyl-β-D-galactoside (IPTG) at 37 ℃ for 4 h, and a fusion protein about 39 000 was obtained which was expressed in supernatant and inclusion bodies. The results of GFP fluorescence observation of transient transformed onion epidermis showed that AsMAPK3 was mainly expressed in the nucleus and plasma membrane. Conclusion The expression and purification of AsMAPK3 in vitro were successfully carried out, and the subcellular localization of AsMAPK3 gene was confirmed. This work provides a substantial foundation for follow-up function study of AsMAPK3.

Construction of an RNAi vector for knockdown of GM-ACS genes in the cotyledonary nodes of soybean

Background: Ethylene plays an important role in the regulation of floral organ development in soybean, and 1-aminocyclopropane-1-carboxylate synthase (ACS) is a rate-limiting enzyme for ethylene biosynthesis. However, whether ACS also regulates floral organ differentiation in soybean remains unknown. To address this, we constructed an RNAi vector to inhibit ACS expression in cotyledonary nodes. Linear DNA cassettes of RNAi-ACS obtained by PCR were used to transform soybean cotyledonary nodes. Results: In total, 131 of 139 transiently transformed plants acquired herbicide resistance and displayed GUS activities in the new buds. In comparison to untransformed seedling controls, a greater number of flower buds were differentiated at the cotyledonary node; GM-ACS1 mRNA expression levels and ethylene emission in the transformed buds were reduced. Conclusion: These results indicate that the cotyledonary node transient transformation system may be suitable for stable transformation and that the inhibition of ACS expression may be an effective strategy for promoting floral organ differentiation in soybean.

Detection of Serum Antibodies to Hepatitis E Virus Based on HEV Genotype 3 ORF2 Capsid Protein Expressed in Nicotiana benthamiana

BACKGROUND: Hepatitis E virus (HEV) causes epidemics in developing countries and is primarily transmitted through the fecal-oral route. There have been recent reports on the zoonotic spread of the virus, and several animal species, primarily pigs, have been recognized as reservoirs of HEV. Because of its possible spread, there is an urgent need of a method for the cost-effective production of HEV proteins that can be used as diagnostic antigens for the serological detection of anti-HEV antibodies. METHODS: The HEV open reading frame (ORF)2 protein was purified from plant tissue by using immobilized metal-anion chromatography (IMAC). The recombinant protein was used to develop an in-house ELISA for testing anti-HEV antibodies in both human and swine sera. Thirty-six serum samples collected from patients with serologically proven HEV infection with commercial kits were tested for anti-HEV IgG antibodies by using the plant-expressed protein. Forty-five serum samples collected from apparently healthy pigs in Bulgarian farms were also tested. RESULTS: We confirmed the transient expression and purification of a truncated version of the HEV genotype 3 capsid protein in Nicotiana benthamiana and its usefulness as a diagnostic antigen. ELISA showed the presence of anti-HEV IgG antibodies in 29 of the 36 human samples. The in-house ELISA showed anti-HEV IgG antibodies in 34 of the 45 pigs. CONCLUSIONS: We describe a method for the production of HEV ORF2 protein in N. benthamiana and the usefulness of this protein for the serological detection of anti-HEV antibodies in both humans and swine.

Establishment of genetic transformation system for Sophra alopecuroides and deletion analysis of SaLDC promoter / 中国中药杂志

Establishing the genetic transformation system of medicinal plant is important to study their functional genes. Based on the established regeneration system of Sophra alopecuroides, 6 factors of genetic transformation were optimized, that was the concentration of Agrobacterium tumefaciens, the infection time, the co-cultivation time of agrobacterium tumefaciensand S.alopecuroides callus, the preculture time of S.alopecuroides callus, the adding method ofacetosyringone (AS) and the concentration of AS, respectively. The results showed that a maximum genetic transformation efficiency of 83.33% was achieved with 15d-precultured of S.alopecuroides callus, which was infected by A600=0.9 A. tumefaciens for 15 minutes and then co-cultivated for 48 hours with 200 μmol•L-1AS. The promoter sequence (1 260 bp) of upstream SaLDC was cloned from S.alopecuroides genomic DNA (gene bank accession number: KY038928). The deletion fragment of SaLDC promoter with different length (310,594,765,924,1 260 bp) were ligated with the GUS reporter gene to form five plant expression vectors named P310,P594,P765,P924,P1260, which were then transferred into S.alopecuroides callus. The GUS transient expression showed that all 5 different deletion fragment of SaLDC promoter can drive the GUS gene expression in S. alopecuroides callus. The SaLDC promoter we cloned has high promoter activity, and they may facilitate its function analysis in the future.

Evaluación del gen que codifica la enzima βHPMEH para la inhibición de la marchitez bacteriana causada por Ralstonia solanacearum

La marchitez bacteriana causada por Ralstonia solanacearum E.F. Smith es una de las enfermedades bacterianas más importantes que ataca a cultivos agrícolas como papa, tomate, banana, entre otros, causando grandes pérdidas en la producción. Desafortunadamente, su control ha sido difícil por su amplio rango de hospederos alternativos, su supervivencia en el suelo y su variación biológica y genética; así como porque no hay variedades con altos niveles de resistencia y porque no existe un control químico efectivo. Quorum sensing (percepción de quorum) es el fenómeno mediante el cual la acumulación de unas moléculas permite a una bacteria saber el número de bacterias que se encuentran en el medio es decir la densidad poblacional. La bacteria R. solanacearum posee un sistema quorum sensing para la regulación de la expresión de genes de virulencia, y en la cual la molécula 3-OH-PAME es el autoregulador de esta señal. Se conoce que la molécula ΒHPMEH hidroliza a 3-OH-PAME, anulando así la señal de autorregulación y por tanto la comunicación quorum sensing en R. solanacearum. Con el objetivo de evaluar el gen βhpmeh, se diseñaron dos vectores que expresen este gen bajo el control de dos diferentes promotores, los cuales fueron verificados por análisis de restricción, secuenciamiento y posteriormente mediante técnicas de agroinfiltración, se observó su expresión y su efecto frente a R. solanacearum en hojas de papa de la variedad Desiree. Los resultados de la expresión transitoria, muestran que el gen βhpmeh retrasó la aparición de síntomas de la marchitez bacteriana y sería un candidato potencial para transformación genética de la planta entera.

Ralstonia solanacearum is the causal agent of the devastating bacterial wilt disease that attacks important agricultural crops such as potato, tomato, banana, among others, causing serious yield losses. Control of R. solanacearum is difficult because of its wide range of alternate hosts, its long survival in soil, its biological and genetic variation, the lack of natural resistance sources and the insufficiency of the appropriate chemical control measures. Quorum sensing is the term that describes the phenomenon whereby the accumulation of molecules allows bacteria to know the number of bacteria found in the environment (population density). R. solanacearum has a quorum sensing system for the regulation of the expression of virulence genes; the molecule 3-OH-PAME is the self-regulatory signal. The molecule ΒHPMEH hydrolyzes 3-OH-PAME nullifying the signal of virulence, and thus, the quorum sensing communication in R. solanacearum. In order to evaluate the βhpmeh gene we designed two vectors that express this gene under the control of two different promoters. Both vectors were verified by restriction analysis and sequencing. Agroinfiltration assays were used to analyze gene expression and the effect against R. solanacearum in potato (Solanum tuberosum) leaves. The results of the transient expression experiments showed that the expression of gene βhpmeh caused a delay in the appearance of symptoms of bacterial wilt and thus is a good candidate for whole genetic plant transformation.

A novel chloroplastic isopentenyl diphosphate isomerase gene from Jatropha curcas: Cloning, characterization and subcellular localization

Background Jatropha curcas is a rich reservoir of pharmaceutically active terpenoids. More than 25 terpenoids have been isolated from this plant, and their activities are anti-bacterial, anti-fungal, anti-cancer, insecticidal, rodenticidal, cytotoxic and molluscicidal. But not much is known about the pathway involved in the biosynthesis of terpenoids. The present investigation describes the cloning, characterization and subcellular localization of isopentenyl diphosphate isomerase (IPI) gene from J. curcas. IPI is one of the rate limiting enzymes in the biosynthesis of terpenoids, catalyzing the crucial interconversion of isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP). Results A full-length JcIPI cDNA consisting of 1355 bp was cloned. It encoded a protein of 305 amino acids. Analysis of deduced amino acid sequence predicted the presence of conserved active sites, metal binding sites and the NUDIX motif, which were consistent with other IPIs. Phylogenetic analysis indicated a significant evolutionary relatedness with Ricinus communis. Southern blot analysis showed the presence of an IPI multigene family in J. curcas. Comparative expression analysis of tissue specific JcIPI demonstrated the highest transcript level in flowers. Abiotic factors could induce the expression of JcIPI. Subcellular distribution showed that JcIPI was localized in chloroplasts. Conclusion This is the first report of cloning and characterization of IPI from J. curcas. Our study will be of significant interest to understanding the regulatory role of IPI in the biosynthesis of terpenoids, although its function still needs further confirmation.

Genomic cloning, bioinformatics analysis, and subcellular localization of an agglutinin gene from Pinellia ternate / 中草药

Objective: To clone an agglutinin gene from Pinellia ternata and to analyze its bioinformatics and subcellular location. Methods: Based on the published sequence GU593718.1 from Genbank, P. ternata agglutinin (PTA) was amplified and cloned from genomic DNA of the fresh leaves of P. ternata. The cloned PTA gene was further fused to the plant expression vector pI1300-CaMV35S-GFP to construct pI1300-CaMV35S-PTA-GFP, then transfered into cells of Agrobacterium tumefaciens GV3101. Its transient expression was observed in Nicotiana tabacum. Results: The full length of PTA contained 810 bp with the deduced 269 amino acid residues; It contained one signal peptide, two conversation B-lectin domains and three mannose binding sites; PTA shared 97%, 85%, and 83% identity with the amino acid sequence from PTA, and Pinellia pedatisecta agglutinin (PPA), Pinellia cordata agglutinin (PCA), respectively; The PTA was localized to the plasma membrane; Its registration number is KF154979 in NCBI. Conclusion: It would provide a stable foundation for the study on its effect against fungi, insects, and bacterium.

Semliki forest virus as a vector: pros and cons for its use in biopharmaceuticals production

The number of biopharmaceuticals for medical and veterinarian use produced in mammalian cells is increasing year after year. All of them are obtained by stable recombinant cell lines. However, it is recognized that transient gene expression produces high level expression in a short time. In that sense, viral vectors have been extensively used for producing recombinant proteins on lab-scale. Among them, Semliki Forest virus is commonly employed for this purpose. This review discusses the main aspects related to the use of Semliki Forest virus technology as well as its advantages and drawbacks which limit currently its utilization in biopharmaceutical industry on large-scale.

DsRed2 transient expression in Culex quinquefasciatus mosquitoes

Culex quinquefasciatus mosquitoes have been successfully genetically modified only once, despite the efforts of several laboratories to transform and establish a stable strain. We have developed a transient gene expression method, in Culex, that delivers plasmid DNA directly to the mosquito haemolymph and additional tissues. We were able to express DsRed2 fluorescent protein in adult Cx. quinquefasciatus mosquitoes by injecting plasmids directly into their thorax. The expression of DsRed2 in adult Cx. quinquefasciatus mosquitoes is an important stepping stone to genetic transformation and the potential use of new control strategies and genetic interactions.

Subcellular localization of a sesquiterpene synthase from Aquilaria sinensis utilizing three transient expression systems / 中草药

Objective: To investigate the subcellular localization of a sesquiterpene synthase from Aquilaria sinensis (ASS) and compare three transient expression systems in the subcellular localization study. Methods: An ASS gene was amplified by RT-PCR using specific primers and cloned into the pEZS-NL and pFGC5941GFP to generate two plant expression vectors: pEZS-NL-ASS- GFP and p5941-GFP-ASS. Three transient expression systems, agroinfiltration of tobacco leaves, particle bombardment of onion epidermal cells, and PEG transformation of protoplasts isolated from ASS calli were adopted and compared. The expression of the GFP fusion proteins were observed by a confocal laser scanning microscopy. Results: The green fluorescence could be observed in all the three systems. However, the results were comparatively different. The cytoplasm and plastid localization of the GFP fusion protein observed in protoplasts was comparatively clear and was consistent with the studies in other plant species. Conclusion: The results demonstrate that the ASS is located in the cytoplasm and plastid in the ASS protoplasts and the different locations in three systems might be caused by the distinct characters of the heterologous or homologous cells.

Human sulfatase transiently and functionally active expressed in E. coli K12

The recombinant human iduronate 2-sulfate sulfatase (hrIDS) was transiently and functionally active expressed in E. coli K12. The enzyme activity (crude extract) at 100 ml and 400 ml oscillated between 0.25 and 10.58 nmol h-1 mg-1. The wide Western-blot peptide profile suggest that hrIDS is proteolitically processed randomly which agrees with the ultrafiltration assay in which the hrIDS activity was found in all fractions (<30kDa, 30-100kDa and >100kDa). No glycation sites were found by computer analysis of the hIDS sequence; discarding the possibility of marks for glycation and proteolytic processing.

Expression pattern and apoptosis-inducing activity to murine macrophages and hepatocytes of Leptospira interrogans Sph2 hemolysin / 中华微生物学和免疫学杂志

Objective To determine the change of expression level of Leptospira interrogans sph2 gene, and hemolytic and cell apoptosis-inducing activities of sphingomyelinase hemolysin Sph2. Methods Entire sph2 gene fragment was amplified by PCR from genomic DNA of L. Interrogans serovar serogroup Icterohaemorrhagiae serovar Lai strain Lai, and sequenced after T-A cloning. Subsequently, a prokaryotic expression system of sph2 gene was constructed. The expression of target recombinant Sph2( rSph2 ) was examined by SDS-PAGE and the expressed rSph2 was extracted by Ni-NTA affinity chromatogaphy. The hemolytic activity of rSph2 was measured by hemolytic test in sheep blood agar plate and spectrophotometry-based hemoglobin measurement, and the apoptosis-inducing activity of rSph2 to murine mononuclear-macrophagelike cell line(J774A. 1) and hepatic cell line(IAR20) was determined by flow cytometry. A real-time fluorescence quantitative RT-PCR was applied to detect the change of sph2 mRNA levels before and after L. Interrogans strain Lai infecting J774A. 1 and IAR20 cells. Results The cloned sph2 gene had 100% sequence identity to the corresponding gene in GenBank. The constructed prokaryotic expression system was able to efficiently express rSph2. The rSph2 could lyse sheep erythrocytes in concentration-dependent pattern. 10μg/ml rSph2 could induce the apoptosis of J774A. 1 cells and IAR20 cells, and the peak apoptotic rates were 23.96% and 32.92%, respectively. The mRNA level of sph2 gene was significantly elevated within 0.5-2 h of L. Interrogans strain Lai infecting either J774A. 1 or IAR20 cells, and then the mRNA level was quickly descended. Conclusion The sph2 gene of L. Interrogans strain Lai has a transient expression when the microbe contacts host cells. rSph2 possesses activities of sheep erythrocyte lysis and inducing macrophage and hepatocyte apoptosis, indicating Sph2 as an important virulence factor during pathogenic process of Leptospira.

Cloning of high molecular weight gluten subunit promoter and study on its function in wheat

The aim of this work was to study the cloning and characterization of HMW-GS 1Dx2 promoter from Triticum aestivum. A 1050 bp partial promoter fragment including a putative TATA box and 5' encoding sequence of the gene was cloned by amplifying the upstream sequences using the nest-PCR with appropriate primers. The analysis of the promoter sequence against the PLACE (Plant cis-acting Regulatory DNA Elements) database showed the presence of certain putative endosperm-specific regulatory cis-elements in the sequence along with the TATA and CAAT boxes. The histochemical method detected the transient expressions of GUS in the seeds of wheat. The results showed that HMW-GS 1Dx2 promoter had the endosperm-specific transcription activity in the wheat seeds.

Construction and expression of humanized anti-EGFR antibody / 军事医学科学院院刊

Objective:To express rationally engineered antibodies against EGFR and assess their affinity to EGFR and anti-tumor cell migration effect. Methods:L and V_H genes of humanized antibodies against EGFR were designed and synthesized. Genes encoding V_H and C_H were connected and then cloned into a pIRES based bicistronic expression vector. Gene encoding the corresponding L gene was also cloned into the same vector. 293T cells were transfected with the recombinant plasmid and the antibody expression was confirmed by Western blotting. The antibodies were purified by protein A based affinity chromatography. Binding of the humanized antibody to the EGFR was assessed by Surface Plasmon Renainance with Biacore3000, and the biological activity of the humanized antibody was determined by tumor cell invasion test.Results:Three expression vectors were constructed and the humanized anti-EGFR antibodies were expressed and purified successfully. In reducing SDS-PAGE, the antibodies exhibited two bands of approximately 25×10~3and 50×10~3, respectively. Western blot assay showed that the humanized antibodies had recognition specificity to goat-human IgG antiserum. Biacore assay revealed that the humanized antibody C3 binds to EGFR with high affinity(6.13×10~(-10)M). Cell migration test showed that C2,C3 and C5 could suppress growth and migration of tumor cells.Conclusion:Three anti-EGFR humanized antibodies (C2,C3 and C5) have been constructed and expressed successfully, and the C3 antibody retained high affinity for EGFR and showed improved inhibitory effect on tumor cell growth and migration.