Construction and self-activation detection of yeast two-hybrid bait plasmid of human programmed cell death ligand 1 immunoglobulin variable region domain gene / 中国生物制品学杂志

@#Objective To construct a yeast two-hybrid recombinant bait plasmid of human programmed cell death ligand 1(PD-L1)immunoglobulin variable region(IgV)domain gene,detect its expression in yeast and detect the cytotoxicity and self-activation of PD-L1 IgV protein as well as the interaction between PD-L1 IgV and human thioredoxin(hTrx).Methods Human PD-L1 was analyzed by bioinformatics method,and primers were designed to amplify PD-L1 IgV domain based on the coding region of PD-L1 gene registered in NCBI GenBank database. PCR amplification was carried out with pENTERPD-L1 plasmid as template,and then cloned into yeast two-hybrid bait vector pGBKT7. The recombinant bait plasmid and pGBKT7 empty vector were transformed into Y2HGold yeast cells respectively,and the PD-L1 IgV gene and its expression were detected by PCR and Western blot;Meanwhile,the protein toxicity and self-activation of PD-L1 IgV were detected,and the interaction between PD-L1 IgV and hTrx was detected by drip plate method.Results The bioinformatics analysis results of PD-L1 were consistent with related reports. The recombinant bait plasmid pGBKT7-PD-L1 IgV was correctly constructed,and Y2HGold positive clone was obtained,in which PD-L1 IgV was stably expressed. The empty vector pGBKT7 and recombinant bait plasmid pGBKT7-PD-L1 IgV grew well on SD/-Trp and SD/-Trp/X-α-Gal plates with the same colony size and number and white colony,but they did not grow on SD/-Trp/X-α-Gal/AbA plates,which indicated that PD-L1 IgV protein had no toxicity and no self-activation effect on yeast. The results of drip plates test showed that all experimental groups grew well on SD/-Trp/-Leu plate,while only positive control group grew on SD/-Trp/-Leu/X-α-Gal/AbA plate and showed blue color,which indicated that bait protein PD-L1 IgV and hTrx did not self-activate,and there was no interaction between them.Conclusion Recombinant human PD-L1 IgV bait plasmid was successfully constructed. PD-L1 IgV protein showed no toxicity and self-activation effect on yeast cells,and there was no interaction between PD-L1 IgV and hTrx. Subsequently,hTrx can be used to construct a peptide aptamer library,from which peptide aptamers that specifically bind to PD-L1 IgV can be screened.

Preparation and identification of the nano antibodies of anti-programmed death receptor-1 / 国际生物医学工程杂志

Objective To prepare camelid-derived nano antibodies with high affinity binding to programmed death receptor-1 (PD-1) antigen,and to provide experimental basis for subsequent functional studies.Methods The PD-1-Fc recombinant protein expressed in eukaryotic expression was used to immunize Xinjiang Bactrian camel 6 times.The peripheral blood was collected and the lymphocytes were isolated.Nested PCR amplification was performed to obtain the genes in variable region of camelid heavy chain antibody (VHH),and to construct a phage display library.The phage display library was screened by solid phase enzyme-linked immunosorbent assay (ELISA).The PD-1 antigen,which was sequentially reduced in mass concentration (5.00、2.50、1.00 μg/ml),was coated in an ELISA plate,and the phage display library was subjected to 3 rounds of affinity selection.Individual clones that bind to PD-1 were further screened by soluble monoclonal ELISA.According to the results of DNA sequencing,three VHH monoclonals with multiple repeats were selected and ligated into pET22b vector,and transformed into E.coli BL21 (DE3) competent cells,and then induced by isopropyl-β3-D-thiogalactoside.The recombinant VHH antibody protein was purified by nickel column affinity chromatography,and its binding activity and affinity to PD-1 antigen were detected by Western Blot and ELISA.Results After immunization of Bactrian camel 6 times with recombinant protein PD-1-Fc,high titer specific antibody was stimulated,and the immune serum titer reached 1∶32 000.A VHH phage display library with a reservoir size of 2.6×108 cfu/ml was constructed from the immunized camel lymphocytes.After 3 rounds of affinity selection,46 VHH monoclonals with absorbance (A600) values above 0.6 were obtained by soluble monoclonal ELISA.Among them,three clones of VHH-B7,VHH-H5 and VHH-H12 had higher repeats,indicating that significant enrichment was obtained.The results of Western Blot and ELISA showed that the purified B7,H5 and H12 nanobodies had good binding activity to PD-1 antigen and had high affinity.Their affinity constants were 1.19×1011 and 1.63×1011,1.59×1011 L/mol,respectively.Conclusion The anti-PD-1 camelid-derived nanobodies were obtained by affinity selection of VHH phage display library,which can bind to the PD-1 antigen with high affinity.This study can provide an experimental basis for subsequent functional studies.

Prokaryotic expression and purification of anti-AEG-1 single-chain variable antibody / 国际检验医学杂志

Objective To construct anti-astrocyte elevated gene-1(AEG-1)single-chain variable antibody (V23)prokaryotic ex-pression vector,and to conduct the protein purification and immunological activity detection.Methods The Primer5 software was applied to design the primers aiming at the gene sequence of the antibody anti-AEG-1 single-chain variable region for constructing the prokaryotic expression plasmid of PRsetC/V23.After the enzyme digestion by the restriction enzyme Pst1 and correct DNA se-quencing,the prokaryotic expression plasmid was led to E.coli BL21 ,the prokaryotic expression engineering strain containing the V23 gene was constructed.After the induction with IPTG,the interest protein was purified by the magnetic beads with the HIS tag,and the content of interest protein was determined by the SDS-PAGE electrophoresis.Western blot and ELISA were adopted to detect the immune activity of the nti-AEG-1 single-chain variable region antibody.Results For the constructed prokaryotic expres-sion plasmid PRsetC/V23,the single enzyme digestion and sequencing analysis displayed that the constructed V23 gene was com-pletely consistent to the designing sequences.After IPTG induction,SDS-PAGE electrophoresis showed an apparent protein band at 31×103 ,the Western blot detection showed a specific AEG-1 response band at 80 ×103 ,the ELISA test showed the positive re-sults.Conclusion The PRsetC/V23 prokaryotic expression plasmid and the V23 prokaryotic expression engineering strain are suc-cessfully constructed,this engineering strain can express anti-AEG-1 single-chain variable region antibody protein,and the protein has good immune activity.

Evaluation and applications of antibody variable stability / 军事医学

Objective To study the intrinsic relationships between the binding energy of the antibody light and heavy chains and the conformational characteristics , physical and chemical properties , and to establish a corresponding mathemat-ical model and evaluate the thermal stability of the antibody molecules , which contribute to the antibody design , optimiza-tion and affinity maturation .Methods Based on bioinformatics and computational biology methods , the antibody′s structur-al information with the crystal diffraction data was analyzed .The conformational character of the variable domain of the antibody was studied using distance geometry and computer graphics technology .With the aid of the intermolecular hydrogen bond formation theory and the reaction free energy theory , the dynamic structure and energy characteristics be-tween the heavy and light chain variable regions of the antibody were studied .Furthermore , using nonlinear fitting and regression analysis, a mathematical model was set up .Results According to simulation and statistic analysis , there was a linear relationship between the binding energy and the number of the intermolecular hydrogen bonding , Van der Waals interaction of the heavy and light chains of the antibody .There was polynomial correlation between the binding energy and the physicochemical properties of the antibody .Using the frequency of amino acid position and the established model , the humanized anti-ricin antibody , which could not obtain the stable engineering cell line , was evaluated and optimized .The stable engineering cell line of the humanized anti-ricin antibody was obtained in the experiment .Conclusion The self structure of the antibody variable region ( conformation and physicochemical properties ) has much effect on its stability . The antibody stability can be improved by structural optimization .

Recent advances in chronic lymphocytic leukemia.

Chronic lymphocytic leukemia (CLL) was largely considered to be a disease of slow progression, standard treatment with Chlorambucil and having almost similar prognosis. With the introduction of molecular methods for understanding the disease pathophysiology in CLL there has been a remarkable change in the approach towards the disease. The variation in B-cell receptor response and immunoglobulin heavy chain variable region (IGHV) mutation, genetic aberration and defect in apoptosis and proliferation has had an impact on therapy initiation and prognosis. Early diagnosis of molecular variant is therefore necessary in CLL.

Role of scavenger receptor class B type I in cell entry of hepatitis C virus / 第二军医大学学报

Hepatitis C virus (HCV) cell entry is a multi step process mediated by various receptors. Among these receptors, the scavenger receptor class B type I (SR-BI) is the one considered to be the first to interact with HCV. SR-B I can bind to HCV envelope glycoprotein E2, in which the hyper variable region 1 (HVR1) segment locating at the N-terminus of E2 protein plays a critical role. The interaction of SR-BI with HCV can not only mediate HCV cell entry, but also attenuate neutralization activity of antibodies against E2 protein, contributing to the HCV immune evasion. Therefore, studying the mechanism of HCV/SR-B I interaction during cell entry can help to identify important targets in the initial step of viral infection, contributing to prevention and treatment of HCV infection. This paper reviews the current knowledge on the biological characteristics of SR-B I and mechanisms of HCV cell entry mediated by virus/SR-B I interaction.

Células T y sus receptores αß y γδ en biopsias de pacientes con enfermedad periodontal / T cells and its receptords and αß and γδ in biopsies from patients with periodontal disease

El propósito de este estudio es determinar la presencia y localización de las células T y de sus receptores αβ y γδ en biopsias de tejido gingival de pacientes con enfermedad periodontal. Se evaluaron 60 biopsias de 12 pacientes, 4 con diagnostico de periodontitis agresiva, 4 con periodontitis crónica y 4 con gingivitis, las cuales fueron procesados para su análisis histológico, inmunohistoquímico e histomorfometrico. Al analizar los resultados por diagnostico los marcadores que mas predominaron fueron, en Gingivitis CD3, CD8 y TCR γδ en tejido conectivo. En Periodontitis crónica CD3, CD8 y TCR γδ en epitelio oral y CD4 el cual presentó una expresión homogénea en los tejidos analizados. En periodontitis agresiva CD3 y CD8 en epitelio crevicular, con una distribución similar entre CD4 y CD8 tanto en epitelio oral como en tejido conectivo y TCR γδ en conectivo. En cuanto a las cadenas variables del TCR Vβ los más expresados en las diferentes patologías estudiadas fueron el 6.7, 8.1 y 12 a nivel del tejido conectivo. Los estudios sobre la expresión de estas familias parecen indicar que es otra vía de activación a tener en cuenta dentro del modelo de la patogenia de la enfermedad y que debe ser estudiado en modelos longitudinales en pacientes con pérdida de inserción progresiva.

T the purpose of this study is identifying the presence and localization of T cells and their receptor αβ and γδ in biopsies of gingival tissue in patients with periodontal disease. 60 biopsies were evaluated in 12 patients, 4 patients with diagnosis of gingivitis, 4 patients with chronic periodontitis and 4 with aggressive periodontitis, which were processed for the histological, immunohistochemical and histomorphometric analysis. The results by diagnosis showed that in gingivitis the more predominant markers were CD3, CD8 and TCR γδ in connective tissue. In chronic periodontitis the markers with bigger expression were CD3, CD8 and TCR γδ in oral epithelium and CD4 that showed a homogeneous behavior in the analized tissues. In aggressive periodontitis CD3 and CD8 in surcular epithelium, TCR γδ in connective tissue and CD4 and CD8 with a similar distribution in oral epithelium and connective tissue. In relation with variable chains of TCR Vβ, the most predominat in the different diagnosis were 6.7,8.1 and 12 in connective tissue. The investigations about the expression of these families indicate that it can be other important via of activation in the pathogenesis of periodontal disease and it should be study in longitudinal models in patients with progressive loss of attachment level.

Construction and eukaryotic expression of recombinant plasmid pEGFP-N1-SrV+encoding the variable region of the surface protein of Streptococcus mutans / 中华微生物学和免疫学杂志

Objective To construct the recombinant plasmid pEGFP-N1-SrV+ and evaluate the expression of SrV+in mammalian 293T cells.nethods srv+.a gene encoding the vailable region of the surface protein of the Streptococcus mutans OMZ175.was cloned chemically based on its reported nucleotide sequence.The eukaryotic expression plasmid,pEGFP-N1-SrV+,was constructed by introducing the srv+ gene into the Kpn Ⅰ/Xho Ⅰ site of pEGFP-NI.The recombinant plasmid pEGFP-N1-SrV+was transfected into 293T cells with lipofectamine and the expression level of SrV+was evaluated.Results The eukaryotic expression plasmid pEGFP-N1-SrV+was constructed successfully.GFP was observed by green fluorescent microscope.and a 72 × 1 03 protein was detected bv Westem blot.Real-time RT-PCR analysis revealed that the expression of the pEGFP-N1-SrV+in 293T was excellent and significant compared the control group. Conclusion The recombinant plasmid pEGFP-N1-SrV+was successfully constructed.which could encode the expression of SrV+after transfected into the mammalian 293T Cells.

Genotyping of White Spot Syndrome Virus in Chinese Cultured Shrimp during 1998-1999 / 中国病毒学·英文版

Recent studies showed that white spot syndrome virus(WSSV)isolates from different geographic locations share a high genetic similarity except the variable regions in ORF23/24 and ORF14/15,and variable number of tandem repeats(VNTR)within ORF94.In this study,genotyping was performed according to these three variable regions among WSSV isolates collected during 1998/1999 from Southern China.These WSSV isolates contain a deletion of 1168,5657,5898,9316 and 11093 bp,respectively in the variable region ORF23/24compared with WSSV-TW,and a deletion of 4749 or 5622 bp in the variable region ORF14/15 relative to TH-96-II.Four types of repeat units(RUs)(6,8,9 and 13 RUs)in ORF94 were detected in these isolates,with the shortest 6 RUs as the most prevalent type.Our results provide important information for a better understanding of the spatio-temporal transmission mode and the WSSV genetic evolution lineage.

Overexpression and unique rearrangement of VH2 transcripts in immunoglobulin variable heavy chain genes in ankylosing spondylitis patients

To identify immunoglobulin variable heavy chain (VH) gene usages in Korean ankylosing spondylitis (AS) patients, expression level of VH2 genes from peripheral blood mononuclear cells (PBMCs) of 8 AS patients and 9 healthy donors was analysed by quantitative real-time PCR (Q-PCR). Q-PCR results demonstrated VH2 genes were overexpressed in AS patients (Relative amount of mRNA of VH2 genes to a house-keeping gene, 7.13 +/- 7.77 vs, 0.68 +/- 0.55; P < 0.0001). The sequence analysis revealed the majority of them contained CDC42 binding protein kinase beta (CDC42 BPB) genes. The insertion of CDC42 BPB gene was confirmed by PCR with primers corresponding CDC42 BPB and CH genes. Our study revealed VH2 overexpression and unique rearrangement in Ig VH genes from peripheral blood of AS patients. This may imply aberrant immunoglobulin gene rearrangement in B cell occurs in Korean AS patients, which requires further investigation.

Variable fragment of heavy chain antibody against the terminal protein region of hepatitis B virus polymerase introduced by Tat protein transduction domain inhibits the replication of hepatitis B virus in vitro / 中华传染病杂志

Objective To study a functional variable fragment of heavy chain(VH)antibody against the terminal protein(TP)region of hepatitis B virus(HBV)polymerase introduced by human immunodeficiency virus Tat protein transduction domain(TAT)and the inhibitive activity of TAT-VH on the replication of HBV in vitro.Methods The gene encoding TAT-VH was cloned into prokaryotic expression vector pET28a(+).Recombinant plasmid was transduced into E coli BL21(DE3)LysS,then the protein was expressed and purified.The purified TAT-VH fusion protein was added into HepG2.2.15 cell culture.The transduction efficiency was evaluated by indirect fluorescence assay(IFA).The cytotoxicity of TAT-VH was detected by Methabenzthiazuron(MTT)assay.HBV DNA level in HepG2.2.15 cell culture was measured using quantitative polymerase chain reaction(PCR).The data were analyzed by one-factor analysis of variance and t test.Results TAT-VH fusion protein was successfully expressed and purified.It was confirmed by IFA and MTT assay that TAT-VH was introduced into HepG2.2.15 cells and the cell growth was not affected.The level of HBV DNA in supernatant of HeDG2.2.15 cell culture with 5 000 nmol/L TAT-VH was(1.211±0.132)lg copy/mL,which was significantly lower than control group[(5.325±0.041)lg copy/mL,t=72.91,P＜0.05].Meanwhile,the level of intracellular HBV DNA was(3.521±0.411)lg copy/mL,which was significantly lower than control group[(8.532±0.132)lg copy/mL.t=28.41,P＜0.05].Conclusion The HBV replication is inhibited by anti-TP TAT-VH antibodies in vitro,which provides valuable experimemal basis for developing therapy of HBV infection with intracellular antibody.

The hyper-variable region genotypes of methicillin-resistant Staphylococcus aureus and their relationship with the resistance / 中华微生物学和免疫学杂志

Objective To investigate the hyper-variable region-polymerase chain reaction(HVRPCR) genotype of methicillin-resistant Staphylococcus aureus (MRSA) in local hospitals in Hunan province, and to compare it with the antibiograms, and to preliminarily discuss its role in molecular epidemiology of MRSA. Methods A total of 80 MRSA clinical isolates were collected from three affiliated hospitals of Central South University. Their DNA were extracted and amplified by PCR. The genotype was classified by the fragments of amplified products based on the size of HVR. The drug sensitivity test of MRSA was performed, and the correlation of genotypes and antibacterial resistance was analyzed. Results Eighty strains of MRSA were divided into 5 HVR genotypes which were named as A, B, C, D and E respectively according to the size of the PCR products. The most common types were D (61.25%) and E (21.25%), followed by A (3.75%), B (5.00%) and C (8.75%). Most strains of genotypes were multi-drug resistant but all strains were sensitive to vancomycin. Conclusion These results suggest that HVR-PCR genotype method is a rapid, convenient and effective method for epidemiological investigation of infections caused by MRSA, and it is also helpful for clinical selection of antibacterial agents in effective treatment of MRSA infection.

Production, Characterization, and Variable Region Analysis of Monoclonal Antibodies Specific for Hepatitis B Virus S Antigen

BACKGROUND: Hepatitis B virus (HBV) infection is one of the worldwide public health problem affecting about 300 million people. The envelope protein of HBV consists of three components known as preS1, preS2, and S antigen. According to the recent study, anti-HBs Ab showed effective neutralization ability against HBV from chronic hepatitis B and liver transplant patients, suggesting the possible development of therapeutic antibody. METHODS: Spleen cells immunized with S antigen of HBV were fused with myeloma cell line to obtain HBsAg specific monoclonal antibodies. High affinity antibodies against HBsAg (adr, ad and ay type) were selected by competitive ELISA method. Nucleotide sequence of the variable regions of monoclonal antibodies was analyzed by RT-PCR followed by conventional sequencing method. RESULTS: We produced 14 murine monoclonal antibodies which recognize S antigen of HBV. Two of them, A9-11 and C6-9 showed the highest affinity. The sequence analysis of A9-11 revealed that variable regions of the heavy chain and light chains are members of mouse heavy chain I (B) and light chain lambda 1, respectively. Likewise, the sequence analysis of C6-9 revealed that variable regions of the heavy chain and light chains are members of mouse heavy chain II (B) and light chain kappa 1, respectively. Neutralization assay showed that A9-11 and C6-9 effectively neutralize the HBV infection. CONCLUSION: These results suggest that A9-11 and C6-9 mouse monoclonal antibodies can be used for the development of therapeutic antibody for HBV infection.

Amplification and sequence analysis of anti-D variable region gene with leader peptide sequence / 中国病理生理杂志

AIM: To amplify from leader peptide region an d obtain human monoclonal anti-D variable region gene with high specificity and affinity, and analyze the nucleotide and deduced amino acid sequences.ME THODS: The total RNA was extracted from an Epstein-Barr-virus-transforme d cell line secreting monoclonal anti- (rhesus D) antibody. The leader region pri mers containing a ribosome recognition site were designed. By using PCR method, the cDNA of human anti-(rhesus D) antibody (IgM ?) variable region gene was amp lified. Cloning and subsequent sequence analysis of the variable region gene was performed. The deduced amino acid sequence was also compared and analyzed with previ ously published sequences.RESULTS: A band of approximate 440 and 410 base pairs were amplified using heavy chain primers and light chain primer s, respectively. Sequence analysis indicated that the deduced amino acid sequenc e w as in agreement with the characterization of the amino acid present in the human Ig variable region. CONCLUSION: The cloning and sequencing of a human anti- (Rhesus D) antibody variable region cDNA will make benefits for pro duction of recombinant anti-(Rhesus D) antibody and prevention of Rh haemolytic disease in newborns.

Cloning and sequencing of the VL and VH genes from the anti-hCD154 McAb hybridoma cell line / 中国病理生理杂志

AIM:To obtain the light chain region(VL) and heavy chain region(VH) genes from the hybridoma cell line and analyse their sequence for construction of the engineering antibody against hCD154. METHODS: In this research ,total RNA was extracted from the hybridoma cell line 7E8, which secretes McAb against hCD154, and subjected to reverse transcription. The VL gene and VH gene were amplified by PCR, cloned into puc18 vector and sequenced by Sanger's dideoxymediated chain-termination method. RESULTS: The VL cDNA of 7E8 McAb consists of 341 bp encoding 113 amino acid residues. Compared with mouse Ig database, the VL region is in accord with the characterization of DNA sequence present in the mouse Ig Vk region , it belong to mouse V?2 light chain. The VH cDNA of 7E8 McAb consists of 354 bp encoding 118 amino acid residues. Compared with mouse Ig database, the VH region is in accord with the characterization of DNA sequence present in the mouse Ig VH region. CONCLUSION: The DNA squence analysis showed that the cloned genes code the light and heavy chain variable region of mouse respectively.

Cloning and expression of Fab gene of anti-p185 monoclonal antibody / 中国免疫学杂志

Objective:To clone Fab genes of anti-p185 monoclonal antibody 5E12 and express it in E.coli.Methods:Fd and ? genes were cloned by RT-PCR, inserted into Fab expression vector and expressed in E.coli. The N-terminal sequences of V regions was resumed by PCR mediated mutagenesis. The antigen-binding activity of the Fab were tested by ELISA and immunohistochemistry.Results:Fd and ? genes were cloned and expressed in E.coli. But the bacterially expressed Fab fragments showed no antigen binding activity. After the N-terminal sequences of V regions was corrected to original sequences, the Fab expressed in bacterial was able to target HER2/neu-expressing cells(NIH3T3/erbB-2 cells). Correction of Fd N-terminal sequences could partially resume the antigen binding activity. But correction of ? chain N terminal sequences was shown no expected result.Conclusion:Successful in constructing and expressing anti-p185 Fab, which will benefit the construction of other engineering antibody and humanization of murine anti-p185 McAb. We also found that the V region N terminal changes introduced with PCR primers may affect antigen binding activity seriously, to which more attention should be paid during antibody engineering.

Production and Gene Cloning of Monoclonal Antibodies Directed Against S Antigen of Hepatitis B virus / 대한면역학회지

We produced two murine monoclonal antibodies designated S2E1 and S2C11, which recognize S antigen of hepatitis B virus (HBsAg). S2E1 could bind to denatured form of recombinant HBsAg as well as native form of HBsAg, but S2C11 could bind only to native form of HBsAg. Both antibodies reacted with HBsAg in the hepatocyte of patient infected with hepatitis B virus. Analyses of the nucleotide sequences encoding the variable regions of these antibodies revealed that S2E1 and S2C11 utilize variable gene segment which belong to V4/5 gene family and utilize the J5 and Jk4 gene segments, respectively. In addition, the heavy chain of S2E1 express a member of V14 gene family and a member of DSP2.9 and Jh3 gene families. S2C11 is related to the V1 gene family and expresses DFL16.1 gene regions in conjunction with the Jh3 gene segment.

Cloning of Fab Gene of an Anti-Human Bladder Cancer Monoclonal Antibody and Its Expression in E. coli / 中国肿瘤生物治疗杂志

Objective: To clone the Fab gene of a monoclonal antibody (mAb) BDI against human bladder cancer and its expression in E. coli. Methods: Fd and K genes of mAb BDI were cloned by RT-PCR and inserted into an Fab expression vector. Phage displaying Fab and soluble Fab were expressed in E. coli. The N-terminal sequence of VH region was corrected by PCR mediated mutagenesis. The antigen-binding characteristics of the Fab were tested by ELISA and immu-nohistochemistry. Results: Fd and K genes were cloned into the expressing vector p3MH and the phage displaying antibody and soluble Fab were expressed in E. coli, which showed weak binding activity to bladder cancer cells. Correction of the N-terminal sequence of the VHimproved the biding activity dramatically. The feasibility of the application of the Fab in phage antibody library screening was confirmed by a simulated panning procedure. Conclusion: The Fab gene of an anti-human bladder cancer mAb was expressed in E. coli. The importance of the N-terminal sequence on antibody binding activity was suggested.

Dynamic variation of HCV hypervariable region gene and homology analysis / 中国输血杂志

Objective To find out the dynamic variation on HCV genome to give the theoretic basis for diagnosis of post transfusion hepatitis C,and demonstrate the causality between donors and recipients suffering from post transfusion hepatitis C.Methods The high variable envelope gene fragments (HVR1 and HVR2) of HCV was selected for a cross sectional study and a one year follow up study was carried out on 3 HCV patients at every 3 month period to understand the dynamic variation of HCV HVR in each patient.Results The homology of HCV HVR genes among different types is 56.99%～60.88%,65.09%～68.80% among different subtypes,and 80.05%～96.40% among the same subtypes.It consists with the geographic distribution of HCV.The homology between the different strains isolated from the same patients is 92.10%～99.73%,95.36%～97.54 and 97.07%～99.20%,respectively.Conclusion The variation of different HCV strains in HVR is great.However,the variation of the same virus in the same patient during short infectious period is rather limited,which is no more than 7.9% per year.So the transmission pathway of some hepatitis C cases can be trailed at the molecular level by this way,and some non transfusion transmitted hepatitis C cases can be exclude

Gene Cloning, Construction and Expression of Single-Chain Fv (scFv) Against the Membrane Protein of Schisotosoma japonicum / 中国寄生虫学与寄生虫病杂志

Objective To construct single chain antibody specific to membrane protein of Schistosoma japonicum by gonetic engineering technique. Methods The V\-H (heavy-chain variable region) and V\-L (light-chain variable region) genes were amplified by PCR from the genomic DNA of NP11-4 cell line, and sequenced by Sanger's method. The ScFv was constructed in pTHA90 vector using V\-H and V\-L genes, then expressed by IPTG. Results The V\-H and V\-L genes were obtained through PCR. The DNA sequences showed that V\-H and V\-L were new variable region genes of antibody. They were registered by GenBank. A ScFv gene with (Gly4Ser) 3 intralinker in the pTHA90 vector was successfully constructed. The ScFv was expressed as thioredoxin-fused proteins about 36^2 kDa. Conclusion A specific ScFv against the membrane protein of Schistosoma japonicum was constructed and expressed.