Screening of human phage single chain antibody against the recombinant surface protein of Streptococcus mutans and identification of its biological activities / 中国人兽共患病学报

To isolate human phage single chain antibody against surface protein of Streptococcus mutans,the recombinant surface protein of S.mutans(rAP) was used to coat the immune tubes and the phage single chain antibody was prepared through pDAN5 phage antibody library after 5 rounds of panning.The eluted phage was enriched nearly 30 times.In these ways,13 positive clones were obtained and found to be able to bind with rAP in ELISA assay.Then one of the 13 positive clone phage plasmid was used to infect E.coli HB2151 to induce the expression of the non-fusion single chain antibody (ScFv) with IPTG induction.As demonstrated by SDS-PAGE,the molecular mass of this single chain antibody was proved to be 30 kDa and the amount of expression constituted to 30% of the total bacterial proteins.Apparently,the human phage single chain antibody against surface protein of S.mutans with biological activity was successfully screened.

Construction of human phage display antibody ScFv library and identification of antibody ScFv against lung adenocarcinoma / 基础医学与临床

Objective To construct a human phage single chain-antibody library, and to sieve out the antibody ScFv against lung cancer from the library. Methods Total RNA was abstracted from lymph node tissue of the lung cancer, and was used to amplify V_H and V_L gene by RT-PCR. V_H and V_L were joined by a DNA linker by SOE-PCR to form the single chain variable fragment ( ScFv) gene. ScFv gene was coloned into the phage vector pCANT-AB5E. Panning against lung cancer cell line A549 was performed and positive clones were chosen for soluble expression. Results A recombination phage single chain-antibody library was constructed. After 4 rounds panning, the number of eluted phages increased by 115 times. Positive reactions to A549 were detected in 7 of 10 random clones. The human ScFvs against lung cancer were produced and confirmed by SDS-PAGE and ELISA analysis. Conclusion ScFvs against lung cancer were acquired by the construction of phage single chain-antibody library. The soluble ScFvs has specificall avidity to human lung cancer cells.

Screening and preliminary identification of liver cancer specific peptide from a phage display peptide library / 中华肝胆外科杂志

Objective To obtain short peptides which specifically binds to HepG2 cell line from 12 peptide libraries, and lay foundation for screening and identifying the new liver cancer markers for early diagnosis and treatment of liver cancer. Methods The liver cancer cell line HepG2 was used as the antigen and LO-2 as the absorber cells for subtraction biopanning from a phage display peptide library at 37℃. The positive phage clones were identified by cell enzyme-linked immunosorbentassay (EL1SA), and the identity of DNA sequence and amino acids were analyzed. Results After 3 rounds of screening, 30 phage clones were identified by EL1SA, ZS-9 of them bind to the HepG2 specifically. The amino acid sequence was blast in NCBI and Swiss-Prot, the results show that the sequence has not identity with the known genes and proteins in the database, and the sequence was not reported in literature. All these show that we had discovered several novel liver cancer associated antigen ligand. Conclusion Several candidate peptide binding to liver cancer specifically have been selected by phage display technology, providing the potential uses for early diagnosis of liver cancer or targeted therapy for liver cancer.

Construction of human phage display antibody ScFv library and identification of antibody ScFv against lung adenocarcinoma / 基础医学与临床

Objective To construct a human phage single chain-antibody library,and to sieve out the antibody ScFv against lung cancer from the library.Methods Total RNA was abstracted from lymph node tissue of the lung cancer,and was used to amplify VH and VL gene by RT-PCR. VH and VL were joined by a DNA linker by SOE-PCR to form the single chain variable fragment (ScFv) gene. ScFv gene was coloned into the phage vector pCANTAB5E. Panning against lung cancer cell line A549 was performed and positive clones were chosen for soluble expression.Results A recombination phage single chain-antibody library was constructed. After 4 rounds panning,the number of eluted phages increased by 115 times. Positive reactions to A549 were detected in 7 of 10 random clones. The human ScFvs against lung cancer were produced and confirmed by SDS-PAGE and ELISA analysis.Conclusion ScFvs against lung cancer were acquired by the construction of phage single chain-antibody library. The soluble ScFvs has specificall avidity to human lung cancer cells.

Screening of human anti-ricin ScFv from large phage library / 中国免疫学杂志

Objective:To clone human anti-ricin antibodies from large phage antibody library.Methods:Panning for a large phage library against ricin toxin was conducted to select specific antibodies against ricin. The binding activities and specificities were tested by ELISA method. Soluble ScFvs were prepared through infecting E coli. HB2151 with the selected phage antibodies and induction with IPTG. Results:Forty positive clones were obtained after 5 rounds of panning, and 12 clones had specific binding ability to ricin toxin. DNA fingerprinting showed 7 different band patterns indicating 7 different positive clones. DNA sequencing showed that variable regions of these ScFvs belonged to different subgroups.Conclusion:Human anti-ricin antibodies were successfully obtained from large phage antibody library.

Gene Cloning of the Epstein-Barr Virus (EBV) Antigen Reactive with the Serum from EBV-infected Patients / 감염

BACKGROUND: Epstein-Barr virus (EBV) is causative agent of infectious mononucleosis and nasopharyngeal carcinoma and associated with Burkitt lymphoma and other tumors. The recombinant protein is needed for the rapid and sensitive serodiagnosis of EBV infection. METHODS: EBV gene encoding the protein reactive with the sera of EBV-infected patient was cloned and characterized with lambda gt11 expression library of cDNA of EBV B95-8 strain. RESULTS: The recombinant proteins from clone 12, 15 and 21 were expressed as 120, 118, 160 kDa-usion protein with beta-galactosidase, respectively, which were reactive with IgG anti-EBV antibody-positive sera, but not with anti-EBV antibody-negative sera. A homology search of the DNA sequence of the cloned gene with EBV B95-8 sequences revealed that those were located at 61716~62087, 61898~62085, and 102128~103158, respectively. These positions correspond to BFRF3, BFRF3, and BZLF1, respectively, which were reported as immunogenic protein which evoked strong and longstanding antibody response and had no sequence homology with the proteins of other herpesviruses. All the patients' sera were reactive with clone 12 protein, but only 5 out of 9 patients' sera were reactive with clone 21 protein. CONCLUSION: Clone 21 protein expressing BFRF3 fragment was immunoreactive in patient sera from natural EBV infection and was regarded as useful candidate for the serodiagnosis of EBV infection.

Affinity Selection of Human ScFv Against Botulinum Neurotoxin Serotype A / 微生物学通报

In this work, rare specific binders to botulinum neurotoxin antigen were selected from human phage immunized library through three rounds of panning in vitro. Of them, clone ScFvB17 is of high affinity and specificity to target antigen. B17 is of 750bp encoding 250 amino acid. Analysis results of gene structure showed V-genes of B17 belonging respectively to VH4 or? chainⅡ family are new antibody Fv genes. More important result showed that recombinant ScFvB17 expressed in E.coli could bind specific for neurotoxin competing with antiserum. Successful selection of human phage antibody library and obtaining specific ScFv against botulinum neurotoxin serotype A provide useful materials for further study on detection of neurotoxin and human engineering therapeutic antitoxin.