Construction, expression and identification of the anti-idiotypic single chain variable fragment against Edwardsiella tarda / 西安交通大学学报(医学版)

Objective To construct, express and identify the anti-idiotypic antibody single chain variable fragment (scFv) against Edwardsiella tarda. Methods By using RT-PCR method, the variable regions of the heavy and light chain of the anti-idiotypic monoclonal antibody (mAb) 1E11 against Edwardsiella tarda were cloned and joined with a (Gly_4ser)_3 linker, and the scFv in the orientation of V_L-linker-V_H was constructed. It was then cloned into vector plasmid pET-28a, expressed in Escherichia coli BL21(DE3), and confirmed by SDS-PAGE, Western blot and ELISA. Results The recombinant scFv could be expressed in E.coli BL21 (DE3) in a fusion protein pattern. The expression product was in the form of an inclusion body and the purified fusion protein was obtained after being purified and refolded. The SDS-PAGE and Western blot analysis showed that the molecular had the binding activity to the antigen. Conclusion The recombinant anti-idiotype scFv has been successfully constructed and expressed in E.coli BL21 (DE3), providing the basis and potential for preparation of genetically engineered vaccine against Edwardsiella tarda.

Construction,expression and identification of the anti-idiotypic single chain variable fragment against Edwardsiella tarda / 西安交通大学学报(医学版)

Objective To construct,express and identify the anti-idiotypic antibody single chain variable fragment(scFv) against Edwardsiella tarda.Methods By using RT-PCR method,the variable regions of the heavy and light chain of the anti-idiotypic monoclonal antibody(mAb) 1E11 against Edwardsiella tarda were cloned and joined with a(Gly4Ser)3 linker,and the scFv in the orientation of VL-linker-VH was constructed.It was then cloned into vector plasmid pET-28a,expressed in Escherichia coli BL21(DE3),and confirmed by SDS-PAGE,Western blot and ELISA.Results The recombinant scFv could be expressed in E.coli BL21(DE3) in a fusion protein pattern.The expression product was in the form of an inclusion body and the purified fusion protein was obtained after being purified and refolded.The SDS-PAGE and Western blot analysis showed that the molecular weight of scFv protein was 27 ku.Indirect ELISA confirmed that the scFv had the binding activity to the antigen.Conclusion The recombinant anti-idiotype scFv has been successfully constructed and expressed in E.coli BL21(DE3),providing the basis and potential for preparation of genetically engineered vaccine against Edwardsiella tarda.

Production and characterisation of monoclonal anti-idiotype antibody to Vibrio anguillarum.

Seven monoclonal anti-idiotype antibodies (mab2) were raised against mouse monoclonal antibody (mab1) 4A6. Identification of subclass showed that 1H5, 1D1, 2B12 and 2F12 belonged to IgG2b, 2H12 and 1H12 to IgG2a and lE10 to IgG3. The titres of these mab2 ascitic fluids ranged from 1 x 10(-4)-1 x 10(-6). The capacity of the mab2 to inhibit the binding between the corresponding rabbit antiserum and Vibrio anguillarum was investigated with the competitive inhibition ELISA. The results showed that mab2 1D1, 1E10, 1H5 and 1H12 were able to inhibit this binding. Another experiment demonstrated that mab2 1D1, 1E10 and 1H5 might induce Balb/c mice to produce Ab3 and these Ab3 competed the same antigen epitopes with Ab1. These results indicate that mab2 1D1, 1E10 and 1H5 are likely to represent an internal image of V. anguillarum and may thus be described as Ab2-beta anti-idiotype antibodies. In protection experiments, Japanese flounders vaccinated with mab21D1, 1E10 and 1H5 showed significantly enhanced survival from challenge with V. anguillarum. Thus. mab21D1, 1E10 and 1H5 may have use as idiotype vaccines for fish in aquaculture.

Effect of human calcyclin binding protein encoding gene on development of multiple drug resistance in gastric cancer / 中华肿瘤杂志

<p><b>OBJECTIVE</b>To study the effect of human calcyclin binding protein (CacyBP) encoding gene on the development of multiple drug resistance in gastric cancer.</p><p><b>METHODS</b>hCacyBP sense nucleic acid eukaryotic expression vector (pcDNA3.1/hCacyBP +) was constructed and then transfected steadily into the gastric cancer drug sensitive cell (SGC7901) mediated by lipofectamine ( trade mark ) 2000. RT-PCR was used to measure the CacyBP mRNA expression level. MTT was used to measure the adriamycin (ADR) drug sensitivity of SGC7901 and SGC7901 after transfection. FCM was used to measure the average ADR accumulation concentration and cell cycle of SGC7901 and SGC7901 after transfection.</p><p><b>RESULTS</b>The hCacyBP mRNA expression level of SGC7901 transfected with pcDNA3.1/hCacyBP + was higher than SGC7901 transfected with pcDNA3.1 or SGC7901, with the higher survival rate in the former. The average ADR accumulation concentration in SGC7901 and SGC7901 transfected with pcDNA3.1 or pcDNA3.1/hCacyBP + was 5.64, 5.49 and 5.17, respectively. The G(1) phase cell proportion of SGC7901 transfected with pcDNA3.1/hCacyBP + or pcDNA3.1 was reduced slightly but G(2) and S phases increased slightly as compared with SGC7901.</p><p><b>CONCLUSION</b>Calcyclin binding protein may play a certain role in gastric cancer drug resistance.</p>