| Background and aims:Anti-glomerular basement membrane diease(GBM)is a rare autoimmune disorder characterized by rapidly progressive glomerulonephritis (RPGN)and alveolar hemorrhage. Despite of low incidence of anti-GBM diease, it needs to be studied further due to its poor prognosis and high mortality. It is reported that anti-GBM antibodies play a central role in the pathogenesis of anti-GBM disease. Binding of anti-GBM antibodies to the GBM leads to autoimmune injury characterized by strong complement activation,leukocyte accumulation and proteinuria. Therefore, anti-GBM antibodies are particularly important to the development of anti-GBM disease.According to thorough studies on anti-GBM disease, the major target antigen of anti-GBM antibodies has been identified as theα3 chain non-collagen 1 domain of typeⅣcollagen [α3(Ⅳ)NC1].The epitopes of anti-GBM antibodies(EA and EB)were usually hidden in the NC1 hexamer complex. Infection and exposure to chemical agents, such as organic solvents and cigarette smoke,appear to have effects on the exposure of epitopes, which induce autoimmune response and produce antibodies. Animal experiments and clinical studies have demonstrated that anti-GBM antibodies contributed to the pathogenesis of anti-GBM disease. In 1967, Lerner et al found the presence of anti-GBM antibodies in the sera and/or kidneys of humans with Goodpasture's nephritis and proved that acute glomerulonephritis can be inducted in monkey by the injection of the antibodies.These observations established the presence and the pathogenic effects of anti-GBM antibodies, confirming the relationship between anti-GBM antibodies and the pathogenesis of Goodpasture's nephritis. Recent studies showed that the immunoglobulin G subclass distribution, avidity and titre of anti-GBM antibodies are associated with the disease severity and prognosis. Natural anti-GBM antibodies can be examined in normal healthy human sera. But the titre and avidity are lower than those of patients with anti-GBM disease and the immunoglobulin G subclass distribution is different between patients and healthy human, which implies characteristics of the antibodies may play a major role in the initiation and progression of the disease. At present, plasma exchange combined with immunosuppressive therapy are considered as the preferred treatments of anti-GBM disease. Plasma exchange is used to eliminate circulating antibodies, while immunosuppressive agents are taken to suppress new antibody production. Based on some foreign studies, in patients who treated with plasma exchange combined with cyclophosphamide and glucocorticoids early, patient survival was 85%,40% of whom reach end-stage renal disease (ESRD). However if the diease is not recognized and treated early, patient mortality will increased by 90%. Consequently, eliminating circulating antibodies and suppressing new antibody production are the key to treatment and prognosis. If the specific antibody to anti-GBM antibodies were obtained,it could prevent and block anti-GBM antibody binding to the target antigen in glomerular basement membrane, which will release the anti-GBM disease.In modern times, phage display technology has played a dominant role in antibody engineering. It allows the presentation of large peptide and protein libraries on the surface of filamentous phage, which leads to the selection of peptides and proteins, including antibodies, with high affinity and specificity to almost any target. Bacause of the advantages of modifying and large-scale production,antibodies from phage display libraries present a great potential capability and possess broad application foreground.In our study,the scFv 3 gene was selected from a human synthetic scFv phage display library of Peking University. The fusion protein scFv 3 was obtained by application of recombinant-DNA technology and affinity chromatography technology and its immunoreactivity was confirmed by indirect ELISA. In order to produce large amounts of high purity protein and analyze its characteristics, we subcloned the scFv 3 gene into pET system and purified highly expressed protein in E. coli.Materials and Methods:The scFv3 gene,as a template,was amplified by PCR and the primer pairs named XP1 and XP2.Afterwards,the amplified fragments were cloned into pGEM-T Easy Vector and transformed into E.coli strain TOP 10. The positive clones selected by blue-white screening were identified by PCR and restriction analysis. Extract recombinant plasmid and digested it with EcoR I and Xho I endonucleases. The target gene was ligated into the expression vector pET28a(+) and transformed into E. coli BL21(DE3).After the recombinant plasmid was determined by restriction endonuclease digestion and DNA sequencing, the target protein expression was induced by IPTG at 30℃for 20h and analyzed by SDS-PAGE.The fusion protein scFv3 was purified by HisLinkTM Spin Protein Purification System. The immunoreactivity were measured by indirect ELISA.Results:1.The amplified DNA fragment was in size of 750bp as expected.2.PCR and restriction analysis showed that the scFv3 gene was inserted in pGEM-T Easy vector,which means the cloning vector pGEM-scFv3 was constructed successfully.3.The scFv3 gene was subcloned into pET28a(+) and transformed into E. coli BL21(DE3).The positive clone was confirmed by PCR and restriction endonucleases digestion. The sequence was confirmed by DNA sequencing and consistent with the result of the scFv3 gene.4.SDS-PAGE profile showed a clear protein band with a relative molecular weight 27 kD.5.The fusion protein scFv3 was purified by affinity chromatography methods.6.The result of indirect ELISA showed the purified fusion protein scFv3 had antigen binding activity.Conclusion:1.The cloning vector pGEM-scFv3 was constructed successfully2.The fusion expression vector pET28a-scFv3 was constructed successfully3.The protein scFv3 appeared as a single band of 27 kDa and can be effectively purified by affinity chromatography4.The purified protein scFv3 showed high immunoreactivity with anti-GBM antibody. |
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