| ObjectAnti-GBM (glomerular basement membrane, GBM) disease refers to a group of autoimmune diseases caused by the anti-GBM antibody in the cycle deposition in the organs. Its characteristics are detected anti-GBM antibodies in the peripheral blood, or after renal biopsy the anti-GBM antibody as line deposition on the glomerular basement membrane. The target antigen of anti-GBM antibody, also known as Goodpasture (GP) antigen, is collagen type??3 chain, non-collagen area 1 [?3 (?) NCI] in the basement membrane. Under normal circumstances, the antigen hide in the collagen type?non-collagen area of basement membrane.Once the epitope exposed to environmental factors or other factors, can induce autoimmune responses. Most anti-GBM disease's condition is dangerous, onset is acute, development is rapid, is one of the worst prognosis kidney disease. Although the clinical use widespread of plasma exchange and corticosteroids combined with cyclophosphamide therapy, there is still a small number of patients early died of pulmonary hemorrhage. In particular, circulating plasma replacement therapy can only remove free anti-GBM antibodies, but can not completely remove the antibody which had been combined with the GBM, and require a large number of normal human plasma, is not only expensive but also may increase the risk of blood-borne diseases. The combination of high-dose corticosteroids and cyclophosphamide for shock treatment, suppressed the body's immune response, will increase or induced infection. So, how can suppress specific immune response of anti-GBM disease will be the promising subject.Phage display antibody engineering technology is an extremely important technology, specific phage antibodies can be directly selected from the antibody library, and antibody fragments presented in the phage surface. The antibody from phage display has many of properties of the polyclonal antibody and of monoclonal antibodies, and can be easily converted by the E. coli, then yeast in the large-scale production. Thus, phage antibodies showed huge potential and broad prospects. This study used a new type of phage display technology, wish that single-chain antibody genes of the anti-GBM antibodies selected from phage antibody library. Then we use recombinant technology to clone the single-chain antibody gene and express vector. And finally through the induction and affinity chromatography technology, neutralizing single chain antibody protein of anti-GBM antibody was purified, and verified whether the single chain antibody protein binded with anti-GBM antibody specificity in vivo and vitro. Thereby this way could improve organ damage generated by the anti-GBM antibody binding target antigen, hoping provide more experimental evidence for overcoming anti-GBM disease.Material and Methods1. The patients'serum anti-GBM antibody were purification. Using phage display technique, the phage antibody library was panned by anti-glomerular basement membrane(GBM) antibody which was coated in a micro-titer plate, one clone was found to have high affinity to anti-GBM antibody. The DNA sequence of the positive clone was determined.2. The scFv3 gene of anti-GBM antibody was amplified by PCR and cloned into pGEM-T Easy Vector.The sequence of cloned scFv3 was confirmed by restriction analysis and DNA sequencing.A scFv3 prokaryotic expression plasmid, pQE80L-scFv3,was then constructed by subclone. pQE80L-scFv3 transformed E.coli Rosetta2 was induced by IPTG. The expression of scFv3 protein was analyzed by SDS-PAGE and Western blot.3. Wistar rats were randomly distributed into five groups:control group I was a negative control and was injected with healty human serum via the caudal vein. Control group II was injected with neutralizing monoclonal antibodies to anti-GBM antibody only.The other three groups were injected with human anti-GBM antibody, neutralizing monoclonal antibodies to anti-GBM antibody at day 7 and 14, respectively. The Blood, urine and kidney tissue were collected on day 7,14,21 for analysis of 24-hour urinary protein, blood urea nitrogen(BUN), creatinine and histological study. Between the two groups were compared with ANOVA, pairwise comparisons between groups using t test to test with a= 0.05 level. Statistical analysis using SPSS 13.0 software for processing. ResuIts1. Along with the increase of rounds anti-GBM antibody specific phage antibody was highly enriched and screening efficiency was increased 137 folds than the firest round. ELISA and competition inhibition assay showed that the scFv had a specific combination character with anti-GBM antibody. DNA sequencing confirmed that the whole gene of scFv was 750 bp, and in accordance with humanized single-chain variable region antibody sequence structure.2. The amplified DNA fragment was in size of 750bp as expected. Restriction analysis showed that the amplified gene was inserted in pQE80L correctly.Sequence showed there was no mutation in both ends of the cloned scFv3.Restrict analysis showed that the recombinant pQE80L-scFv3 was right. A Mr 27x 103 protein could be seen in Rosetta2 induced by IPTG with SDS-PAGE and was positive while reacting with anti-GBM antibody by Western blot.3. On 21th day, there was decreased significantly in intervention group I compared with nephritis model on 24 hours proteinuriaf (16.62±5.53) g/d], blood urea nitrogen?(11.53±2.26) mmol/L] and creatinine[(102.46±16.86)?mol/L] (P <0.05), and also was decreased in intervention group II than nephritis group, but no significant difference (P> 0.05). There was obviously decresed on renal cell proliferation, crescent formation and deposition of immune complexes in Intervention group I and the intervention group II compared with nephritis model group, but was decreased more significantly in the intervention group I. There was no significant change in control group I and control group II.Conclusions1. The results suggested that the scFv fragment to anti-GBM antibody could be successfully selected by recombinant phage antibody technique.2. Anti-GBM antibodies neutralizing scFv protein was constructed and expressed in the prokaryotic, and was verified that can specifically bind with the human anti-GBM antibody.3. The early application of anti-GBM antibodies neutralizing monoclonal antibodies could effectively improve the anti-GBM nephritis rat kidney pathologic changes. |
PDF Full Text Request