| Anti tumor necrosis factor α(TNF-α) antibody can neutralize the deleterious effects caused by high-level TNF-α in vivo. It can also block the pathway of autoimmune diseases such as rheumatoid arthritis (RA) etc. This provides it a broad clinical application in treating TNF-α related diseases. Many experiments have shown that anti-TNF-α monoclonal antibody (mAb) is effective in reducing the symptoms of inflammation and infectious diseases that are related with high-level TNF-α. Here we report the preparation of anti-TNF-α scFv using phage display technique. First, this establishes a massive and simply productive technology of antibody in vitro and resolves the problems of complicated production of anti-TNF-α mAb with low yield. Secondly, with respect to the shortcomings of antigenicity, large molecular mass and powerless penetration of murine mAb, scFv is characterized by its low mass, reduced immunogenicity and powerful penetration due to its structure composed of only VH and VL. This makes anti-TNF-α scFv a wide application in clinical immunological treatment. Thirdly, the physical association of the phenotype and genotype of phage display technique allows it a very valuable tool for exploitation and analysis of laws of protein recognition. In this paper, we present the construction of anti-TNF-α phage scFv antibody library and the isolation, characterization and humanizaiton of specific scFv directed against TNF-α. The expression level of anti-TNF-α scFv was improved using gene engineering technique and the consensus of primary structure of scFv VH was analyzed briefly. I Construction of anti-TNF-α phage antibody scFv libraryWe construct the mouse phage scFv library to obtain anti-TNF-α scFv. First, immunizing mice with rhTNF-α and the titer of antibody in sera was detected using ELISA. The results showed that the titer of antibody of two mice was up to 10-5-10-6 and conforms to the demands of phage antibody library construction. Then total RNA was isolated from splenocytes of these two mice and converted into cDNA. And scFv gene was amplified by the overlap extension PCR method using cDNA as template. This procedure included: ①The VH and VL fragments were separately amplified using the primer pairs that hybridize to the sequence encoding the variable regions of heavy and light chain. The PCR products were purified and the concentrations were determined. ②The VH and VL fragments were assembled into scFv with a specially constructed DNA linker by PCR. ③ScFv was amplified with 5' heavy chain primer with Sfi I site and 3' light chain primer with Not I site. After the analysis of agarose gel electrophoresis, the PCR product of VH formed a band at ~360 bp and that of VL at ~330 bp and the VH and VL were successfully spliced together to generate a fragment of scFv with 750 bp in length. The purified scFv was digested with Sfi I and Not I and cloned into phagemid pCANTAB 5 E. The restriction analysis of the ligation product suggested that scFv fragment was inserted in front of gIIIp of phagemid pCANTAB 5 E. Then the ligation product was named pATF and transferred into competent E.coli TG1 to generate a bacterial form of phage scFv antibody library. The transformation reaction was carried out several times to increase the diversity of the library. The diversity of the library evaluated by means of bacterial colony counts was about 4.6(106. It is medium. The recombinant ratio of the library was 83% by restriction analysis.II Screening and determination of anti-TNF-α positive phage clone The library was screened by rhTNF-α to get the antigen-specific phage clones. Above all, three panning rounds were performed of the library with rhTNF-α. The transformed cells were infected with M13KO7 helper phage to produce a phage form of library. Then the library was added into microtiter wells coating with rhTNF-α and incubated for a period of time and washed several times. The phage clones binding specifically to TNF-α were retained in the wells. These clones were eluted from the we...
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