| The single chain variable fragment (ScFv) is a type of recombinant protein which is spliced with the variable region of heavy chain (VH) and the variable region of light chain (VL) from end to head through peptide connective via the method of gene engineering. The ScFv is the smallest antibody fragment with the antigen binding activity, and has the characteristics of small molecular weight, strong affinity and specificity to the corresponding antigen, mass production through gene engineering. In addition, compared with the monoclonal antibodies, single chain antibody is easy to save. The key of single-chain antibody's preparation is the obtainment of variable region genes.The most commonly used method is amplifying antibody variable region gene from the hybridoma which secretes monoclonal antibodies, and then obtaining the single-chain antibody gene by splicing. In view of the above characteristics of the single-chain antibody, we can obtain antibody fragment variable region from the hybridoma.In the study we attempted to construct single-chain antibody against the two monoclonal antibodies which are prepared and preserved in laboratory.The total RNA is abstracted from the secretion of anti-Goose parvovirus NS1 protein monoclonal antibody and anti-Goose IFN-γmonoclonal antibody hybridoma cell, and OligodT is employed as the reverse primer to synthesize the first chain of cDNA. Gene fragments in the variable region are amplified through VH and VL PCR primers, and VH and VL genes are cloned into the pMD18-T vector to test the sequences. Followed splicing by overlap extension, peptide coding sequences (Gly4Ser)3 of the connecting peptide is leaded in between VH downstream and VL upstream by lap extending splicing method, thereby getting the GPV-NS1-ScFv gene, and the sequence analysis is achieved. Finally, the ScFv gene is recombined in the prokaryotic expression vector pET32a and pET-30a-EAP. After it is proved to be correct by restriction enzyme and sequenceing, the recombinant plasmid was transformed into E coli. Rosetta (DE3) pLysS. The recombinant protein is highly effectively expressed with the form of inclusion body via the IPTG inducing. Through extracting inclusion body protein, the purpose of a purer protein is gained by puring. And then GPV-NS1-ScFv protein and GoIFN-γ-ScFv-EAP protein were renaturated by PBS gradient and by Tris-Hcl gradient dialysis separatedly. Then we analysed the GPV-NS1-ScFv by indirect antigen ELISA and GoIFN-γ-ScFv antigenicity by direct antigen ELISA method.The variable region of two monoclonal antibody genes were successfully amplified in the experiment, which GPV-NS1 protein monoclonal antibody VL gene fragments 324bp, VH gene fragments 354bp; GoIFN-γmonoclonal antibody VL gene fragments 342bp, VH gene fragments 357bp. The GPV-NS1-ScFv gene fragment spliced by SOE-PCR method was 741bp, GoIFN-γ-ScFv gene fragment was 762bp. Two ScFv recombinant gene expression vector were gained, and achieved their expression in Escherichia coli. GPV-NS1-ScFv protein molecular weight is 46Ku around; GoIFN-γ-ScFv-EAP protein molecular weight is about 80Ku, consistent with the expected. The protein expressed was purified and renaturated. Ultimately ScFvs which can react with Goose parvovirus NS1 protein monoclonal antibody and gooseγ-interferon associated antigen monoclonal antibody were obtained. The study the experiment lay the basis of the material for the diagnosis of Gosling Plague and Gooseγ-interferon detection.
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