| We recombined anti-parathion single-chain variable fragment gene (scfv) and the core streptavidin gene(sa) to produce an anti-parathion single-chain variable fragment-core of streptavidin-biotin fusion gene (scfv-sa), and inserted the fusion gene (scfv-sa) into the vector pET28a (+) in E.coli BL21 for transformation by prokaryotic expression.We can obtain the fusion protein, and then we detecte its biological activity were The main contents are:1. Search the related knowledge about the streptavidin, and analysis of its major functional components, retrieve that part of the gene coding sequence, and synthesize the gene. We define the protein with its main function as the core streptavidin protein avidin or streptavidin core protein factors .2. Designed primers to amplified the anti-parathion scFv gene, and then connect the gene with the core streptavidin gene by the method of overlapping extension PCR, we can obtain a new recombinant gene. After the Verification by enzyme digestion and PCR amplification,we believe it is the target recombinant genes ,and then we sent it to the company to confirmed the sequencing.3. We construction the prokaryotic expression vector, and explore the conditions to form soluble proteins in the prokaryotic expression, induction of recombinant gene expression in prokaryotic cells. And retrieve soluble protein, and obtain the purification protein by using Ni +-NTA, detect protein molecular weight by SDS-PAGE, and we identify the expression product as the specific target protein by Western blot. The recombinant gene could express a fusion protein about 46 kDa, which formed a tetravalent domain, tetravalent antibody.4. The biological activity of the recombinant antibody was measured by ELISA results showed that the tetravalent antibody could bound to parathion specifically with the affinity constant of 4.25×107 L/mol and titer of over 1:1×106. The recombining anti-parathion tetravalent antibody could react with parathion specifically with significantly more binding sites with the monoclonal antibody, based on which the detection sensitivity of ELISA would be improved. |
