Construction And Expression Of Bifunctional Molecule Of Detecting CSF Antibody Quickly

Classical swine fever(CSF) caused by Classical swine fever virus(CSFV) is a kind of urgent febricity and fatal disease. It has made a great loss in world's swine industry.monitoring regulaurly CSF antibody has important significance with control of CSF, at present, because there is still no a convenient and fast and stable detective method of CSF antibody to refer to primary level,the study of bifunctional reagent of detecting CSF antibody quickly performed according to the principle of haemagglutination.The main result of study as follow:1. Splicing of bifunctional molecule and construction of prokaryotic vector2E8ScFv (ScFv gene against H antigen of human erythrocyte) and mE2(main antigen region of E2 gene of CSF) were spliced by over lap extension(SOE) PCR and assembled bifunctional molecule named 2E8mE2, then it was cloned into PMD18-T Simple vector,the sequenced recombinant bifunctional molecule was sub-cloned into prokaryotic vector PET-DsbA and indentified by PCR and enzyme digest.Result showed that PCR and enzyme digest detected a 1125bp target band.The result indicated construction of prokaryotic vector PET-DsbA-2E8mE2 had been successful.2.Expression of the 2E8mE2 recombinant proteinRecombinant vector PET-DsbA-2E8mE2 was transformed into the BL21 (DE3) PlysS E coli cell and expressed by common method, sonication of the bacterial cells and solubility analysis of recombinant protein by SDS-PAGE, then choice of expression conditions by grads of temperature and IPTG concentration and induce time.Result indicated the recombinant protein was gained high expression in the form of inclusion body at the level up to 30% total bacterial protein; the best temperature and IPTG concentration and induce time of recombinant protein expression were 37℃and 0.3mmol/L IPTG and 3h,respectively.Detection of the recombinant protein with 6xHis monoclone antibody and positive serum against CSFV respectively by Western-blot, result showed that recombinant protein could react with 6xHis monoclone antibody and positive serum against CSFV respectively and showed limpid and specific reaction band which negative control didn't. The Result indicated expressed recombinant protein was specific.3.Renaturation of inclusion body of the 2E8mE2 recombinant protein and detection of renatured recombinant proteinSonication of the bacterial cells and collection of inclusion body of the recombinant protein, the inclusion body was washed with 0.5% TritonX-100 and 1mol/L NaCl and then subjected to denaturation in 8mol/L urea, then the denatured protein was purified with Ni-NTA resin by affinity chromatograph, at last the purified and denatured protein was renatured by glutathione reoxidation and detection of reactivity of the renatured recombinant protein and positive serum against CSFV by indirect ELISA. Result showed that the purity of interest protein reached 86% after washed and sublimation of purity of the interest protein reached 92% after purified with Ni-NTA resin by affinity chromatograph so that it may utend renature, reactivity of the renatured recombinant protein and positive serum against CSFV was boosted obviously contrasting to the denatured recombinant protein, result indicated renaturation of the recombinant protein was successful.