| Africa classical swine fever (ASF) is an acute infectious disease caused by Africa classical swine fever virus (ASFV) with acute high fever, systemic and general visceral organ bleeding, Once infectde swine morbidity and mortality can reach 100%, is classified as notification disease by OIE. The clinical manifestations of ASF are very similar with CSFV, so it is easy to be misdiagnosed. No ASFV has found in our country at present, need to guard against. The rapid diagnosis and monitoring for imported products have become one of the key technologies to prevent ASF spread into China. In this experiment the use of prokaryotic expression system and immune colloidal gold technique, Feasibility of colloidal gold test strip for detection of ASFV was discussed.Firstly, protein expression of ASFV K205R gene was preparated by prokaryotic expression system:pMD 19-T Simple vector containing ASFV K205R gene was got by artificial synthesis,then restriction enzyme it to got K205R gene fragment use Not I and Bgi Ⅱ, then put K205R gene and Plasmid pET32a enzymed by restriction endonuclease Not Ⅰ and Bgi Ⅱ together, Identified by PCR, double enzyme digestion and sequencing, The results showed that Recombinant Prokaryotic expression system of pET32a-K205R plasmid was successfully constructed.Then get the recombinant plasmid pET32a-K205R was transferred into the expression vector BL21 in Escherichia coli. Induced by IPTG,it can obtain specific protein band at about 44 kDa,and the optimized 37 ℃,1 mmol/L IPTG, induced the expression of 3H was the best inducement conditions. SDS-PAGE analysis showed that K205R gene protein expression recombinant strains were mainly distributed in bacteria’s supernatant, and the expression is far greater than the other cell protein. Purified protein of K205R gene was obtained by The Ni-TED affinity chromatography.The results of ELISA showed that The purified K205R protein can reach with the positive serum (ASFV positive serum was purchased from ID-VET company of France) and the purified protein has immunogenicity, then obtained positive serum by immunizing rabbits.The preliminary preparation and discussion of colloidal gold test strip:Different diameter size of colloidal gold particles were made by citric acid three sodium in this experiment, colloidal gold solution was clear and transparent through the naked eye and and no visible float.The results Scaned by UV Spectrophotometry showed that the colloidal gold was particles when put 2.2 ml 1% citric acid three sodium solution into 100 ml 0.01%gold acid chloride solution. The colloidal solution preparation was red wine and 10 nm-20 nm in diameter,it can be used for marker protein. According to the principle of colloidal gold immunity chromatography, gold particles of rabbit anti K205R antibody was used as the gold standard pad in this experiment, use rabbit anti K205R antibody as a detection line (T line) to Capture K205R protein,use staphylococcal protein A (SPA) as the control line (C line). Through the optimization of the working conditions, the best label quantity of gold labeled Rabbit anti K205R antibody was 40 ng/ml, the optimum pH value is 7.5, the detection line concentration was 1.5 mg/ml, the control line concentration was 2 mg/ml. After testing, colloidal gold test strip can be reached with K205R protein expressed, can’t reached with BL21 Escherichia coli. lysate, classical swine fever virus (CSFV), Porcine reproductive and respiratory syndrome(PRRSV), pseudorabies virus (PRV), porcine circovirus (PCV) and so on. The expression of K205R protein in the minimum detection amount is about 30 ng/ml-40 ng/ml. |
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