The human immunodeficiency virus type 1 gag p20 gene fragment was clonedinto plasmid pBV220 to construct a recombinant expression plasmid pCY7.By induction,the p20 protein was expressed in E.coli,and it was confirmed ...The human immunodeficiency virus type 1 gag p20 gene fragment was clonedinto plasmid pBV220 to construct a recombinant expression plasmid pCY7.By induction,the p20 protein was expressed in E.coli,and it was confirmed by Western blotting thatthe expressed p20 protein could be specifically recognized by anti-p17 monoclonalantibodies.The recombinant bacteria was lysed and fractionated into snpernatant andprecipitate by centrifugation.It was found that the p20 protein was present chiefly inthe precipitate.After p20 protein being washed twice,its purity reached 27.4%.Then theprecipitate was washed with 1 mol/L urea solution and the purity of p20 protein wasraised to 58.1%.Finally,the precipitate was dissolved in 6mol/L of urea and appliedonto a heparin affinity column.Four peaks were obtained and the p20 protein wasfound mainly in peak 3.The purity of p20 protein at this step reached 84.2% as meas-ured by gel scanning.展开更多
基金Supported by the National Science Foundation of China No.39100029
文摘The human immunodeficiency virus type 1 gag p20 gene fragment was clonedinto plasmid pBV220 to construct a recombinant expression plasmid pCY7.By induction,the p20 protein was expressed in E.coli,and it was confirmed by Western blotting thatthe expressed p20 protein could be specifically recognized by anti-p17 monoclonalantibodies.The recombinant bacteria was lysed and fractionated into snpernatant andprecipitate by centrifugation.It was found that the p20 protein was present chiefly inthe precipitate.After p20 protein being washed twice,its purity reached 27.4%.Then theprecipitate was washed with 1 mol/L urea solution and the purity of p20 protein wasraised to 58.1%.Finally,the precipitate was dissolved in 6mol/L of urea and appliedonto a heparin affinity column.Four peaks were obtained and the p20 protein wasfound mainly in peak 3.The purity of p20 protein at this step reached 84.2% as meas-ured by gel scanning.
