Expression,purification and identification of Gag P55 and P24 recombinant protein of HIV-1 strain CN54

Objective To overexpress and purify Gag P55 and P24 recombinant protein of HIV 1 strain CN54 for HIV vaccine research and clinical assay.Methods Coding sequences of Gag P55 and P24 of HIV 1 CN54 were cloned into prokaryotic expression vector pBV220 and pThioHisC respectively to construct non fusion expression cassettes.Recombinant plasmids were transformed into E.coli BL21 codonplus RIL and induced for protein expression.Recombinant proteins were identified by Western blot and purified by DEAE Separose Fast Flow column chromatography.Purified proteins were coated on ELISA plates to test Gag specific antibodies in HIV DNA vaccine immunized mice or control serums from HIV infected individuals.Results P55 was expressed as inclusion bodies and observed about 30% of total cellular proteins.P24 was overproduced as soluble protein and up to about 40% of total cellular proteins.The purity of P55 and P24 reached 80% and 90%,respectively,after purification.Western blot demonstrated that P55 and P24 had good antibody affinity The titration of antibody in HIV DNA vaccine immunized mice was assayed up to 1∶30 000 with P55 ELISA plate,The total positive and negative conformity of 20 control serums was 100% by testing with P24 coated ELISA.Conclusion High level expression of HIV 1 CN54 strain Gag P55 and P24 can be achieved in E.coli.Purified P55 and P24 can be applied in HIV vaccine research and clinical assay.Beijing 100050,China