HIV-1 CN54 Gag P55和P24蛋白的高效表达、纯化和鉴定

目的　探讨HIV 1中国株CN5 4GagP5 5和P2 4重组蛋白的高效表达并纯化 ,为研究HIV疫苗和诊断试剂创造条件。方法　分别将CN5 4GagP5 5和P2 4基因克隆至 pBV2 2 0和 pThioHisC载体 ,构建非融合表达载体 ;将重组质粒转化大肠杆菌BL2 1codonplus RIL ,对工程菌进行诱导表达。Western Blot检测目的蛋白 ,用DEAE SeparoseFF阴离子交换层析柱纯化目的蛋白 ;纯化的P5 5和P2 4抗原蛋白分别包被酶标板作ELISA检测。结果　P5 5以包涵体的形式表达 ,表达量占菌体总蛋白的 30 % ,P2 4实现了可溶性表达 ,表达量占总菌体总蛋白的4 0 % ;纯化后P5 5纯度达到 80 % ,P2 4纯度超过 90 %。Western Blot和ELISA检测均显示了良好的的灵敏度和特异性。结论　HIV 1CN5 4GagP5 5和P2 4抗原蛋白可在大肠杆菌中高效表达 ,纯化的抗原蛋白具有良好抗原性 ,可用于HIV基础研究和临床检验

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