人类疱疹病毒6型U94基因克隆?表达?纯化及抗体制备

目的:制备人类疱疹病毒6型U94蛋白及其抗体.方法:PCR扩增U94基因,经测序后克隆到原核表达载体PGEX-6p-1中.重组质粒转化大肠杆菌Rosetta,诱导蛋白表达.应用酶切鉴定、SDS-PAGE及Western blot等方法确保基因片段的正确性及表达蛋白的特异性.表达的蛋白经亲和层析纯化.纯化的蛋白免疫动物制备抗血清.结果:成功地获得了高纯度的U94融合蛋白,纯度可达90%以上.用其制备多克隆抗体滴度可达1:100 000.结论:获得高纯度的U94融合蛋白及其高效价的抗体.

Cloning,expression and purification of human herpesvirus 6 U94 and its antibodies production

Objective:To prepare human herpesvirus 6 U94 protein and produce its antibodies.Methods:U94 gene of HHV6 was amplified by PCR and sequenced.The resulting DNA construct was cloned into a prokaryotic expression vector(PGEX-6P-1).The recombinant plasmid was transformed into Eserichia coli Rosetta.The accuracy of inserted gene and specificity of proteins were detected by two enzymes digestion,SDS-PAGE,and Western Blot.The fusion U94 protein purified by affinity chromatograph was used to vaccinate rabbit to produce antibodies.Results:The purity of U94 protein was above 90%.The titer of the antibodies was 1: 100 000.Conclusion:HHV6 U94 fusion protein with high purity was obtained and its corresponding polyclonal antibodies with high titer were produced.