HCMV重组嵌合抗原表达质粒构建及免疫反应的初步鉴定  

Construction of HCMV recombination multi-epitope antigen expression vector and preliminary identification of immunogenicity

作　　者：曾宪聪[1] 汪小五[1] 陈伟[1] 王林定[1,2] 

机构地区：[1]安徽医科大学微生物学教研室,合肥230032 [2]安徽医科大学病原与免疫学实验室,合肥230032

出　　处：《安徽医科大学学报》2014年第6期752-756,共5页Acta Universitatis Medicinalis Anhui

基　　金：国家自然科学基金(编号:81271837);安徽省教育厅自然科学基金(编号:KJ2012A161);安徽医科大学博士科研经费资助项目(编号:XJ200914)

摘　　要：目的选用人巨细胞病毒(HCMV)抗原性强且亲水性较高片段构建融合抗原表达质粒,诱导其表达,纯化目的蛋白;并初步鉴定其免疫反应性。方法通过Protean软件分析了HCMV UL32、UL44、UL83序列,设计合理引物;各基因片段用Overlap方法连接,并引入BglⅡ、BamHⅠ酶切位点,使之可通过酶切与质粒pQE-80L连接。重组质粒转化入感受态BL21(DE3)菌株,经PCR、酶切鉴定目的基因后再诱导、表达、纯化,获得目的蛋白,采用Western blot法检测免疫反应性。结果诱导后的重组菌pQE80-L-UL32-UL44-UL83经十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)证实蛋白表达在包涵体。纯化后获得大小约为52 ku的目的蛋白,采用Western blot法检测证实重组嵌合抗原能与HCMV阳性血清有反应。结论构建了重组嵌合抗原表达质粒,并诱导、表达、纯化获得目的蛋白。Western blot法鉴定嵌合抗原是HCMV特异性抗原且有免疫反应性。Objective To construct fusion antigen expression plasmid by choosing strong antigenicity and higher hydrophilic segments of HCMV gene, we induce expression, purify target protein and preliminarily identify immu- nogenicity of HCMV in order to develop fast and sensitive diagnostic kits research laboratory diagnosis. Methods in future. Methods Sequences of HCMV UL-32,UL-44, UL-83, were analyzed by software of Protean to design rea- sonable primers. Each gene fragment was linked by using Overlap method at the same time Pst I , Bgl Ⅱ , BamH I sites were introduced to make it connected to plasmid pQE80-L by enzyme digestion, then the recombinant plasmid was transformed into BI21. Target gene identified by PCR and digestion was induced to express the protein. The protein was purified and its immunoreactivity was assayed by Western blot. Results After inducing, recombinant bacteria, pQE80-L-UL32- UIA4-UL83, had a fusion of 52 ku protein by SDS-PAGE electrophoresis. Recombinant chimeric antigen could react to HCMV positive serum through Western blot. Conclusion antigen expression plasmid is constructed to obtain target protein. Multi-epitope antigen is cific antigen for HCHV and has immunity by Western blot. The recombinant fusion confirmed to be the specific antigen for HCHV and has immunity by Western blot.

关 键 词：人巨细胞病毒 重组融合抗原 免疫反应性 

分 类 号：R373.4[医药卫生—病原生物学]