人疱疹病毒7型被膜蛋白pp85的原核表达和应用

发布时间：2018-05-28 02:11

本文选题：人疱疹病毒7型 + ELISA　；参考：《青岛大学》2005年硕士论文

【摘要】：目的构建人疱疹病毒7型(HHV7)被膜蛋白pp85的原核高效表达克隆,制备用于HHV7感染血清学诊断的基因工程抗原。方法 ①应用HHV7标准株Glasgow感染SupT1细胞,收获病变细胞制备粗提细胞抗原,并用Sephadex G-75层析柱初步纯化,获得HHV7细胞工程抗原。②应用PCR技术扩增HHV7 ORF U14基因的主要抗原决定簇编码区(328～533氨基酸),目的基因与原核表达载体pThioHis A分别双酶切后连接,转化宿主菌E.coli BL21,IPTG诱导融合蛋白表达,经镍-敖合物琼脂糖树脂柱亲和层析纯化获得基因工程抗原。Western blot检测重组融合蛋白的特异性。③选择最适浓度包被96孔ELISA反应板,选择血清标本进行检测,并与Qiagen公司生产的HHV7 IgM和HHV7 IgG抗体ELISA检测试剂盒进行比较。结果 HHV7被膜蛋白pp85可在E.coli BL21中高效表达,Western blot检测结果表明该融合蛋白可与12份HHV7特异性IgM阳性血清中的10份(83.3%)、12份HHV7特异性IgG阳性血清中的11份(91.7%)发生反应,而10份阴性的血清均不与之发生反应。基因工程抗原的各项技术指标与Qiagen公司ELISA检测试剂盒有较好的符合率。结论 HHV7 pp85重组蛋白具有良好的抗原性和特异性,进一步完善后可用于HHV7感染的临床检测,为进一步研制开发新型HHV7诊断试剂提供了重要的理论和实验依据。
[Abstract]:Objective to construct a prokaryotic expression clone of human herpesvirus 7 (HHV7) membrane protein pp85 and to prepare a genetically engineered antigen for serological diagnosis of HHV7 infection. Methods (1) HHV7 standard strain Glasgow was used to infect SupT1 cells, and the diseased cells were harvested and purified by Sephadex G-75 chromatographic column. HHV7 cell engineering antigen .2 was used to amplify the main antigen determining cluster coding region of HHV7 ORF U14 gene by PCR. The target gene was digested with the prokaryotic expression vector pThioHis A, and the fusion protein was induced by E.coli BL21. The recombinant fusion protein was purified by agarose resin affinity chromatography. The specificity of the recombinant fusion protein was determined by Western blot. The optimal concentration of the recombinant fusion protein was coated with 96 well ELISA reaction plate, and the serum samples were selected for detection. The results were compared with HHV7 IgM and HHV7 IgG antibody ELISA kits produced by Qiagen Company. Results HHV7 envelope protein pp85 could be highly expressed in E.coli BL21. The results showed that the fusion protein could react with HHV7 specific IgM in 10 out of 12 HHV7 specific IgM positive sera, and in 11 out of 12 HHV7 specific IgG positive sera. None of the 10 negative sera reacted with it. The technical parameters of genetic engineering antigen were in good agreement with the ELISA kit of Qiagen Company. Conclusion the recombinant HHV7 pp85 protein has good antigenicity and specificity and can be used in the clinical detection of HHV7 infection. It provides an important theoretical and experimental basis for the further development of new HHV7 diagnostic reagent.
【学位授予单位】：青岛大学
【学位级别】：硕士
【学位授予年份】：2005
【分类号】：R346

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