人疱疹病毒8型基因工程抗原的制备和应用

发布时间：2018-11-04 08:41

【摘要】：目的构建人疱疹病毒8型(HHV8)小衣壳蛋白(ORF65)的原核高效表达克隆,制备用于HHV8感染血清学诊断的基因工程抗原。方法应用BCBL-1细胞(HHV8阳性而EBV阴性)制备粗提细胞抗原,并用Sephadex G-75层析柱初步纯化,获得HHV8细胞工程抗原。应用PCR技术扩增HHV8 ORF65编码区,PCR产物与原核表达载体pThioHisA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,经带有6个组氨酸标签的亲和层析柱纯化获得基因工程抗原,Western blot检测其抗原性和特异性。选择最适抗原浓度包被96孔ELISA反应板,对相关病人进行检测,并与Biotrin公司生产的HHV8 IgM和HHV8 IgG抗体IFA检测试剂盒和Qiagen公司ELISA检测试剂盒进行比较。结果 HHV8 ORF65小衣壳蛋白可在E.coli BL21中高效表达,Western blot检测结果表明该融合蛋白可与5份HHV8特异性IgM阳性血清的4份(80%)和所有10份(100%)HHV8特异性IgG阳性血清发生反应,而10份阴性血清均不与之发生反应。基因工程抗原的各项技术指标与Biotrin公司和Qiagen公司检测试剂盒有较好的符合率。结论 HHV8 ORF65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HHV8感染的临床检测,为进一步研制开发新型HHV8诊断试剂提供了有价值的理论和实验依据。
[Abstract]:Objective to construct a prokaryotic expression clone of small capsid protein (ORF65) of human herpesvirus type 8 (HHV8) and to prepare a genetically engineered antigen for serological diagnosis of HHV8 infection. Methods BCBL-1 cells (HHV8 positive but EBV negative) were used to prepare the crude extracellular antigen, and the HHV8 cell engineering antigen was obtained by Sephadex G-75 chromatographic column. The coding region of HHV8 ORF65 was amplified by PCR, and the PCR product was digested with the prokaryotic expression vector pThioHisA, respectively. The expression of fusion protein was induced by transforming the host strain E.coli BL21,IPTG. The genetic engineering antigen (, Western blot) was purified by affinity chromatography with six histidine labels to detect its antigenicity and specificity. The optimal antigen concentration was coated with 96 well ELISA reaction plate to detect the patients concerned and compared with the HHV8 IgM and HHV8 IgG antibody IFA test kit produced by Biotrin and Qiagen ELISA test kit. Results HHV8 ORF65 capsid protein was highly expressed in E.coli BL21. The results showed that the fusion protein could react with 4 (80%) of 5 HHV8 specific IgM positive sera and 10 (100%) HHV8 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 detection kit of Biotrin and Qiagen. Conclusion the recombinant HHV8 ORF65 protein has good antigenicity and specificity and can be used in the clinical detection of HHV8 infection. It provides a valuable theoretical and experimental basis for the further development of a new HHV8 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】：R392

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