人疱疹病毒7型被膜蛋白pp85的原核表达及应用

目的构建人疱疹病毒7型(HHV7)被膜蛋白pp85编码基因(ORF U14)的原核表达载体,并进行原核表达。方法应用HHV7标准株Glasgow感染SupT1细胞,PCR技术扩增HHV7ORFU14基因的主要抗原决定簇编码区(328~533AA),目的基因与原核表达载体pThioHis A连接后,转化宿主菌E.coliBL21,IPTG诱导融合蛋白表达,经镍-螯合物琼脂糖树脂柱亲和层析纯化获得重组抗原。重组蛋白电泳后转至PVDF膜,与HHV7阳性血清进行免疫印迹反应。结果DNA序列分析表明,目的基因序列与HHV7标准毒株Glasgow相应序列完全一致,SDS-PAGE可观察到相对分子质量(Mr)为35.7×103融合蛋白的表达,免疫印迹反应显示重组抗原具有较高的特异性。结论HHV7重组抗原具有较好的抗原性,进一步完善后可用于HHV7抗体的检测。

Construction and application of the expressive clone of human herpes virus 7 tegument protein pp85

Objective To construct prokaryotic expression clone of human herpes virus 7 (HHV7) tegument protein pp85 as an efficient recombinant antigen for serological diagnosis of HHV7 active infection. Methods SupT1 cells were infected with HHV7(Glasgow)and harvested. The major antigen epitopes coding sequence(328-533 AA) of HHV7 ORF U14 gene was amplified by PCR and then inserted into prokaryotic expression vector pThioHis A, the recombinant plasmid was transformed into E.coli BL21 for expression. The products were purified by nickel-chelating Sepharose resin affinity chromatography. The antigenicity and the specificity of the recombinant proteins were identified by Western blot with HHV7-positive serum samples. Results Gene sequencing suggested that the target gene was identical with that of HHV7 standard species(Glasgow), the 35.7 kD fusion protein was found in SDS-PAGE. Conclusion The recombinant proteins showed a high specificity and sensitivity,it is valuable for advanced study on HHV7 specific antibody detection.