CAJ | 학술논문

目的表达2型单纯疱疹病毒(HSV-2)糖蛋白G免疫优势片段gG321-580,探讨重组gG321-580作为检测HSV-2特异性IgG诊断抗原的可行性,为进一步开发诊断试剂盒奠定基础.方法整合有gG321-580基因的重组杆状病毒接种Sf9昆虫细胞,NTA-Ni2+纯化表达His-gG321-580融合蛋白.应用HSV-2感染Vero细胞,制备HSV-2病毒抗原.将gG321-580蛋白和HSV-2病毒抗原分别作为包被抗原,建立间接ELISA法(gG321-580-ELISA和病毒抗原-ELJSA).分别用gG321-580-ELISA和病毒抗原-ELISA检测105份临床样本,两者的检测结果以HerpeSelect 2 ELISA IgG试剂盒作为金标准,在特异性、灵敏性和符合率等方面进行比较.结果 SDS-PAGE和Western印迹结果显示,表达的gG321-580蛋白相对分子质量约为60000,具有良好的免疫学活性.与HerpeSelect 2 ELISA IgG试剂盒相比,gC321-580-ELISA的灵敏度、特异度、符合率分别为83.3％、81.8％、82.9％,病毒抗原-ELISA的灵敏度、特异性和符合率分别为20.8％、93.9％、43.8％.gG321-580-ELISA敏感度和符合率高于病毒抗原-ELISA.结论表达的HSV-2重组gG321-580蛋白作为包被抗原的ELISA法是一种敏感、特异的方法,具有较大的应用价值.
목적 표체2형단순포진병독(HSV-2)당단백G면역우세편단gG321-580,탐토중조gG321-580작위검측HSV-2특이성IgG진단항원적가행성,위진일보개발진단시제합전정기출.방법 정합유gG321-580기인적중조간상병독접충Sf9곤충세포,NTA-Ni2+순화표체His-gG321-580융합단백.응용HSV-2감염Vero세포,제비HSV-2병독항원.장gG321-580단백화HSV-2병독항원분별작위포피항원,건립간접ELISA법(gG321-580-ELISA화병독항원-ELJSA).분별용gG321-580-ELISA화병독항원-ELISA검측105빈림상양본,량자적검측결과이HerpeSelect 2 ELISA IgG시제합작위금표준,재특이성、령민성화부합솔등방면진행비교.결과 SDS-PAGE화Western인적결과현시,표체적gG321-580단백상대분자질량약위60000,구유량호적면역학활성.여HerpeSelect 2 ELISA IgG시제합상비,gC321-580-ELISA적령민도、특이도、부합솔분별위83.3％、81.8％、82.9％,병독항원-ELISA적령민도、특이성화부합솔분별위20.8％、93.9％、43.8％.gG321-580-ELISA민감도화부합솔고우병독항원-ELISA.결론 표체적HSV-2중조gG321-580단백작위포피항원적ELISA법시일충민감、특이적방법,구유교대적응용개치.
Objective To express immunodominant fragment gG321-580 of herpes simplex virus 2 (HSV-2) Glycoprotein G,and explore the feasibility of the gG321-580 as the diagnosis antigen to detect HSV-2 type-specific IgG antibodies,and provide a technological basis for the development of diagnosis kit.Methods Sf9 cells were inoculated with recombinant baculovirus containing gG321.580 gene and the recombinant gG321-580 protein were purified by NTA-Ni2 + columns from the cell lysate.Vero cells were infected with HSV-2 to produce virus antigen.Two indirect ELISA methods (gG321-580-ELISA,HSV-2-ELISA) were established by recombinant gG321-580 and HSV-2 antigen as coating antigens.One hundred and five clinical serum samples were assayed by gG321-580-ELISA,HSV-2-ELISA and HerpeSelect 2 ELISA IgG kit respectively.The results of HerpeSelect 2 ELISA IgG kit as a golden standard were compared with those of the other two methods in sensitivity,specificity and accordance rate.Results The results of SDS-PAGE and Western blotting analysis showed the molecular weight of gG321-580 protein was about 60 000 and had good immunoactivity.Compared to HerpeSelect 2 ELISA IgG kit,the sensitivity,specificity and accondance rate of gG321-580-ELISA and HSV-2-antigen ELISA were 83.3％,81.8％,82.9％ and 20.8％,93.9％,43.8％,respectively.The sensitivity and accordance rate of gG321-580-ELISA were higher than HSV-2-ELISA.Conclusions gG321-580-ELISA is a specific,sensitive,quick and convenient method for diagnosis of HSV-2 infection,which can provide much information for a diagnosis kit.