潜伏相关抗原UL138蛋白的制备及其用于人巨细胞病毒感染血清学检测的评价  

Preparation of human cytomegalovirus latency-associated determinant pUL138 and evaluation of its preliminary use in the serodiagnosis of HCMV infection

作　　者：陈文静[1] 黄崇安[2] 陈静[3] 郭刚强 谢旺凯 林刻智[5] 沈贤[1] 薛向阳[4] 

机构地区：[1]温州医科大学附属第一医院胃肠外科,浙江温州325015 [2]温州医科大学第一临床医学院,浙江温州325035 [3]温州医科大学附属第一医院风湿免疫科,浙江温州325015 [4]温州医科大学分子病毒与免疫研究所,浙江温州325035 [5]温州医科大学基础医学实验中心,浙江温州325035

出　　处：《温州医科大学学报》2015年第10期703-708,共6页Journal of Wenzhou Medical University

基　　金：国家自然科学基金资助项目(81472308;81001343;31470891);浙江省自然科学基金资助项目(Y2100909;Y2012314);浙江省科技厅科研基金资助项目(2012C33126)

摘　　要：目的:探讨基于人巨细胞病毒(HCMV)潜伏相关抗原UL138蛋白的ELISA方法用于HCMV感染血清学检测的意义。方法:生物信息学分析HCMV UL138蛋白的跨膜区结构,选择胞内区序列,经原核密码子优化后全基因合成,克隆至p ET21a(+)原核载体,将成功构建的重组质粒转化至大肠杆菌BL21(DE3),诱导蛋白表达后,SDS-PAGE及Western blot法鉴定目的蛋白表达,采用镍柱亲和层析法纯化目的蛋白。将纯化的UL138蛋白包被ELISA板,检测173例人血清UL138特异性抗体,并与临床上使用的罗氏公司cobas 8000分析系统及配套试剂盒进行血清HCMV Ig G抗体检测比较,评价UL138蛋白作为HCMV感染血清学检测工具的效能。结果:利用原核表达系统成功表达了UL138蛋白,经镍柱亲和纯化后获得高纯度的目的蛋白;健康成人血清UL138特异性抗体的ELISA法检测结果显示,几乎所有的健康成人都存在UL138特异性抗体,与罗氏公司的化学发光(CLIA)法Ig G检测结果比较,灵敏性为100.0%,符合率为98.8%,差异无统计学意义(x2=0.5,P>0.05),而且具有较好的一致性(Kappa=0.496,P<0.001)。结论:潜伏相关抗原UL138是HCMV感染血清学检测重要候选抗原,可望为基于此蛋白的HCMV血清学检测试剂的研制提供依据。Objective： To prepare latency-associated protein UL138 of human cytomegalovirus, determine its antigenicity and evaluate its preliminary use as an antigen in the serodiagnosis of HCMV infection. Methods： Bioinformatics methods were used to analyze the transmembrane domain of UL138 protein, and the complete coding region of p UL138＇s cytoplasmic domain was optimized by the usage of the favorite codons in E.coli, amplified by PCR and cloned into the expression vector p ET21a（＋）. The constructed p ET21a（＋）/UL138 plasmid was transformed into E.coli BL21 cells and induced by IPTG. The recombinant protein p UL138 was purified by NiNTA affinity chromatography and confirmed by SDS-PAGE and Western blot analysis. Then the purified UL138 protein was coated on plates as a diagnosis antigen and an indirect ELISA method was established and optimized to investigate the HCMV-specific Ig G. Finally serum samples from 173 healthy individuals were detected by the established method, and Roche＇s electrochemiluminescence detection kit was considered as the golden standard to evaluate its significance in the serodiagnosis of HCMV infection. Results： HCMV UL138 protein was successfully expressed in prokaryotic system and highly purified by affinity purification. The positive rate of specific Ig G against HCMV was 99.4% by the established ELISA, with no statistically significant difference between the result of p UL138-based ELISA and that of Roche＇s detection kit（x2=0.5, P〉0.05）, and the sensitivity and coincidence rate of the ELISA method were 100.0% and 98.8%. These two results had good coherence（Kappa=0.496, P〈0.001）. Conclusion： Latency-associated protein UL138 is a potential candidate antigen for HCMV detection and can be useful for development of new techniques for the serodiagnosis of HCMV infection.

关 键 词：人巨细胞病毒 UL138蛋白 血清学检测 酶联免疫吸附试验 

分 类 号：R373.9[医药卫生—病原生物学]