人巨细胞病毒gp52-pp65-pp150的融合表达及应用  

The expression and use of the human cytomegalovirus gp52-pp65-pp150 fusion gene

作　　者：张乐海[1] 马丽霞[1] 王世富[1] 姜忠强[1] 彭振居[1] 王素兰[1] 

机构地区：[1]山东大学齐鲁儿童医院,山东济南250022

出　　处：《中国病原生物学杂志》2011年第1期12-16,共5页Journal of Pathogen Biology

基　　金：济南市科技局科技计划专项(No.200807035)。

摘　　要：目的克隆表达人巨细胞病毒(human cytomegalovirus,HCMV)特异性强的抗原决定簇基因,制备并纯化重组蛋白,并通过组装成捕获法ELISA试剂盒对其抗原性进行评价。方法提取人巨细胞病毒AD169株的DNA,用PCR扩增HCMV的pp150(UL32)、gp52(UL44)、pp65(UL83)基因片段,将3个基因片段串联克隆至原核表达载体pGEX-5x-3进行融合表达和纯化,采用SDS-PAGE电泳法和免疫印迹法(Western blot)对融合基因的克隆及重组蛋白的表达进行鉴定,并组装成捕获法ELISA试剂盒对临床标本进行检测,评价试剂盒的各项性能指标。结果经序列测定各基因片段序列正确,成功构建了HCMV的高效表达融合基因的重组载体gp52-pp65-pp150,表达的融合蛋白经Western blot分析,分子量在50 ku,具有良好的抗原性。将该蛋白组装成捕获法ELISA试剂盒,经232份血清标本的检测,与进口试剂盒相比,本试剂盒的灵敏度为93.91%;特异度为97.43%;粗一致性为96.1%;约登指数为0.901;批内变异系数为12.4%;稳定性良好,37℃保存试剂与4℃保存试剂进行对照试验,变异系数为13%。结论本实验通过基因工程技术的方法有效地获取纯度较高、特异性强的HCMV抗原(gp52-pp65-pp150重组蛋白),该融合蛋白构建的捕获法ELISA试剂与进口试剂盒比对,具有较高的灵敏度和特异性,经初步临床应用,具有进一步开发应用的价值,为不同临床感染阶段的HCMV的检测提供了技术基础。Objective To clone and express antigen-determining genes from human cytomegalovirus（HCMV） with a high level of specificity,to prepare and purify the recombinant protein,and to then evaluate antigenicity using an ELISA capture kit.Methods DNA was extracted from the HCMV strain AD169,gene fragments of pp150（UL32）,gp52（UL44）,and pp65（UL83） were amplified with PCR,and then these three genetic fragments were connected to the prokaryotic expression vector pGEX-5x-3 in a series for fusion,expression,and purification.SDS-PAGE electrophoresis and Western blotting were used to evaluate clones with fusion genes and expression of recombinant protein.Clinical samples were tested with an ELISA capture kit to evaluate the performance index of the kit.Results Sequences of the genetic fragments all proved to be correct according to sequencing.The gp52-pp65-pp150 recombinant vector was successfully constructed with efficient expression of fusion genes of HCMV.Analysis of this expression by Western blotting indicated that the molecular weight of the fusion protein was 50 ku and that it had good antigenicity.This protein was subjected to assay with an ELISA capture kit.Testing of 232 serum samples indicated that this kit had a sensitivity of 93.91%,specificity of 97.43 %,crude consistency of 96.1%,Youden index of 0.901,and coefficient of variation of 12.4%.The kit had satisfactory stability compared to an imported kit.In a controlled test with the reagent kept at 37℃ and 4℃,the coefficient of variation was 13%.Conclusion In this experiment,bioengineering effectively yielded HCMV antigen with better purity and specificity（gp52-pp65-pp150 recombinant protein）.Compared to imported reagents,the ELISA capture kit consisting of fusion protein in a capture assay had better sensitivity and specificity.Preliminary clinical use warrants further development and use of this kit.Furthermore,it provides a technological basis for detection of HCMV in different stages of clinical infection.

关 键 词：人巨细胞病毒 gp52-pp65-pp150 免疫球蛋白M 融合基因 原核表达 免疫印迹法 

分 类 号：R373.11[医药卫生—病原生物学]