论文部分内容阅读
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 the antigenic determinant gene of human cytomegalovirus (HCMV), prepare and purify the recombinant protein, and evaluate its antigenicity by assembling into a capture ELISA kit. Methods The DNA of human cytomegalovirus (AD169) was extracted and the pp150 (UL32), gp52 (UL44) and pp65 (UL83) genes of HCMV were amplified by PCR. The three fragments were cloned into the prokaryotic expression vector pGEX-5x-3 The fusion protein was cloned and expressed in E.coli BL21 (DE3). The fusion protein was identified by SDS-PAGE electrophoresis and Western blot, and then assembled into a capture ELISA kit for detection of clinical samples. The kit The performance indicators. Results The sequence of each gene fragment was sequenced successfully. The recombinant vector gp52-pp65-pp150 with high expression of HCMV was successfully constructed. The fusion protein expressed by HCMV was proved to be 50 ku by Western blot. The fusion protein was of good antigenicity. The protein was assembled into a capture ELISA kit, the detection of 232 serum samples, compared with the imported kit, the sensitivity of the kit was 93.91%; the specificity was 97.43%; the crude consistency was 96.1% The index was 0.901. The intra-assay coefficient of variation was 12.4%. The stability was good. The control reagent was stored at 37 ℃ and stored at 4 ℃. The coefficient of variation was 13%. Conclusion In this experiment, the HCMV antigen (gp52-pp65-pp150 recombinant protein) with high purity and specificity was effectively obtained by the genetic engineering method. The capture ELISA kit constructed by the fusion protein was compared with the imported kit, High sensitivity and specificity, preliminary clinical application, with further development and application of value, for different stages of clinical infection HCMV detection provides the technical basis.