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①目的 构建人巨细胞病毒 (HCMV) pp15 0的原核高效表达系统 ,制备用于急性、活动性HCMV感染血清学诊断的基因工程抗原。②方法 PCR扩增HCMVpp15 0抗原决定簇 (84 0～ 10 4 8aa)编码基因片段 ,分别插入原核表达载体 pMAL p2、pET 2 8a ,转化宿主菌E .coli.诱导表达 ,经麦芽糖亲和层析树脂纯化 ,以Western Blot及间接ELISA法鉴定其抗原性。③结果 重组表达质粒 pMAL p2 pp15 0可在E .coli.DH5α中有效表达 ,表达产物经SDS PAGE电泳后 ,凝胶成像系统分析显示相对分子质量约为 6 .4万 ,约占菌体蛋白的 10 %。而重组质粒pET 2 8a pp15 0未见明显表达带。Western Blot检测 ,阳性识别率为 80 % (12 / 15 )。用此蛋白包被ELISA板 ,检测正常孕妇血清 ,阳性率为 6 .5 % (17/ 2 6 3)。与本室制备的全病毒抗原ELISA的诊断符合率为 96 %。④结论此重组蛋白具有良好的抗原性 ,进一步完善后可用于HCMV急性和活动性感染的诊断。
Objective To construct a prokaryotic expression system of human cytomegalovirus (HCMV) pp15 0 and to prepare a genetically engineered antigen for serological diagnosis of acute and active HCMV infection. Methods The gene fragment of HCMVpp15 0 (84 0-10 4 8 aa) was amplified by PCR and inserted into the prokaryotic expression vector pMAL p2 and pET 2 8a respectively. The recombinant plasmid was transformed into E.coli and induced by maltose affinity chromatography The resin was purified and its antigenicity was identified by Western Blot and indirect ELISA. Results The recombinant plasmid pMAL p2 pp15 0 was expressed efficiently in E.coli DH5α, and SDS PAGE electrophoresis showed that the molecular weight of the recombinant protein was about 64,000, 10%. The recombinant plasmid pET 2 8a pp15 0 no significant expression. Western Blot test, the positive recognition rate was 80% (12/15). The protein was used to coat the ELISA plate to detect the serum of normal pregnant women. The positive rate was 6.5% (17/263). The diagnostic accuracy of the whole virus antigen ELISA prepared in our laboratory was 96%. ④ Conclusion This recombinant protein has good antigenicity and can be further used for the diagnosis of acute and active HCMV infection.