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目的 :克隆表达人巨细胞病毒 (humancytomegalovirus,HCMV)gp5 2基因 ,制备特异性抗原 ,用于HCMV早期诊断。方法 :从感染的HCMV细胞上清液中提取病毒的DNA ,用PCR扩增gp5 2蛋白IgM抗原决定簇编码区DNA片段 ,并将其克隆到pGEM_Teasy载体测序 ,然后将其克隆到含有谷胱甘肽转移酶 (GST)融合蛋白表达载体pGEX_5X_3中表达 ,表达产物经GST亲和层析柱纯化后 ,采用ELISA和免疫印迹 (Westernblotting)检测重组蛋白的抗原性。结果 :经序列测定gp5 2基因片段的序列正确 ,并在pGEX_5X_3表达载体中得到了高效表达。表达的融合蛋白用亲和层析柱纯化后经免疫印迹和ELISA分析具有良好的抗原性 ,能够与HCMVIgM阳性血清呈特异性反应。结论 :通过基因工程制备的重组gp5 2蛋白可作为抗原用于HCMV感染者的早期诊断。
OBJECTIVE: To clone and express gp5 2 gene of human cytomegalovirus (HCMV) for the preparation of specific antigen for the early diagnosis of HCMV. Methods: The DNA of virus was extracted from supernatant of infected HCMV cells. The DNA fragment encoding gp5 2 protein IgM epitope was amplified by PCR and cloned into pGEM_Teasy vector and then cloned into pGEM_Teasy vector containing glutathione (GST) fusion protein expression vector pGEX_5X_3. The expressed product was purified by GST affinity chromatography and the antigenicity of the recombinant protein was detected by ELISA and Western blotting. Results: The sequence of gp5 2 gene was sequenced correctly and was highly expressed in pGEX_5X_3 expression vector. The expressed fusion protein was purified by affinity chromatography, and then showed good antigenicity by Western blotting and ELISA. It could specifically react with HCMVIgM positive sera. Conclusion: Recombinant gp5 2 protein prepared by genetic engineering can be used as antigen for the early diagnosis of HCMV infection.