人巨细胞病毒pp65蛋白的原核表达及免疫学特性分析

目的应用生物工程方法研制人巨细胞病毒包膜蛋白pp65,并行相应抗体制备的探讨性研究。方法利用DNA重组技术,以HCMVAD169病毒株基因组为模板,通过PCR克隆HCMVpp65基因。以pET32a( )为表达载体,构建重组质粒,并在E.Coli(DE3plys)中诱导表达。然后经柱层析获得纯化蛋白,由蛋白质印迹法验证蛋白的特异性。最后,免疫小鼠获取抗血清,检测抗体产生的效价。结果重组质粒经测序与Genebank序列一致,转染的细菌能高效表达目的蛋白,纯化后的蛋白能与已知单克隆抗体结合,免疫后的小鼠抗血清能同时识别重组蛋白和病毒抗原。结论重组表达的HCMVpp65全蛋白经纯化获得较高纯度,且保留良好的免疫原特性。为HCMV感染诊断用单克隆抗体的研制奠定了基础,并为HCMV感染的免疫诊断和治疗提供了新的研究方向。

Expression and Purification of Human Cytomegalovirus Tegument Protein pp65 and Analysis of its Immunologic Characteristics

Objective: To express and purify Human Cytomegalovirus tegument phosphoprotein pp65 in prokaryotic expression system. Methods: Primers designed based on the gene sequence and the gene of pp65 amplified by PCR was cloned into the vector pet32a( ), the expressed protein was purified by two steps of Ni~2 chelate affinity chromatography and anion exchange chromatography respectively. Use Western blot to identify the purified protein and vaccinated mice to test the antibody titer by indirect ELISA. ~Results : Recombinant expression plasmid pp65-pet32a was constructed successfully. The expressed protein existed in the form of inclusion body. Western blot analysis indicated protein had certain antigenicity. The mice can produce high titer antibody. Conclusion: High-level expression of pp65 was achieved in E.coli, the purified protein had strong immunoresponse. It indicated that expressed pp65 had a potential value for immunodiagnosis and immunotherapy.