人巨细胞病毒pp65蛋白的原核表达及免疫学特性分析

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

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。

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. [