戊型肝炎病毒ORF2 C端抗原片段的表达及其免疫学特性研究

The expression and characterization of the C terminal fragment of HEV ORF2

目的 表达戊型肝炎病毒 (hepatitisEvirus ,HEV)ORF2C端 12 8个氨基酸残基的抗原片段ORF2 3 ,并进行其免疫学特性的研究。方法 用pBV2 2 0载体进行抗原的非融合表达 ,包涵体经变性凝胶过滤层析及离子交换层析纯化后透析复性 ,以Westernblot、ELISA、动物免疫与抗原捕获RT nPCR等方法研究其免疫学特性。结果 获得了ORF2 3的高效表达 ,表达量占菌体总蛋白 5 0 %左右 ,纯化后纯度达 98%以上 ,Westernblot表明表达抗原可与戊肝患者阳性血清特异性结合 ;在HEV感染恒河猴实验中用于IgG抗体的检测 ,具有较好灵敏度与特异性 ;用其免疫豚鼠 ,抗体经ELISA法测定效价为 1∶80 0 0 ;用制备的豚鼠抗血清捕获戊型肝炎病毒颗粒 ,经RT nPCR扩增出特异性片段。结论 ORF2 3抗原片段具有HEV抗体识别的抗原表位 ,能够刺激机体产生抗体 。

Objective To express and characterize the C terminal fragment of HEV ORF2. Methods The gene encoding C terminal 128 amino acid fragment of HEV ORF2 was inserted into nonfusion expression vector pBV220. The resultant pBVORF2.3 was transformed into E.coli strain DH5α. Results High level expression was achieved 4 hours after induction at 42℃. The recombinant protein, with molecular weight about 14?000, was up to 50 percent of total protein and presented in inclusion bodies. The recombinant protein ORF2.3 was purified to near purity after denatured size exclusion chromatography and ionic exchange chromatography. It was recognized by serum from HEV positive patients and Macaque in Western blot assay. Furthermore, if induced antibodies in Guinea pig specific for HEV particles. Conclusion The fragment ORF2.3 can be recognized by anti HEV antibody, it can induce the production of antibody capable of recognizing HEV particles.