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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 ,抗血清具有一定的识别与结合戊型肝炎病毒颗粒的能力
Objective To express the ORF2 3 antigen fragment of 12 8 amino acids at the ORF2C end of hepatitis E virus (HEV) and study its immunological properties. Methods The non-fusion antigen was expressed in pBV220 vector. The inclusion bodies were purified by denaturing gel filtration chromatography and ion exchange chromatography. The immunoprecipitation was studied by Western blot, ELISA, animal immunization and RTnPCR Learn characteristics. Results High expression of ORF2 3 was obtained. The expression level of ORF2 3 was about 50% of the total bacterial proteins, and the purity of purified ORF2 was over 98%. Western blot showed that the expressed antigens could specifically bind to the positive serum of hepatitis E patients. Experiment used for IgG antibody detection, with good sensitivity and specificity; with its guinea pigs immunized, the antibody titer determined by ELISA 1:80 0; captured guinea pig antisera seized hepatitis E virus particles, the RT nPCR amplified specific fragments. Conclusion The ORF2 3 antigen fragment has the antigenic epitope recognized by HEV antibody and can stimulate the body to produce antibodies. The antiserum has certain ability of recognizing and binding to hepatitis E virus particles