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目的 :研究乙型肝炎病毒表面抗原 (HBsAg)人源单链可变区抗体 (ScFv)细胞内免疫抗乙型肝炎病毒(HBV)基因治疗的作用。方法 :用噬菌体表面展示技术筛选特异性的HBsAg人源单链可变区抗体 ,聚合酶链反应(PCR)法扩增HBsAg单链抗体基因 ,并构建表达HBsAgScFv基因的重组逆转录病毒载体pLXSN HBsAgScFv,转染PA317细胞 ,将转染细胞分泌的假病毒颗粒感染 2 2 15细胞 ,酶联免疫吸附法 (ELISA)检测其上清HBsAg和HBeAg ,定量检测HBVDNA。结果 :成功筛选出HBsAgScFv ,PCR扩增出 75 0bp的全基因 ,构建HBsAgScFv基因的逆转录病毒载体 ,转染PA317细胞 ,在上清中检测出含HBsAgScFv假病毒颗粒的存在 ,上清感染 2 2 15细胞后第 3、5、7、14天 ,HBsAg、HBeAg逐渐下降 ,到第 14天时HBsAg已变为阴性 ,DNA定量检测无明显变化。结论 :HBsAgScFv能成功地在逆转录病毒载体中表达 ,并有抑制HBsAg、HBeAg表达的作用
Objective: To study the effect of anti-hepatitis B virus (HBV) gene therapy in Hepatitis B virus surface antigen (HBsAg) human single chain variable region antibody (ScFv) cells. Methods: Specific HBsAg human single-chain variable region antibody was screened by phage display technique. HBsAg single-chain antibody gene was amplified by polymerase chain reaction (PCR) and recombinant retroviral vector pLXSN HBsAgScFv , And transfected into PA317 cells. The virus particles secreted by the transfected cells were infected with 2 2 15 cells. HBsAg and HBeAg were detected by enzyme-linked immunosorbent assay (ELISA) and HBVDNA was detected quantitatively. Results: The HBsAgScFv was successfully screened. The 75 0bp gene was amplified by PCR. The retroviral vector containing HBsAgScFv gene was constructed and transfected into PA317 cells. The presence of HBsAgScFv pseudovirions was detected in the supernatant. The supernatant was infected 2 2 15th, 5th, 5th, 14th and 14th days, HBsAg and HBeAg gradually decreased. On the 14th day, the HBsAg had become negative, and there was no significant change in DNA quantitative detection. Conclusion: HBsAgScFv can be successfully expressed in retroviral vector and inhibit the expression of HBsAg and HBeAg