乙型肝炎病毒表面抗原单链抗体细胞内免疫抗乙型肝炎病毒基因治疗研究

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

Inhibitory effect on hepatitis B virus of HBsAg ScFv

Objective:To study the inhibitory effect on hepatitis B virus of HBsAg ScFv, HBsAg ScFv gene Methods: HBsAg ScFv gene was screened and identified from a semi synthetic phage library by phage display technique and amplified and cloned into the retroviral vector pLXSN The reconstructed plasmid pLXSN HBsAg ScFv was transfected into PA317 cells and infected 2 2 15 cells by using the supernatant containing the pseudovirus Then, HBsAg and HBeAg were detected by ELISA and the density of HBV DNA was tested by quantitative determination Results: The specific HBsAg ScFv was screened and cloned into retroviral vector pLXSN HBsAg ScFv pseudovirus contained in the supernatant of transfected PA317 cells were identified by RT PCR When the HBsAg ScFv pseudovirus were infected into 2 2 15 cells and incubated for 3, 5, 7, 14 days, the P/N value of HBsAg and HBeAg were decreased gradually and turn to negative at day 14 The density of HBV DNA had not changed apparently Conclusion: The results indicated that HBsAg ScFv gene expressing in 2 2 15 cells can inhibit the expression of HBV significantly and it may be a potential candidate for gene therapy against HBV infection