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

Inhibitory effect on hepatitis B virus of HBsAg ScFv

  • 摘要: 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HBsAg ScFv基因的重组逆转录病毒载体pLXSN-HBsAg ScFv,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HBV DNA。结果:成功筛选出HBsAg ScFv,PCR扩增出750bp的全基因,构建HBsAg ScFv基因的逆转录病毒载体,转染PA317细胞,在上清中检测出含HBsAg ScFv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:HBsAg ScFv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。

     

    Abstract: 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 sem-i 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 2.2.15cells 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 3,5,7,14days,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 2.2.15cells can inhibit the expression of HBV significantly and it may be a potential candidate for gene therapy against HBV infection.

     

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