人干扰素-α基因转移、表达及其抗乙型肝炎病毒的实验研究被引量：11

Transfer and expression of human interferon -α gene and its effect on HBV inhibition

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摘要目的　研究人干扰素 α(hIFN α)基因的转移、表达及其抗乙型肝炎病毒的作用。方法　利用聚合酶链反应 (PCR)技术和基因重组技术 ,体外扩增hIFN α基因并构建重组表达hIFN α基因的逆转录病毒表达载体 (pDOR IFN α) ,用NIH 3T3细胞扩增法测定其假病毒颗粒滴度 ,用染料吸收法检测hIFN α活性 ;将假病毒颗粒感染 2 .2 .15细胞 ,用固相放免法检测其上清HBsAg和HBeAg ,用斑点杂交法结合计算机扫描检测其细胞HBVDNA密度。结果　PCR克隆出 6 48bphIFN α全基因 ;假病毒颗粒滴度为 1× 10 6CFU/ml;hIFN α生物活性为 2 39IU/ml/1× 10 6/4 8h ;对HBsAg的抑制率随培养时间 (2、4、6和 8d)的延长而逐渐增高 ,抑制率分别为 33%、5 5 %、5 5 %和6 2 % ,而对HBeAg的抑制则在转染后的第 2天抑制率最高 ,以后随培养时间延长而逐渐下降 ,其抑制率分别为 6 7.7%、40 %、19.5 %和 2 5 .3%。而对照组对HBsAg第 2、4、6、8天的抑制率分别为 0 %、10 .8%、0 %和 13% ,对HBeAg的抑制率分别为 0 %、0 %、0 %和 1.2 %。而且对HBVDNA亦有明显的抑制作用 ,抑制率达 6 6 % ;而对照组 (空白载体 )抑制作用不明显。结论　hIFN α基因可以成功地转移和表达。 Objective Interferon α(IFN α) is a confirmed drug against hepatitis viruses. However, its anti HBV effect in patients is not so good as expected. In order to study the full play of IFN α, human IFN α(hIFN α) gene was transferred and expressed in 2.2.15 cells. Meanwhile, the inhibition of hepatitis B virus(HBV) replication and expression was investigated. Methods hIFN α gene was amplified from PBMC DNA of healthy donor. The pDOR hIFN α vector expressing hIFN α was constructed with retroviral vector pDOR. Both pDOR hIFN α and pDOR vector were transfected into PA317 cells respectively and infected NIH 3T3 and 2.2.15 cells using their supernatant containing the pseudovirus. HBsAg and HBeAg were detected by RIA and the density of HBV DNA was tested by dot blot hybridization in 2.2.15 cell line.Results The 648 bp of hIFN α gene were cloned from peripheral blood mononuclear cells(PBMC) DNA by polymerase chain reaction(PCR). Pseudovirion titer was 1×10 6 CFU/ml. The hIFN α activity in NIH 3T3 cells was 239 IU/ml/l×10 6/48 hs. When pDOR hIFN α and pDOR vector(control) were transfected into 2.2.15 cells respectively and incubated for 2,4,6 and 8 days, the inhibition rate of HBsAg were 33%, 55%, 55% and 62% respectively, compared to 0%,10.8%,0% and 13% in control. Moreover, the inhibition rate of HBeAg in pDOR hIFN α transferred cell lines were 67.7%, 40%, 19.5% and 25.3% respectively, and 0%, 0%,0% and 1.2% respectively in control cells. The HBV DNA decreased 50% more over than that of control(pDOR). Conclusion The results indicated that hIFN α gene expressing in 2.2.15 cells can inhibit the replication and expression of HBV significantly and it may be a potential candidate for gene therapy against HBV infection.