白细胞介素-18逆转录病毒载体的构建及抗乙型肝炎病毒的研究

INHIBITORY EFFECT OF IL-18 ON HEPATITIS B VIRUS IN VITRO

研究鼠白细胞介素 18(IL 18)基因的转移、表达及其抗乙型肝炎病毒 (HBV)的作用。以逆转录聚合酶链反应 (RT PCR)技术从受植物凝血素(PHA)和脂多糖 (LPS)刺激BALB/c小鼠脾脏细胞中扩增出IL 18的cDNA ,并构建表达IL 18基因的重组逆转录病毒载体pLXSN IL 18,转染PA317细胞 ,以逆转录巢式RT PCR方法 ,验证细胞培养液上清分泌的假病毒颗粒中IL 18的表达 ,将假病毒颗粒感染 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。成功克隆出 5 80bp的小鼠IL 18全基因并构建逆转录病毒载体 ,在转染PA317细胞的上清中检测出含IL 18假病毒颗粒 ,上清感染 2 .2 .15细胞后第 3、5、7、14天 ,HBsAg、HBeAg逐渐下降 ,到14天时HBsAg已变为阴性 ,对HBVDNA有明显抑制作用。IL 18能成功地在逆转录病毒载体中表达 ,并有抑制HBsAg。

Interleukin 18 (IL 18) is emerging as a powerful, pleiotropic cytokine involved in determining the polarization of T cell responses. To identify the effect of IL 18 on hepatitis B virus, mouse IL 18 gene was transferred and expressed in 2.2.15 cells. Meanwhile, the inhibition of HBsAg, HBeAg and HBV DNA was investigated. Reverse transcription polymerase chain reaction (RT PCR) was used to amplify the mouse IL 18 from spleen cell of Balb/c mouse challenged with lipopolysaccharide (LPS) and phytahematoagglutinin (PHA), and reconstruct plasmid pLXSN IL18 with the retroviral vector pLXSN. Both pLXSN IL18 and pLXSN were transfected into PA317 cells and then 2.2.15 cells were infected by using the supernatant containing pseudovirus released by PA317. HBsAg and HBeAg were detected by ELISA and the HBV DNA was determined by quantitative PCR. The 580bp of mIL 18 was cloned into retroviral vector pLXSN. The pseudovirus contained in the supernatant of transfected PA317 cells was identified by RT PCR. When the pseudovirus was infected into 2.2.15 cells and incubated for 3, 5, 7, 14 days, the P/N value of HBsAg and HBeAg decreased gradually and became negative on day 14. The copies of HBV DNA reduced markedly. The results indicate that mIL 18 gene expressed in 2.2.15 cells can significantly inhibit the replication expression of HBV and may be used as a potential agent for gene therapy against HBV infection.