PDL_1Ig基因修饰的恒河猴肝细胞在混合淋巴细胞反应中的效应研究

Effects of PDL_1Ig gene-modified Rhesus' hepatocyte on the mixed lymphocyte reaction

目的探讨恒河猴来源的PDL1Ig在体外混合淋巴细胞反应中的作用。方法在Gen Bank中检索到恒河猴目的基因PDL1和Ig G1Fc的序列,采用重叠延伸PCR法合成该目的基因,与p GEM-T连接转化感受态E.coli TOP10,经鉴定得到PDL1Ig基因,p Shuttle-CMV线性化后连接目的基因转化感受态E.coli TOP10,克隆p Shuttle-CMV/PDL1Ig重组穿梭质粒。将p Ad Easy-1和线性p Shuttle-CMV/PDL1Ig共电转至BJ5183细胞中同源重组,构建重组腺病毒载体p Ad Easy-1/PDL1Ig骨架,以脂质体2000将p Ad Easy-1/PDL1Ig转染至293细胞中包装成有活性的重组腺病毒。感染恒河猴肝细胞,RT-PCR和Western blotting检测目的基因的表达。混合淋巴细胞反应(MLR)测定其生物学活性。结果成功构建了PDL1Ig重组腺病毒载体,能感染恒河猴肝细胞,RT-PCR和Western blotting检测有目的基因表达,在混合淋巴细胞反应中其能抑制T细胞增殖。结论成功构建了p Ad Easy-1/PDL1Ig重组腺病毒载体,其可在恒河猴肝细胞中高效表达,该重组蛋白可在体外通过PD-1/PDL1通路抑制T淋巴细胞增殖。

The study designed to explore the roles of rhesus＇ PDL1 Ig in mixed lymphocyte reaction. The sequence of PDL1 and Ig G1 Fc of rhesus gene were obtained from Genebank and PDL1 Ig was synthetized by overlap extension PCR. Then PDL1 Ig gene was connected with the p GEM-T and transformed into E. coli TOP10 to identify the gene by restricted digestion and sequencing. The target gene was connected to linearized p Shuttle-CMV and transformed into E. coli TOP10 to clone p Shuttle-CMV/PDL1 Ig recombinant shuttle plasmid. The p Ad Easy-1 and linearized p Shuttle-CMV/PDL1 Ig were electric-cotransformed to BJ5183 cells to obtain recombinant adenovirus vector p Ad Easy-1/PDL1 Ig skeleton by homologous recombination. Then p Ad Easy-1/PDL1 Ig was transfected to 293 cells for package of the active recombinant adenovirus, which was then used to infect rhesus＇ hepatocytes. RT-PCR and Western bloting were applied to identify the gene expression, while mixed lymphocyte reaction（MLR） was used to mesure the biological activity. We observed that T cells could not largely proliferate in MLR. In conclusion,p Ad Easy-1/PDL1 Ig could inhibit the proliferation of T lymphocytes through the PD-1/PDL1 pathway.