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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。
Objective To investigate the role of rhesus monkey-derived PDL1Ig in mixed lymphocyte reaction in vitro. Methods The gene sequences of PDL1 and Ig G1Fc of rhesus macaques were retrieved from Gen Bank. The target gene was amplified by overlap extension PCR and transformed into pCMD-T competent E.coli TOP10. The pL1Ig gene was identified by p After shuttle-CMV was linearized, the target gene was transformed into competent E.coli TOP10 and cloned p Shuttle-CMV / PDL1Ig recombinant shuttle plasmid. The pAd Easy-1 / PDL1Ig scaffold was constructed by co-electroporation of pAd Easy-1 and linear p Shuttle-CMV / PDL1Ig into homologous recombination in BJ5183 cells, pAd Easy-1 / PDL1Ig was transfected into 293 cells and packaged into an active recombinant adenovirus. Infection of rhesus monkey hepatocytes, the expression of the target gene was detected by RT-PCR and Western blotting. Mixed lymphocyte reaction (MLR) was used to determine its biological activity. Results PDL1Ig recombinant adenovirus vector was successfully constructed and infected with rhesus monkey hepatocytes. RT-PCR and Western blotting were used to detect the expression of target genes. T lymphocyte proliferation was inhibited in mixed lymphocyte reaction. Conclusion The pAd Easy-1 / PDL1Ig recombinant adenovirus vector was successfully constructed and was highly expressed in rhesus monkey hepatocytes. The recombinant protein could inhibit the proliferation of T lymphocytes through the PD-1 / PDL1 pathway in vitro.