【摘要】 目的 将细胞毒性T淋巴细胞抗原4免疫球蛋白(CTLA-4Ig)cDNA克隆到腺相关病毒载体pSNAV中,获得目的基因在移植肝表达,为器官移植免疫耐受基因治疗奠定基础。 方法 应用基因重组技术和限制性内切酶酶切构建pSNAV-CTLA-4Ig,转化DH-5α,得到抗性克隆,BamH Ⅰ酶切鉴定。用脂质体将psNAV-CTLA-4Ig导入BHK-21中,G418筛选,以HSV1-rc感染此细胞株,产生pSNAV-CTLA-4Ig病毒。斑点杂交法测定病毒滴度, PCR鉴定CTLA-4Ig基因的整合并测序鉴定CTLA-4Ig全长,免疫组织化学方法检测CTLA-4Ig蛋白在大鼠移植肝中的表达。 结果 重组质粒经BamH Ⅰ酶切得到CTLA-4Ig全长cDNA片段,表明CTLA-4Ig成功地插入pSNAV内;病毒滴度测定的病毒颗粒数为8.5×1011／ml;PCR扩增产物为500~600 bp的基因片段,证实有CTLA~4Ig基因的整合;所测得的DNA序列与已知的CTLA-4Ig基因序列完全一致。经pSNAV-CTLA-4Ig病毒灌注的肝脏可见CTLA-4Ig蛋白的表达。 结论将CTLA-4Ig cDNA成功克隆到腺相关病毒载体pSNAV内,证实CTLA~4Ig cDNA正确插入pSNAV多克隆位点内,CTLA-4Ig蛋白可在移植肝中表达。更多还原

【Abstract】 Objective To construct a recombinant adeno-associated virus vector pSNAV expressing CTLA-4Ig and to demonstrate its expression in transplanted liver allografts and to see if a long term inhibitive effect of CTLA-4Ig could be obtained though its use. Methods After AAVCTLA-4Ig and PUC18 were cut with BamHI, CTLA-4Ig cDNA was inserted into the plasmid PUC18 by T4DNA ligase and PUC18-CTLA-4Ig was constructed. The obtained PUC18-CTLA-4Ig and pSNAV cut with Kpn I and EcoR I, CTLA-4Ig cDNA was inserted into plasmid pSNAV to construct the recombinant vector pSNAV-CTLA-4Ig, which was transfected into BHK-21 packaging cells by lipofectine-mediated transfection. Then the BHK-21 cell line was infected with HSVl-rc to produce a large amount of pSNAV- CTLA-4Ig. The specificity of the expressed product was identified by digestion with BamHI, PCR and sequence determination. The titer of the virus was detected. The product was infused into rats liver allografts via portal vein and its expression in the transplanted livers was detected immunohistochemically. Results Recombinant adeno-associated virus vector pSNAV-CTLA-4Ig was generated and purified into 8.5 × 1011/ml. Agarose gel analysis of PCR products verified the presence of CTLA-4Ig. Digestion with BamHI and sequence determination confirmed that pSNAV-CTLA-4Ig was constructed. Expression of CTLA-4Ig in the transplanted livers was detected successfully. Conclusion Prepared pSNAV-CTLA-4Igwas constructed correctly and can express CTLA-4Ig effectively. Besides this, it can express CTLA-4Ig in rat liver allografts. It may be used in the study of transplant tolerance.更多还原