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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。
Objective To clone the CTLA-4Ig cDNA into the adeno-associated virus vector pSNAV and obtain the target gene expression in the transplanted liver, which will lay the foundation for gene therapy of immune tolerance in organ transplantation. Methods pSNAV-CTLA-4Ig was constructed by gene recombination and restriction endonuclease digestion, and transformed into DH-5α. The resistant clone was obtained and digested with BamH Ⅰ. PsNAV-CTLA-4Ig was introduced into BHK-21 by lipofectamine and selected by G418. The cell strain was infected with HSV1-rc to generate pSNAV-CTLA-4Ig virus. The virus titer was determined by dot blot hybridization. The CTLA-4Ig gene was identified by PCR and the full-length CTLA-4Ig was sequenced. The expression of CTLA-4Ig protein in rat liver was detected by immunohistochemistry. Results The full-length CTLA-4Ig cDNA fragment was digested with BamH Ⅰ, which indicated that CTLA-4Ig was inserted into pSNAV successfully. The number of virus particles detected by virus titer was 8.5 × 1011 / ml. The PCR products were 500-600 bp gene fragment confirmed CTLA ~ 4Ig gene integration; measured DNA sequence and known CTLA-4Ig gene sequence is exactly the same. CTLA-4Ig protein expression was seen in the liver perfused with pSNAV-CTLA-4Ig virus. Conclusion The CTLA-4Ig cDNA was successfully cloned into the adeno-associated virus vector pSNAV. The CTLA-4Ig cDNA was inserted into the pSNAV multi-cloning site correctly and the CTLA-4Ig protein was expressed in the transplanted liver.