摘要
目的构建猪MHCⅡ分子的反转录病毒表达载体,建立产生病毒颗粒的包装细胞株,体外转导小型猪骨髓细胞并观察目的基因的表达情况,为研究小型猪转导同种异体MHCⅡ分子对于肝脏移植特异性免疫耐受的诱导提供实验基础和实验材料。方法利用基因重组技术将目的基因SLA-DRBdd、SLA—DQAdd和SLA-DQBdd克隆到反转录病毒载体pLNCX2上,通过脂质体转染包装细胞AmphoPackTM-293细胞,从G418筛选阳性克隆细胞培养上清中获得了有感染性的病毒颗粒;并对体外培养的猪骨髓细胞进行了感染,采用Nothern杂交和逆转录-聚合酶链反应(RT- PCR)等方法检测了目的基因在骨髓细胞中的表达。结果经过酶切和测序验证已成功构建了 MHCⅡ分子的反转录病毒表达载体,在转染AmphoPackTM-293细胞后可以产生有感染性的重组病毒颗粒,稳定的CFM-GFG418抗性率为6％～11％,RT结果显示抗性细胞中有目的基因的RNA 转录产物存在,而转染空载体的对照组没有。结论重组有目的基因的逆转录病毒载体pLNCX2 经过包装细胞AmphoPackTM-293产生的病毒颗粒可以有效地将猪MHCⅡ分子转导入猪的骨髓细胞并获得长期稳定的表达。
Objective To construct retroviral vector carrying swine MHC Class Ⅱ, to select a package cell line that can produce infective virus and to dected the expression of target gene in bone marrow cells of miniature swine for further investigating the role of swine MHC Class Ⅱ molecules in allograft of liver and providing infective virus to the following animal experiment. Methods The recombinant vector pLNCX2-DRB^dd, pLNCX2-DQA^dd and pLNCX2-DQB^dd was constructed respectively by cloning SLA-DRB^dd, SLA-DQA^dd and SLA-DQB^dd into pLNCX2. Recombinant plasrnids were transfected into package cell line AmphoPack^TM-293 by Lipofectamine. G418-resistant cell line was selected to get cell line which was stable G418-resistant and virus titer was determined by counting the number of G418- resistant forming unit by infecting NIH3T3 cells. Recombinant virus was used to infect swine bone marrow cells and the expression of SLA-DRB^dd, SLA-DQA^dd and SLA-DQB^dd was detected by Northern blot and RT-PCR. Results The correct recombinant plasmid was conformed by restriction enzyme digestion and infective virus particles was released by G418-resistant package cell line. Using infective recombinant virus, 10%-15 % G18-resistant CSF-GM was obtained. Conclusion Recombinant pLNCX2 vector produced by package cell AmphoPackTM-293 can transfer effectively swine MHC class Ⅱ genes into swine bone marrow cells and promote their expression.
出处
《中华实验外科杂志》
CAS
CSCD
北大核心
2005年第12期1559-1561,共3页
Chinese Journal of Experimental Surgery
基金
国家自然科学基金资助项目(C03020504)
