慢病毒介导HLA-G转基因小鼠的建立  

Preparation of lentivirus-mediated HLA-G transgenic mice

作　　者：王露露[1] 刘勤[1] 周晓杨[1] 刘昌峨[1] 王勇[1] 魏泓[1] 

机构地区：[1]第三军医大学基础医学部实验动物学教研室,重庆400038

出　　处：《第三军医大学学报》2012年第14期1392-1396,共5页Journal of Third Military Medical University

基　　金：国家自然科学基金(30700076)~~

摘　　要：目的以慢病毒作为载体建立HLA-G转基因小鼠,为研究HLA-G与移植免疫提供模型动物。方法利用FUW载体,构建含有HLA-G编码序列的慢病毒表达载体FUW-HLA-G。用磷酸钙沉淀法将包装质粒psPAX2、PMD2.G与重组慢病毒载体质粒FUW-HLA-G共转染293FT细胞,包装成慢病毒。通过同步转染含有绿色荧光标记基因的慢病毒质粒FUGW为参照,测定FUW-HLA-G病毒液的滴度。用显微注射法将浓缩后的病毒液注射至FVB/N小鼠1-细胞期胚胎透明带下,建立HLA-G转基因小鼠。PCR鉴定转基因小鼠的基因整合,用RT-PCR检测转基因在转录水平的表达,用Western blot检测HLA-G蛋白的表达。结果载体BamHⅠ、EcoRⅠ双酶切结果和测序结果显示重组的慢病毒质粒与所设计的序列一致。浓缩和纯化后的病毒液滴度达到109TU/ml以上,符合用慢病毒制备转基因小鼠的要求。PCR筛选出阳性鼠6只,RT-PCR与Western blot检测结果表明HLA-G基因在F0和F1代转基因小鼠中均有表达。结论通过慢病毒介导的基因转移,成功建立了表达人HLA-G蛋白的转基因小鼠,为研究HLA-G的功能及其在器官或组织移植中的效应提供了动物模型。Objective To provide the animal model for studying HLA-G and transplantation immunity by generating HLA-G transgenic mice with lentivirus as a vector. Methods A lentivirus expression vector, FUW-HLA-G containing HLA-G encoding sequences, was constructed and 293FT cells were transfected with packaged plasmids （psPAX2 and pMD2. G） and recombinant lentivus vector plasmid FUW-HLA-G, and packed into lentivirus. Synchronically transfected lentivus vector plasmid FUGW containing green fluorescence- marked gene was used as a control. Titer of the FUW-HLA-G viral suspension was measured. The concentrated FUW-HLA-G viral suspension was injected into the vitellary membrane of mice murine 1-cell eggs to generate HLA-G transgenic mice. Genetic interaction in the generated HLA-G transgenic mice was identified by PCR, transcriptional expression level of transgenic gene was measured by RT-PCR and expression of HLA-G protein was detected by Western blotting. Results Double BamH I and EcoR I digestion and sequencing showed that the sequences of recombinant lentivirus vector, FUW-HLA-G, were identical with its designed sequences. The concentrated and purified biological titre of FUW-HLA-G viral suspension reached over 1 ×10^9 WU/ml, which met the requirements of generating lentivirus transgenic mice. Six mice with positive FUW-HLA-G were screened by PCR. RT-PCR and Western blotting showed that the HLA-G was expressed in F0 and F1 transgenic mice. Conclusion Transgenie mice expressing HLA-G are successfully generated by lentivirus mediated gene transfer, which can thus provide an animal model for studying the function of HLA-G and its efficiency in organ or tissue transplantation.

关 键 词：HLA-G 慢病毒 转基因小鼠 

分 类 号：R-332[医药卫生]