中文摘要：

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293Fr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10^9TU／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。

英文摘要：

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.