HLA-C和HLA-G基因真核细胞双表达载体的克隆及鉴定分析  

Clone and identification of the eukaryotic co-expression vector of human HLA-G and HLA-C genes with bicistronic vector pVITRO2

作　　者：张磊[1] 杨珏琴[1] 姚芳娟[1] 许玲娣[1] 范丽安[1] 

机构地区：[1]上海市免疫学研究所,上海第二医科大学上海200025

出　　处：《免疫学杂志》2004年第5期334-337,342,共5页Immunological Journal

基　　金：国家自然科学基金 (30 0 70 784;30 371 350 );上海市科委重点项目 (0 2DJ1 4 0 54)资助

摘　　要：目的　构建人HLA C和HLA G基因真核细胞双表达载体。方法　用RT PCR从孕妇蜕膜组织有核细胞总mRNA中逆转录扩增全长HLA C和HLA GcDNA ,首先将HLA G经BamHⅠ和ClaⅠ双酶切 ,HLA C经EcoRⅠ和XhoⅠ双酶切后 ,分别定向插入真核细胞双顺反子载体pVITRO2的多克隆位点 1(mcs1)和 2 (mcs2 )中 ,然后经酶切和测序鉴定 ,确定已建立pVITRO2的单表达质粒pVITRO2 HLA G和pVITRO2 HLA C ,再将HLA CcDNA经EcoRⅠ和XhoⅠ双酶切 ,定向插入单表达质粒pVITRO2 HLA G的多克隆位点 2 (mcs2 )中 ,经PCR反应初筛 ,再经双酶切鉴定。结果　限制性内切酶和测序分析表明已成功构建了HLA Cw 0 10 3和HLA G 0 10 1的单表达质粒pVITRO2 HLA C和pVITRO2 HLA G以及双表达质粒pVITRO2 HLA CG。结论　本研究成功构建了真核细胞双顺反子载体pVITRO2的双表达质粒pVITRO2 HLA CG ,以及单表达质粒pVITRO2 HLA C和pVITRO2 HLA G。Objective To construct eukaryotic co-expression vector of human HLA-C and HLA-G genes. Methods RT-PCR was used to amplify the full-length HLA-C and HLA-G cDNA from total RNA of cytotrophoblast karyote in pregnant women. The HLA-G cDNA fragment (digested with BamHⅠand ClaⅠ) and the HLA-C cDNA fragment (digested with EcoRⅠand XhoⅠ) were inserted into the multiple cloning sites 1 and 2 (mcs1 and mcs2) of the eukaryotic bicistronic co-expression vector pVITRO2,respectively. The construction of the single expression plasmid pVITRO2-HLA-Cw0103 and pVITRO2-HLA-G*0101 was been confirmed by restriction endonuclease treatment and sequence identification. Then the HLA-Cw0103 cDNA fragment was directionally inserted into the mcs2 of the vector pVITRO2-HLA-G*0101, and the recombined plasmid was identified by PCR and restriction endonucleases digestion. Results The restriction endonucleases digestion and sequencing suggested that the eukaryotic co-expression vector pVITRO2-HLA-CG and the single gene expression plasmids pVITRO2-HLA-C and pVITRO2-HLA-G were constructed successfully. Conclusion The recombinant eukaryotic expression vector pVITRO2-HLA-CG and the single gene expression plasmids pVITRO2-HLA-C and pVITRO2-HLA-G were constructed successfully.

关 键 词：HLA-C HLA-G 双顺反子载体pVITR02 双表达质粒 

分 类 号：R392.11[医药卫生—免疫学]