BALB/c小鼠MHCI类基因的克隆与逆转录病毒载体的构建

Cloning of MHC I gene of BALB/c mice and its construction of retroviral expression vector

目的 构建BALB/c小鼠MHCI类分子的逆转录病毒表达载体。方法 用RT -PCR从BALB/c小鼠脾细胞基因组中扩增BALB/c小鼠MHCI类分子H -2Dd的编码基因 ,克隆于载体pGEM -T中。经EcoRI和XhoI双酶切后 ,亚克隆于逆转录病毒载体pMSCV ,构建pMSCV -H2Dd重组逆转录病毒表达质粒 ,并进行酶切和序列测定鉴定。结果 用EcoRI和XhoI双酶切鉴定证实 ,H -2Dd基因正确插入载体pMSCV。H -2Dd的编码基因经序列测定证实 ,与文献报道完全一致 ,阅读框架与设计相符。结论 成功构建BALB/c小鼠MHCI类分子编码基因的逆转录病毒载体 。

Objective To construct the retroviral expression vector of BALB/c mice MHC I gene. Methods The gene H-2Dd gene, encoding BALB/c mice MHC I, was amplified by reverse transcription polymerase chain reaction from genome of BALB/c mice spleen cells and inserted into cloning vector pGEM-T. After restriction endonuclease digestion, gene fragment encoding H-2Dd was subcloned to retroviral expression vector pMSCV and was confirmed by restriction endonuclease digestion and nucleotide sequencing.Results The cloned gene, gene encoding H-2Dd, was correctly inserted into vector pMSCV,which was confirmed by restriction endonuclease digestion and nucleotide sequencing.Conclusion Successful construction of recombinant retroviral expression vector based on the gene encoding MHC I of BALB/c mice, lays the foundation for further research of the function of MHC in transplantation immunology.