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目的构建BALB/c小鼠MHCⅠ类基因的逆转录病毒表达载体并探讨该基因在C57BL/6小鼠造血细胞中的表达。方法由逆转录病毒载体pMSCV介导,将BALB/c小鼠的H-2Dd基因导入C57BL/6小鼠的造血细胞中,并用逆转录聚合酶链反应和流式细胞仪检测转染后基因的表达。结果用EcoRⅠ和XhoⅠ双酶切鉴定证实,H2Dd基因正确插入载体pMSCV。重组逆转录病毒感染的C57BL/6小鼠造血细胞整合了BALB/c小鼠的H2Dd基因,且能转录为mRNA,在其细胞膜上有H2Dd分子的表达。结论成功构建小鼠H-2Dd编码基因的逆转录病毒载体,该基因已稳定地整合进C57BL/6小鼠造血细胞的基因组内,且在细胞中得到表达。
Objective To construct the retrovirus expression vector of MHC class Ⅰ gene of BALB / c mice and to explore the expression of this gene in hematopoietic cells of C57BL / 6 mice. Methods The retroviral vector pMSCV was used to transduce the H-2Dd gene of BALB / c mice into the hematopoietic cells of C57BL / 6 mice. The expression of the gene after transfection was detected by reverse transcription polymerase chain reaction and flow cytometry expression. Results The double digestion with EcoR Ⅰ and Xho Ⅰ confirmed that the H2Dd gene was correctly inserted into the vector pMSCV. Recombinant retrovirus-infected C57BL / 6 mouse hematopoietic cells integrate the H2Dd gene of BALB / c mice and are transcribed into mRNA with H2Dd expression on their cell membranes. Conclusion The retrovirus vector encoding mouse H-2Dd gene was successfully constructed and has been stably integrated into the genome of hematopoietic cells of C57BL / 6 mice and expressed in the cells.