大鼠心肌肌凝蛋白a重链基因逆转录病毒载体的构建

Construction of retrovirus vector containing rat cardiac myosin α heavy chain cDNA

目的 :克隆编码大鼠心肌肌凝蛋白α重链 aa736～ 96 0的 c DNA片段 ,构建携带 hu IL - 2 / m yosin融合基因的逆转录病毒载体。方法 :以 RT- PCR方法从幼龄大鼠心肌组织中扩增出 6 81bp的目的基因 ,与 hu IL - 2 c DNA序列拼接 ,构建融合基因 hu IL - 2 / m yosin及其逆转录病毒载体。采用脂质体法转染 NIH 3T3细胞 ,经 G418筛选后 ,用 RT- PCR和免疫组化法分析重组逆转录病毒的表达。 结果 :核苷酸序列测定显示 ,克隆基因的核苷酸及推导的氨基酸序列与报告序列一致 ,开放阅读框正确 ;逆转录病毒载体 p L NC- hu IL - 2 - m yosin酶切结果与预期一致。RT- PCR检测提示转染细胞有 p L NC- hu IL - 2 - myosin m RNA转录 ;免疫组化分析表明转染细胞胞质内有大量肌凝蛋白表达。 结论 :大鼠心肌肌凝蛋白α重链基因逆转录病毒载体构建成功 ,并可在 NIH3T3细胞中表达 。

Objective: To clone rat cardiac myosin α heavy chain cDNA fragment encoding aa736 960 and construct its recombinant retrovirus vector(RV). Methods: The 681 bp target gene was amplified from heart tissue of young rats with RT PCR, fusion gene of huIL 2/myosin was constructed by splicing with huIL 2 cDNA using ligation methods and its RV was constructed. RT PCR and immunohistochemical assay were used to identify the expression of myosin protein in transfected cell. Results: The determination of nucleotide sequence showed that the nucleotide and amino acid sequence of gene clone was the same as reported, its opening reading frame was correct, the digesting result of pLNC huIL 2 myosin was identical with the predicted. NIH3T3 cell was transfected with recombinant RV, and G418 resistant NIH3T3 cell was established.RT PCR analysis indicated that mRNA of pLNC huIL 2 myosin was present in cell transfected with RV. The immunohistochemical assay also showed that the myosin protein expression was higher in the cell transfected with constructed RV. Conclusion: Rat cardiac myosin α heavy chain cDNA has been cloned and its RV has also been constructed and expressed in NIH3T3 successfully, it will contribute to research of prevention and treatment of heart immune injury by cardiac myosin gene transfer to induce specific tolerance.