Construction of retrovirus vector containing rat cardiac myosin α heavy chain cDNA
- VernacularTitle:大鼠心肌肌凝蛋白α重链基因逆转录病毒载体的构建
- Author:
Xinhua Lü
;
Qian SHEN
- Publication Type:Journal Article
- From:
Academic Journal of Second Military Medical University
2001;22(1):50-53
- CountryChina
- Language:Chinese
-
Abstract:
Objective： To clone rat cardiac myosin α he avy chain cDNA fragment encoding aa736-960 and construct its recombinant retrov irus vector(RV). Methods: The 681 bp target gene was amplified f rom heart tissue of young rats with RT-PCR, fusion gene of huIL-2/myosin was c onstructed by splicing with huIL-2 cDNA using ligation methods and its RV was constructed. RT-PCR and immunohistochemical assay were used to iden tify the expression of myosin protein in transfected cell. Results: The determination of nucleotide sequence showed that the nucleotide and ami no acid sequence of gene clone was the same as reported, its openin g reading frame was correct, the digesting result of pLNC-huIL-2-myosin was i dentical with the predicted. NIH3T3 cell was transfected with recombinant RV, and G418-resistant NIH3T3 cell was established.RT-PCR analysis indicated tha t mRNA of pLNC-huIL-2-myosin was present in cell transfected with RV. The im munohistochemical assay also showed that the myosin protein expression was highe r in the cell transfected with constructed RV. Conclusion: Rat cardiac myosin α heavy chain cDNA has been cloned and its RV has also been cons tructed 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.
