Construction and secretive expression of fused gene containing partial rat cardiac myosin ? heavy chain cDNA sequence and signal peptide of human IL-2 / 中国免疫学杂志

ABSTRACT

Objective:To clone the rat cardiac myosin ? heavy chain cDNA fragment encoding aa736 960 and construct its recombinant retrovirus vector Methods:The 681 bp target gene was amplified from heart tissue of young rats with RT PCR Fusion gene of hIL 2/myosin was constructed by splicing with preserved region of hIL 2 cDNA using ligation methods and subsequently the plasmid pLNC hIL 2 myosin was constructed NIH3T3 cells and PA317 cells were transfected with plasmid pLNC hIL 2 myosin using Lipofectamine After screening with medium containing G418, the positive clone was chosen and was detected using RT PCR, immunohistochemistry, immune electron microscope and dot blot Results:The determination of nucleotide sequence showed that the nucleotide and amino acid sequence of the gene cloned was the same as the reported sequence, and its open reading frame was correct RT PCR analysis indicated that mRNA of the fused gene was present in the positive clone Immunohistochemistry, immune electron microscope and dot blot showed that the fused gene IL 2 myosin was successfully expressed Conclusion:The fused gene of rat cardiac ? heavy chain fragment and the preserved region of human IL 2 was constructed and expressed successfully