Cloning And Related Adenovirus Vector Construction Of Human Cardiac-specific Tnni3k Homologue Gene From Rattus Norvegicus

Background and Objectives:TNNI3K is a novel cardiac-specific protein kinase, isolated on the basis of constructed human adult cardiovascular cDNA library. The effect of the kinase in cardiac hypertrophy should be employed in cardiomyocytes, however, homo sapiens cardiomyocyte cell lines could not be used. Further exploration has to be turnd to study rat homologou gene by the use of neonatal rat cardiomyocytes. The problem we have to face is that there is no rat TNNI3K gene for further research in our group. Hence, the principal objective is to clone rat TNNI3K gene coding sequence, and then to screen out the valid RNA interfere fragment. Finally, construction the recombinant adenovirus with rat gene TNNI3K and the valid RNAi fragment.Methods and Results:1 Bioinformatics was employed to identify rat gene TNNI3K sequence. Two overlap rTNNI3K fragments were obtained by PCR from the rat cardiomyocytes cDNA, and then the fragments were digested by restriction endonuclease XspI, ligated to be a full rTNNI3K coding sequence and cloned into pGEM-T Easy vector. The plasmid containing TNNI3K was identified by sequencing.2 Four specific interference sequences targeting rat TNNI3K were designed, synthesized and cloned into the pGCSIL-GFP vector. RNAi plasmid (pGCSIL-RNAi-1,-2,-3 and -4, respectively) and recombinant rTNNI3K expression plasmid were co-transfected into 293T cells, and the efficiency of RNAi were detected by Western Blot and Real-time PCR. The pGCSIL-RNAi-1 had the excellent interference effect, which contained the target site of +880～+898. The recombinant adenovirus vector with the valid RNAi fragment were constructed.3 rTNNI3K gene was inserted into the shuttle vector of adenovirus, and then rTNNI3K adenovirus plasmid were obtained through homologou recombination in vivo in bacteria. The recombinant adenovirus with rTNNI3K was constructed in 293A cell line.4 The rat neonatal cardiomyocytes were infected by recombinant adenovirus rTNNI3K and RNAi, and tthe phosphorylation level of JNK,p38 and Erk were detected by Western Blot. The primary result showed that both rTNNI3K overexpression and RNAi could increase JNK and p38 phosphorylation level.Conclusion:1. Rat TNNI3K gene was cloned correctly;2.The valid RNAi fragment for rTNNI3K was screened out; 3. rTNNI3K and RNAi recombinant adenovirus were constructed successfully in this study, as effective tools, could be used in the future research.