Cloning, Eukaryotic Expression And Identification Of Nuclear Respiratory Factor-1

Objective:The NRF-1gene was cloned, and analyzed by bioinformatics tools. To recognize thestructural and functional features of NRF-1, the relationship between NRF-1and associatedproteins. It can provide experimental support and ideas for further research on function ofNRF-1and the mechanism of NRF-1in heart failure.To infect the rat cardiomyocyte cell line(H9c2(2-1) cells) by constructed NRF-1recombinant lentivirus, and screen out highexpression of NRF-1gene in transfection H9c2(2-1) cells. It lays a foundation for furtherresearch on gene therapy and the regulation of NRF-1gene in heart failure.Methods:1.Bioinformatics analysis of NRF-1: We analyzed the amino acid composition, thesecondary structure of the protein, protein modification sites, signal peptide, three-dimensional structures of NRF-1,and the relationship of NRF-1with related protein analysisthrough online tools EXPASY, PSSFinder, PSITE, SignalP, Swiss-model, STRING of NRF-1.2.The clone and characterization of NRF-1gene:2.1Cloning of NRF-1gene The geneof NRF-1was cloned by the biosynthesis and the overlap extension PCR, then recombinantedinto vector pMD18-T. The NRF-1/pMD18-T recombinant was transformed into competent E.coli DH5.2.2The idenification of NRF-1gene The sequencing of recombinant plasmidsNRF-1/pMD18-T was identified by PCR.3.Construction of NRF-1recombinant lentivirus and stability expression of gene NRF-1in H9c2(2-1) cells:3.1The packaging and titer determination of NRF-1recombinantlentivirus NRF-1gene was reorganized to vector pLenti6.3-MCS-IRES2-EGFP. The plasmid of pLenti6.3-NRF1-IRES2-EGFP and two packaging plasmids (pLP1, pLP2and pLP/VSVG)were packaged into recombinant lentivirus in293T cells by lipofectamine2000.Then the viraltiter of recombinant lentivirus was measured.3.2To measure the optimal concentration ofNRF-1recombinant lentivirus when infecting the H9c2(2-1) cells The appropriate MOIvalues was selected with the virus infecting the H9c2(2-1) cells according to the concentrationgradient.3.3The measurement of the optimal concentration of antibiotic(Blasticidin) Afterinfected the H9c2(2-1) cells with appropriate recombinant lentivirus, we filtered the positivetransfection H9c2(2-1) cells with Blasticidin according to the concentration gradient, thenpick out suitable concentration of Blasticidin.3.4The identification of the positivetransfection H9c2(2-1) cells To obtain high expression of gene NRF-1in H9c2(2-1) cells, thepositive transfection cells was continually screened with the appropriate concentration ofBlasticidin. The expression of NRF-1in positive transfection cells was identificated by PCR,QPCR and Western blot.Results:1.Bioinformatics analysis showed that the molecular weight of NRF-1protein is57280.1Da, isoelectric point is5.28, formula for C2498H3976N704O808S15,534amino acids, ofwhich46basic amino acids,64acidic amino acids,148npolar amino a cids,191hydrophobicamino acids. There are46(Arg+Lys) positively charged residue and64(Asp+Glu)negatively charged residue in NRF-1, the instability coefficient is35.74, the total hydropathyindex is-0.334, above results illustrated that the NRF-1is stable hydrophilic protein. Thereare amount of alpha helix and beta–fold structure uniformly distributed in the secondarystructure of NRF-1protein.There are2glycosylation sites,5protein kinase C phosphorylationsites,13casein kinase II phosphorylation sites,9terminal acylation sites,1amidation sites,11carboxy-terminal micro-targeting signal. The result of analysis by STRING showed the NRF-1has different degrees of correlation with ten factors.2.The sequencing identification of recombinant plasmid NRF-1/pMD18-T showed that the NRF-1gene sequence in recombinant plasmid was consistent with the original sequenceon GenBank.3.The NRF-1recombinant lentivirus was constructed. The viral titer of NRF-1recombinant lentivirus was5.5×107TU/ml. The appropriate MOI values of the virus infectingthe H9c2(2-1) cells was100. The suitable Blasticidin concentration in the screening ofpositive transfection cells was3μg/ml. The NRF-1recombinant lentivirus infected H9c2(2-1)cells according to MOI=100, after continually screened with the3μg/ml Blasticidin, weobtained stable transfection H9c2(2-1) cells. PCR results showed that lentiviruses carryingNRF-1inserted into the target cell genome, QPCR results showed that the mRNA expressionof NRF-1in stable transfection group was2.25times higher than that in the control group,Western blot results showed that the protein expression of NRF-1in stable transfection groupwas1.28times higher than that in the control group.Conclusion:1.We have a knowledge of the amino acid composition and the protein moleculecharacteristics of NRF-1, also the relationship between NRF-1and associated proteins bybioinformatic analysis.2.NRF-1gene is successfully cloned, and E.coil DH5containning recombinant plasmidNRF-1/pMD18-T is also successfully constructed.3.NRF-1recombinant lentivirus eukaryotic expression system is successfullyconstructed.We successfully make NRF-1recombinant lentivirus transfected into the H9c2(2-1) cells, after identified by PCR, QPCR and Western blot, the results shows that the highexpression of gene NRF-1in rat cardiomyocyte cell line H9c2(2-1) is successfullyconstructed. It can provides the research idea and experimental support for the research on themechanism of NRF-1in heart failure, and the regulation function of NRF-1in the energymetabolism.