Objective To analyze the physicochemical properties of nicotinamide adenine dinucleotide dehy-drogenase(ubiquinone)iron-sulfur protein 6(NDUFS6),a structural subunit of mitochondrial respiratory chain complex Ⅰ,by applying bioinformatics approaches,and to construct and identify the pCV702-NDUFS6 overex-pression plasmid.Achievement of these work is helpful for further study on the protein's function.Methods The physicochemical properties and secondary structure of NDUFS6 protein were analyzed by employing bioin-formatics tools such as Expasy,UniProtKB,NCBI and SOPMA.According to the NDUFS6 cDNA sequence,the overexpression plasmid pCV702-NDUFS6 carrying the NDUFS6 gene was constructed and transfected into rat cardiomyocytes H9C2.The negative control(NC)and the corresponding empty vector CON520 were used as positive control(PC)groups.After screening with puromycin,the expression levels of NDUFS6 mRNA and pro-tein were detected by RT-qPCR and Western blot.Results NDUFS6 protein was composed of 116 amino acids,and the theoretical isoelectric point pI was 9.37.The secondary structure of the protein was mainly composed of random coils(50％).The results of enzyme digestion and gene sequencing showed that the pCV702-NDUFS6 expression plasmid was successfully constructed.RT-qPCR and Western blot results showed that the expression level of NDUFS6 in the overexpression group was up-regulated compared with the NC group and the PC group(P all＜0.05).Conclusion Successfully constructed an overexpression plasmid that can effectively overexpress NDUFS6 gene in cardiomyocytes H9C2.
关键词
烟酰胺腺嘌呤二核苷酸脱氢酶(泛素)铁硫蛋白6/生物信息学分析/心肌细胞/质粒构建
Key words
nicotinamide adenine dinucleotide dehydrogenase(ubiquinone)iron-sulfur protein 6/bioinformat-ics analysis/cardiomyocyte/plasmid construction
维普
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