NDUFS6蛋白生物信息学分析及过表达质粒的构建与鉴定  

Bioinformatics Analysis of NDUFS6 Protein and Construction and Identification of Overexpression Plasmid

作　　者：张瑜[1] 孙美琪 徐方晶 王洁[3] 方克宝 王一帆[3] 何军 ZHANG Yu;SUN Meiqi;XU Fangjing;WANG Jie;FANG Kebao;WANG Yifan;HE Jun(First Clinical Medical College of Ningxia Medical University,Yinchuan 750004,China;Yinchuan Hospital of Traditional Chinese Medicine Affiliated to Ningxia Medical University,Yinchuan 750000;General Hospital of Ningxia Medical University,First Clinical Medical College of Ningxia Medical University,Yinchuan 750004,China)

机构地区：[1]宁夏医科大学第一临床医学院,银川750004 [2]宁夏医科大学附属银川市中医医院,银川750000 [3]宁夏医科大学总医院,宁夏医科大学第一临床医学院,银川750004

出　　处：《宁夏医科大学学报》2024年第4期353-359,共7页Journal of Ningxia Medical University

基　　金：宁夏自然科学基金项目(2023AAC02070,2023A2237)。

摘　　要：目的 应用生物信息学方法分析线粒体呼吸链复合体Ⅰ结构亚基烟酰胺腺嘌呤二核苷酸脱氢酶(泛素)铁硫蛋白6(NDUFS6)的理化性质,构建pCV702-NDUFS6过表达质粒并进行鉴定,为进一步研究NDUFS6蛋白功能奠定基础。方法 利用Expasy、UniProtKB、NCBI、SOPMA等生物信息学工具分析NDUFS6蛋白的理化性质、二级结构等;根据NDUFS6 cDNA序列构建携带NDUFS6基因的过表达质粒pCV702-NDUFS6,转染大鼠心肌细胞H9C2,并设置阴性对照(NC)组和相应空载体CON520作为阳性对照(PC)组,经嘌呤霉素筛选后,采用RT-qPCR和Western blot检测NDUFS6 mRNA和蛋白表达水平。结果 NDUFS6蛋白由116个氨基酸组成,理论等电点pI为9.37。蛋白二级结构以无规则卷曲(占50%)为主。酶切鉴定和基因测序结果显示,pCV702-NDUFS6表达质粒构建成功。RT-qPCR和Western blot结果显示,相较于NC组和PC组,过表达组NDUFS6表达水平均上调(P均<0.05)。结论 成功构建了能在心肌细胞H9C2中有效过表达NDUFS6基因的过表达质粒。Objective To analyze the physicochemical properties of nicotinamide adenine dinucleotide dehydrogenase(ubiquinone)iron-sulfur protein 6(NDUFS6),a structural subunit of mitochondrial respiratory chain complexⅠ,by applying bioinformatics approaches,and to construct and identify the pCV702-NDUFS6 overexpression 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 bioinformatics 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 protein 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 

分 类 号：R34[医药卫生—基础医学]