金黄色葡萄球菌Cna基因的生物信息学分析及原核表达

Bioinformatics analysis and prokaryotic expression of Cna gene in Staphylococcus aureus

[目的]对金黄色葡萄球菌的Cna基因进行生物信息学分析,并进行克隆和原核表达,为金黄色葡萄球菌重组疫苗的研发提供参考数据。[方法]使用Protparam、PSORT、Signal P-5.0、TMpred、Net Phos 3.1 Server、PSIPRED、SWISSMODEL等在线生物信息软件,分析Cna蛋白的理化性质、亚细胞定位、跨膜区域、磷酸化修饰位点、二级结构和三维结构。以基因组DNA为模板扩增Cna基因编码N1-N2子域的片段,扩增产物经酶切回收后与载体pET-32a(+)相连接,构建重组表达质粒,将该重组质粒转化E.coli TG1,经菌落PCR、测序鉴定后,增菌培养并抽提质粒,再转化E.coli BL21(DE3),经IPTG诱导表达后进行SDS-PAGE分析。[结果]Cna蛋白由1 183个氨基酸残基组成,相对分子质量为133 006.82 Da,等电点为5.89,负电荷氨基酸残基数为180个,正电荷氨基酸残基数为167个,分子式:C5863H9241-N1569O1936S10,原子总数18 619个,是一种稳定的亲水蛋白质,定位于细胞膜上,第4~23、1 158~1 177位氨基酸残基分别形成一个典型的跨膜区,具有148个潜在的磷酸化修饰位点,二级结构中无规则卷曲和β折叠占较大比例,三维结构与二级结构预测结果相符。成功构建了重组表达质粒pET-32a(+)-Cna,经IPTG诱导后重组蛋白(N1-N2子域)获得了表达,重组蛋白相对分子质量为52.1 kDa。[结论]Cna基因的生物信息学分析及该基因的成功表达,为研究Cna蛋白在免疫保护中的作用奠定了一定的基础。

[Objective] Bioinformatics analysis of the Cna gene of Staphylococcus aureus,cloning and prokaryotic expression were performed to provide reference data for the development of recombinant Staphylococcus aureus vaccine.[Method]Physicochemical properties,subcellular localization,transmembrane region,phosphorylation of Cna protein,secondary structures,and three-dimensional structures were analyzed using online bioinformatics software such as Protparam,PSORT,Signal P-5. 0,TMpred,Net Phos 3. 1 Server,PSIPRED,SWISS-MODEL. The Cna gene was amplified by self-designed primers,and the amplified product was digested with the prokaryotic expression vector pET-32 a(+) to construct a recombinant plasmid. The recombinant expression plasmid was transformed into TG1 and identified by colony PCR and gene sequencing. The plasmid was cultured and extracted,and then transformed into E. coli BL21(DE3),and the expression was induced and subjected to SDS-PAGE analysis.[Result] Cna protein consists of 1 183 amino acids with a relative molecular mass of 133 006. 82 Da,an isoelectric point of 5. 89,a total of 180 negatively charged amino acid residues,and a total of 167 positively charged amino acids.The molecular formula is C5863 H9241 N1569 O1936 S10,and the total number of atoms is 18 619. The 4-23 and 1 158-1 177 amino acids form a typical transmembrane region with 148 potential phosphorylation sites,the secondary structure has the high proportion of β-sheet and random curl,and the three-dimensional structure is consistent with the prediction of secondary structure.The expression vector pET-32 a(+)-Cna was successfully constructed,and the recombinant Cna protein was expressed by IPTG. The relative molecular mass of the recombinant protein was 52. 1 kDa.[Conclusion] The bioinformatics analysis of Cna gene and its successful expression lay a foundation for studying the role of Cna protein in immune protection.