牦牛源产气荚膜梭菌β2基因的原核表达及生物信息学分析

Prokaryotic Expression and Bioinformatics Analysis of β2 Gene of Clostridium perfringens from Yaks

为了原核表达一株牦牛源产气荚膜梭菌Qinghai-1的β2基因及了解其编码蛋白的生物信息。首先利用PCR扩增技术、基因克隆技术构建原核表达质粒并通过高通量测序、双酶切技术、SDS-PAGE检测和Western blotting方法对表达情况进行鉴定,其次利用生物信息学分析的方法对β2毒素蛋白的一级结构、二级结构、蛋白特殊区域、三级结构及B细胞线性抗原表位等多方面进行预测分析。原核表达结果显示:牦牛源产气荚膜梭菌β2基因在温度为37℃,IPTG浓度为1.5 mmoL/L,培养时间为8 h~10 h的条件下可得到大小约为28KD的可溶于细胞质的不含信号肽的目的蛋白。生物信息学分析结果显示:牦牛源产气荚膜梭菌β2基因大小为798 bp,共编码265个氨基酸,与印度的产气荚膜梭(FR687302.1)的β2毒素基因相似性最高;其分子式为C_(1392)H_(2136)N_(356)O_(418)S_(11),分子量为30.9 ku,pI=6.04,负电荷(Asp+Glu)残基总数36个,正电荷(Arg+Lys)残基总数35个,脂肪指数:74.64,不稳定指数:28.52,亲水性总平均值(GRAVY):-0.478;拥有1个跨膜螺旋和35个潜在磷酸化位点;为含跨膜结构的、含信号肽的、含磷酸化位点的、含多个B细胞线性抗原表位的、稳定的亲水性蛋白。研究结果旨在准确预测牦牛源产气荚膜梭菌β2毒素抗原表位,为进一步研究牦牛源产气荚膜梭菌β2毒素致病机理提供重要理论依据。

In order to express the β2 gene of Clostridium perfringens of Qinghai-1 strain,derived from yaks,in a prokaryotic system and understand the biological information of its encoded protein,PCR amplification technology and gene cloning technology were firstly used to construct a prokaryotic expression plasmid,and the expression was identified by high-throughput sequencing,double restriction enzyme digestion,SDS-PAGE detection and western blotting methods.Furthermore,bioinformatics analysis was employed to predict and analyze various aspects of the β2 toxin protein,including primary and secondary structures,protein domains,tertiary structure,and B-cell linear antigenic epitopes.The prokaryotic expression results showed that the β2 gene of Clostridium perfringens from yaks could obtain a cytoplasmic target protein without signal peptide of approximately 28 KD in size,soluble in the cytoplasm and devoid of signal peptides,under conditions of 37°C temperature,1.5 mmol/L IPTG concentration,and 8 to10 hours of cultivation.The bioinformatics analysis revealed that the β2 gene of Clostridium perfringens from yaks was 798 bp in size,encoding 265 amino acids,and exhibited the highest similarity to the β2 toxin gene of an Indian Clostridium perfringens strain (FR687302.1).The protein had a molecular formula of C_(1392)H_(2136)N_(356)O_(418)S_(11),a molecular weight of 30.9 ku,a pI of6.04,a total of 36 negative charge residues (Asp+Glu),a total of 35 positive charge residues (Arg+Lys),a GRAVY score of-0.478,a instability index of 28.52,and a hydrophilicity index of 74.64.The protein harbored a single transmembrane helix and presented 35 potential phosphorylation sites.Notably,it exhibited structural stability,containing a signal peptide,multiple B-cell linear antigenic epitopes,and transmembrane regions.The study aimed to accurately predict the antigenic epitopes of the β2 toxin of yak-derived Clostridium perfringens and provide an important theoretical basis for further research on the pathogenic mechanism of the β2 toxin in yak-derived Clostridium perfringens.