新疆羊种布鲁氏菌分离株的鉴定与L7/L12蛋白的原核表达和生物信息学分析

Identification of Isolate Strain of Brucella Melitensis in Xingjiang and Prokaryotic Expression and Bioinformatics Analysis of Its L7/L12 Protein

【目的】分离并鉴定新疆羊种布鲁氏菌。原核表达该菌的L7/L12蛋白,检测该蛋白的反应原性及进行部分生物学分析。【方法】采用细菌划线培养、形态学观察、PCR检测以及生化试验进行布鲁氏菌分离鉴定。利用常规分子生物学方法表达并纯化羊种布鲁氏菌分离株的L7/L12蛋白,应用Western Blot分析融合蛋白的反应原性。使用生物信息学软件对该蛋白进行了生物信息学分析。【结果】分离鉴定确定该菌株为羊种布鲁氏菌。经过测序与酶切鉴定,正确构建了表达载体p ET-28a-L7/L12.SDS-PAGE试验显示纯化的L7/L12蛋白为单一条带;经过Western Blot检测,该融合蛋白具有良好的反应原性。生物信息学分析显示,该蛋白无跨膜区结构,不存在信号肽,二级结构α-螺旋为主并利用Phyre2服务器成功构建了该蛋白的三维模型。【结论】鉴定出该分离菌株,表达并纯化了该菌的L7/L12融合蛋白,Western Blot证明该蛋白具有良好的反应原性,为后续该蛋白的亚单位疫苗研究奠定了基础。

【Objective】To isolate and identify Xinjiang Brucella melitensis,this project focuses on prokaryotic expression of the L7/L12 protein of the bacteria,detection of its reactionogenicity and and partial biological analysis. 【Method】Isolation and identification of Brucella melitensis by using bacterial streak culture,morphological observation,biochemical test and PCR detection. Using conventional and molecular biological methods to express and purify the L7/L12 protein of this Brucella melitensis. Expression and purification isolate strain L7/L12 protein by using the conventional molecular biological methods,and analysis of the fusion protein reactionogenicity by Western Blot. Using bioinformatics software to analyze some functions of this protein. 【Result】After identification,the strain was identified as Brucella melitensis. After enzyme digestion and sequencing,the expression vector p ET-28a-L7/L12 was correctly constructed. SDS-PAGE tests showed that the purified L7/L12 protein was a single band. The fusion protein had good reactionogenicity by Western Blot detection. Bioinformatics analysis showed the protein had no trans-membrane domain and no signal peptide. Its secondary structure was mainlyα-helix. And the three-dimensional structure of the protein was constructed by Phyre2 Server. 【Conclusion】The isolated strain was identified successfully and L7/L12 fusion protein of this isolate strain was expressed and purified. Blot Western test proved that the protein had a good reactionogenicity,which laid the foundation for the protein follow-up research of the subunit vaccine.