Abstract: [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 pET-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.

摘要： [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pET-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.