Prokaryotic Expression and Identification of the WbkC Gene of Brucella melitensis

[ Objective] In order to clone and express the formyltransferase of Brucella abortus in E. coli, purify the expressed protein and detect its immunogenicity. EMethods] A gene recoding formyltransferase 27 -35 ku （Wbkc） was amplified from the genomic DNA of Brucella abortus PCR. The amplified fragments were digested with BamH I and Sal I, and then cloned to the vector pET28a. The constructed recombinant plasmid pET28a-Wbkc was transformed to E. coil BL21 and was induced to express the fusion protein. Then the protein was purified by histidine-binding res- in column chromatography, and the immunogenicity was detected by Western blot. The PCR product of Wbkc gene was cloned to the vector pET28a, and the recombinant vector was confirmed by colony PCR identification, recombinant vector digested identification and sequencing analy- sis. [ Results] It was successfully expressed in E. coil BL21 as a fusion protein with histidine at the presence of IPTG, and a specific protein band of 29 ku was found when identified by SDS-PAGE. Western blot showed good immunoreactivity of the expressed product. Wbkc was successfully cloned and expressed, and the purified fusion protein had immunogenicity. This study provides a solid foundation for the further study on the function of proteins and brucellosis diagnostic antigens. [ Conclusion] Amplification of formyltransferase gene of Brucella abortus and prokaryotic expression of WbkC_ indicatino the formvItransferase has specific immune reactivitv.

Prokaryotic Expression and Antigenic Analysis of Wbkc Gene from Brucella abortus

[ Objective ] This study aimed to clone and express formyltransferase （Wbkc） gene from Brucella abortus in E. coli, purify the expressed protein and analyze its immunogenicity. [Method] A gene encoding 27 -35 ku formyltransferase （Wbkc） was amplified from the genomic DNA of BruceUa abortus by PCR. The amplified fragments were digested with BamH I and Sal I, and inserted into pET28a vector. The constructed recombinant plasmid pET 28a-Wbkc was trans- formed into E. coli BL21 and was induced to express the fusion protein. Subsequently, the protein was purified by histidine-binding resin column chromatography, and the immunogenicity was detected by Western blot assay. The recombinant plasmid was identified by colony PCR, double digestion and sequencing analysis. [ Result] Wbkc was successfully cloned and expressed in E. coli. A specific protein band of 29 ku was detected by SDS-PAGE. Western blot showed specific im- munoreactivity of the purified fusion protein. [ Conclusion] This study provided a solid foundation for further investigating diagnostic proteins for brucellosis and developing Brucella gene-deletion vaccines.

牛布鲁氏菌Wbkc基因原核表达及抗原性分析

克隆牛布鲁氏菌的甲酰基转移酶基因(Wbkc),在大肠杆菌中表达/纯化,并对甲酰基转移酶的抗原性进行分析。以牛布鲁氏菌的染色体DNA为模板,扩增Wbkc基因,双酶切后克隆至pET-28a上,在大肠杆菌BL21中诱导表达,His Trap FF层析柱纯化,Western blot鉴定甲酰基转移酶的抗原性。提取的重组质粒经PCR鉴定、双酶切鉴定和测序分析确定目的基因成功插入到了克隆载体中。SDS-PAGE分析证明,表达产物为相对分子质量29 000的融合蛋白。Western blot结果表明,表达的蛋白具有特异的免疫反应原性。

带有氯霉素抗性基因标记的重组布氏杆菌S2的构建及其生物学特性

无法区分免疫接种和自然感染是限制布氏杆菌疫苗应用的主要原因。本研究通过基因同源重组技术,用氯霉素抗性基因(Cmr)替代布氏杆菌弱毒S2株的WbkC基因,筛选获得重组布氏杆菌rS2-WbkC株。试验发现,重组菌rS2-WbkC由原先的光滑型转变成粗糙型。rS2-WbkC在TSA培养基上传25代后仍能稳定表达氯霉素抗性蛋白。小鼠动物试验模型表明,rS2-WbkC与S2有相似的保护性,但rS2比S2具有更高的安全性。rS2-WbkC免疫小鼠后,其抗血清可通过平板凝集试验与S2免疫的血清相区分。本研究为布氏杆菌标记疫苗的研制提供了技术平台。

羊型布氏菌WbkC基因真核表达质粒的构建及鉴定

目的构建羊型布氏菌脂多糖O-侧链合成必需的甲酰基转移酶WbkC基因真核表达质粒,并进行鉴定。方法提取16M株羊型布氏菌基因组DNA,PCR扩增WbkC基因,克隆至psos载体上,构建诱饵重组质粒psos-WbkC,转化酵母菌株cdc25Hα,进行菌落PCR鉴定,并检测其在酵母双杂交系统中的自激活和定位情况。结果诱饵重组质粒psos-WbkC经PCR双酶切及测序证明构建正确;重组酵母菌经PCR鉴定,可扩增出约780 bp的目的基因;诱饵质粒在酵母双杂交系统中无自激活且定位正确。结论已构建了羊型布氏菌WbkC基因真核表达质粒psos-WbkC,可应用于酵母双杂交系统中,用于寻找巨噬细胞cDNA文库中与WbkC相互作用的蛋白质。