以内蒙古科尔沁地区牛场布病疫情流行性病学检测资料为基础,重点进行布病的流行病学调查,了解布病流行情况。本研究采用虎红平板凝集试验(RBT)、试管凝集试验(SAT)、全乳环状试验(MRT)、补体结合试验(CFT)、竞争酶联免疫吸附试验(c ELISA)、奶液间接酶联免疫吸附试验(i ELISA)方法进行筛选和确诊。结果表明,2009年共采集1360份牛血清,其中阳性血清57份,感染率为4.19%;2010年采集2175份牛血清,感染布病的牛共有129头,发病率为5.10%;2011年采集2340份牛血清,发病率为7.78%。2010年在通辽市牛布鲁氏菌病流行病学调查时共采集奶样1931份,经MRT和OIE i ELISA检测阳性率为24.39%。屠宰场牛布病总体阳性率为7.34%,而散户牛布病的总体阳性率为12.84%。通辽地区奶牛布病感染率呈现出明显的群间和区域间不均衡性。屠宰场由于检疫制度严格,阳性率明显低于散户。
[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 Brucella 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 transformed 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 immunoreactivity 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.
