摘要
为了探索PPE13蛋白在牛分枝杆菌感染宿主细胞中所发挥的具体调控作用,本研究利用分子克隆技术构建了表达PPE13蛋白的耻垢分枝杆菌重组菌株Ms_PPE13。首先采用PCR技术扩增mb0902c（PPE13）基因片段,将其克隆于载体pMN437中。挑取阳性克隆测序后,将重组质粒电转至耻垢分枝杆菌中,通过Western-blot方法检测PPE13蛋白是否在耻垢分枝杆菌中表达。然后测定重组菌株Ms_PPE13的体外生长曲线、感染该菌的巨噬细胞的死亡情况以及使用荧光定量PCR方法检测巨噬细胞内炎症相关因子IL-6、TNF-α和i NOS的表达情况,以初步探索PPE13的功能。结果显示,PPE13蛋白可在耻垢分枝杆菌中表达,并且PPE13的表达不影响耻垢分枝杆菌在体外的生长。另外,使用耻垢分枝杆菌感染巨噬细胞后,发现PPE13可促进巨噬细胞的死亡,并且促进IL-6的表达。上述研究结果为进一步探索PPE13蛋白调控牛分枝杆菌感染的具体机制提供了新的线索。
To investigate the role of PPE13 in Mycobacterium bovis infecting host cells,the recombinant Mycobacterium smegmatis expressing PPE13 was constructed by molecular cloning technology in the present study.Firstly,the mb0902 c gene encoding PPE13 was amplified by PCR and inserted into the vector p MN437.The positive colonies were screened and sequenced to obtain the correct recombinant vector,and then which was transformed into Mycobacterium smegmatis.Western-blot was used to examine if PPE13 could express in Mycobacterium smegmatis.To investigate the function of PPE13,the growth curve of recombinant Mycobacterium smegmatis named Ms_PPE13 and the death of macrophages infected with Ms_PPE13 were tested,and the expression of IL-6,TNF-α and i NOS were examined by the quantitative real-time PCR.In result,PPE13 could express in Mycobacterium smegmatis and does not influence the growth of bacteria in vivo.In addition,PPE13 enhanced the death of macrophages infected with Mycobacterium smegmatis and the expression of IL-6.The above-mentioned results provide a new clue for the investigation of the pathogenicity of PPE13.
作者
何萍
徐翩翩
杨杨
宋厚辉
HE Ping;XU Pian-pian;YANG Yang;SONG Hou-hui(College of Animal Science and Technology,Zhejiang A ＆F University,Lin＇an 311300,China)
出处
《中国兽医科学》
CAS
CSCD
北大核心
2018年第12期1548-1553,共6页
Chinese Veterinary Science
基金
国家自然科学基金资助项目(31502034)
浙江省自然科学基金资助项目(LQ15C180002)
浙江农林大学校科研发展基金人才启动项目(2014FR069)
