摘要
为探究牛分枝杆菌(M.bovis)PE/PPE家族成员PE16蛋白的生物学功能,本研究以M.bovis基因组DNA为模板扩增PE16基因片段并克隆至pMN437载体中,构建原核表达质粒pMN437-PE16,经PCR和测序鉴定正确后将该重组质粒及对照质粒PMS2电转入耻垢分枝杆菌中构建重组耻垢分枝杆菌MS_PE16及对照重组耻垢分枝杆菌MS_Vec。经western blot鉴定结果显示,重组耻垢分枝杆菌中PE16在55 ku左右正确表达,而MS_Vec则无该特异性条带。对MS_PE16及MS_Vec进行生长曲线测定,并利用倒置显微镜观察MS_PE16及MS_Vec的菌落形态;将培养至对数生长期的MS_PE16及MS_Vec分别以MOI 10感染人白血病单核细胞(THP-1细胞),感染后0(感染4 h后经庆大霉素处理2 h,此时计为0)、24 h、48 h时采用平板计数法检测胞内菌数量;细菌感染后24 h和48 h时收集各组细胞培养上清,利用ELISA试剂盒检测各组细胞IL-6、TNF-α、IL-1β、IFN-β的表达分泌水平。生长曲线测定结果显示,PE16对分枝杆菌的生长无显著影响;菌落形态观察结果显示,MS_PE16与MS_Vec的菌落形态无明显区别;胞内活菌数量检测结果显示,细菌感染后0和24 h,MS_PE16在THP-1细胞中的数量极显著低于MS_Vec(P<0.01),但在感染后48 h,MS_PE16在THP-1细胞中的存活率极显著高于MS_Vec组在THP-1细胞中的存活率(P<0.001);细胞因子检测结果显示,MS_PE16感染组细胞中IFN-β的分泌水平极显著高于MS_Vec组(P<0.01)。上述结果表明PE16对分枝杆菌的生长和菌落形态均无显著影响,但其可提高分枝杆菌在巨噬细胞内的存活率,且可促进巨噬细胞中IFN-β的分泌水平。本研究初步揭示了分枝杆菌PE16蛋白的功能,为M.bovis感染机制的进一步研究提供了实验数据。
To explore the biological function of PE16 protein,a member of PE/PPE family in Mycobacterium bovis(M.bovis),the PE16 gene fragment was amplified from M.bovis and cloned into pMN437 vector to construct the prokaryotic expression plasmid pMN437-PE16.After identification by PCR and sequencing,the recombinant plasmid was electroporated into Mycobacterium smegmatis(M.smegmatis)to construct the recombinant M.smegmatis MS_PE16.The results of western blot showed that PE16 was correctly expressed at about 55ku in MS_PE16.The growth curve of MS_PE16 was plotted and the colony morphology of MS_PE16 was observed under an inverted microscope.THP-1 cells were infected with MS_PE16 in the logarithmic growth phase at MOI 10,and the number of intracellular bacteria was detected at 0(treated with gentamicin for 2 hours after 4 hours infection,counted as 0),24 and 48 hours post infection,and the expression levels of IL-6,TNF-α,IL-1βand IFN-βin THP-1 cells infected with MS_PE16 were detected by ELISA at 24 and 48 hours post infection.The results of growth curve showed that PE16 had no significant effect on the growth of recombinant M.smegmatis.There was no significant difference in colony morphology between MS_PE16 and MS_Vec.The number of MS_PE16 in THP-1 cells was significantly lower than that in MS_Vec group at 0 and 24 hours post infection,however,the survival rate of MS_PE16 in THP-1 cells was significantly higher than that in MS_Vec group at 48 hours post infection(P<0.001).The results of cytokine detection showed that the level of IFN-βin MS_PE16 group was significantly higher than that in MS_Vec group(P<0.01).These results indicated that PE16 overexpressing in M.smegmatis had no significant effect on the growth and colony morphology of the bacteria,which was beneficial to the survival of mycobacteria in macrophages,and could promote the expression level of IFN-βin macrophages.This study preliminarily revealed the function of PE16 protein and provided experimental data for further understanding of the mechanism of M.bovis in the process of infection.
作者
毕斯琪
杨可
徐阿慧
宋厚辉
BI Si-qi;YANG Ke;XU A-hui;SONG Hou-hui(Key Laboratory of Applied Technology on Green-Eco-Healthy Animal Husbandry of Zhejiang Province,Zhejiang Provincial Engineering Research Center for Animal Health Diagnostics&Advanced Technology,Zhejiang International Science and Technology Cooperation Base for Veterinary Medicine and Health Management,China-Australia Joint Laboratory for Animal Health Big Data Analytics,College of Animal Science and Technology&College of Veterinary Medicine of Zhejiang A&F University,Hangzhou 311300,China)
出处
《中国预防兽医学报》
CAS
CSCD
北大核心
2023年第11期1158-1164,共7页
Chinese Journal of Preventive Veterinary Medicine
基金
浙江农林大学学校科研发展基金项目(2021LFR024)。
关键词
牛分枝杆菌
PE16
胞内菌
免疫反应
Mycobacterium bovis
PE16
intracellular bacteria
immune response
