表达促凋亡蛋白Bid的重组耻垢分枝杆菌的构建及其对巨噬细胞凋亡的影响  

Construction of A Recombinant Mycobacterium Smegmatis Expressing the Pro-apoptotic Protein BID and its Effect on the Apoptosis of Macrophages

作　　者：逄亚宁 杨国平 孟余 吴利先 李光华 PANG Ya-ning;YANG Guo-ping;MENG Yu;WU Li-xian;LI Guang-hua(Department of Medical Microbiology and Immunology,Basic Medical College,Dali University,Dali 671000,Yunnan,China)

机构地区：[1]大理大学基础医学院医学微生物与免疫学教研室,云南大理671000

出　　处：《医学信息》2022年第23期41-49,共9页Journal of Medical Information

基　　金：大理大学博士科研启动费项目(编号:KYBS201710);大理大学临床分子免疫学创新团队(编号:ZKLX2019105)。

摘　　要：目的构建表达并分泌外源性促凋亡蛋白Bid的重组耻垢分枝杆菌,并研究其对小鼠巨噬细胞RAW264.7凋亡的影响。方法运用生物信息技术对小鼠Bid蛋白质的理化性质、分泌属性、保守结构域及蛋白相互作用网络等进行分析,应用无缝克隆技术及传统酶切酶连方式构建pMV261-ASP(BamHI)-10His、pMV261-ASP(BamHI)-Bid-10His、pMV261-ASP(EcoRI)-10His、pMV261-ASP(EcoRI)-Bid-10His重组质粒,并电转化入耻垢分枝杆菌;通过BCA蛋白定量及免疫印迹技术分析蛋白表达情况,采用AnnexinV-FITC/PI双染法流式检测Bid重组耻垢分枝杆菌对RAW264.7小鼠巨噬细胞凋亡的影响。结果小鼠Bid蛋白是一种等电点为4.80的亲水性蛋白质,其中亮氨酸所占比例最高,为11.3%,色氨酸含量最低,为0.5%;二级结构中,α-螺旋(Hh)、无规则卷曲(Cc)、β-折叠(Ee)、β-转角(Tt)所占比例依次为65.13%、27.69%、4.10%、3.08%;Bid蛋白不具有跨膜结构域及信号肽,属于非分泌性蛋白质,具有死亡保守结构域,可与Caspase8、Bcl-2、Granzyme B等凋亡相关蛋白发生相互作用;本次构建的ASP-Bid重组耻垢分枝杆菌可以表达并分泌目标蛋白,且可增强RAW264.7小鼠巨噬细胞凋亡的比例。结论表达外源基因Bid的重组耻垢分枝杆菌可以促进巨噬细胞凋亡发生,这可为新型结核病预防疫苗的研发提供新的思路与技术。Objective To construct the recombinant Mycobacterium Smegmatis expressing and secreting the exogenous pro-apoptotic protein Bid,and to study its effect on the apoptosis of mouse macrophages RAW264.7.Methods Bioinformatics was used to analyse the physicochemical properties,secretory attributes,conserved structural domains and protein interaction networks of Bid protein from mouse.Recombinant plasmids pMV261-ASP(BamHI)-10His,pMV261-ASP(BamHI)-Bid-10His,pMV261-ASP(EcoRI)-10His,pMV261-ASP(EcoRI)-Bid-10His were constructed by applying seamless cloning techniques and conventional enzymatic digestion and enzyme linkage.Then the recombinant plasmids were electrotransferred into Mycobacterium Smegmatis.BCA protein quantification and immunoblotting techniques were applied to analyze protein expression.The effect of recombinant Mycobacterium Smegmatis expressing the Bid gene on apoptosis in RAW264.7 mouse macrophages was examined by AnnexinV-FITC/PI.Results The Bid protein of mouse was a hydrophilic protein with an isoelectric point of 4.80,with the highest proportion of leucine at 11.3%and the lowest content of tryptophan at 0.5%.The proportions ofα-helix(Hh),irregular curl(Cc),β-fold(Ee)andβ-turn(Tt)in the secondary structure were 65.13%,27.69%,4.10%and 3.08%respectively.Bid protein did not possess transmembrane domain or signal peptide,but was a non-secretory protein with death conserved domain,which could interact with Caspase8,Bcl-2,Granzyme B and other apoptosis-related proteins.The constructed recombinant Mycobacterium Smegmatis ASP-Bid expressed and secreted the target protein and enhanced the rate of apoptosis in RAW264.7 mouse macrophages.Conclusion Recombinant Mycobacterium smegmatis expressing foreign gene Bid can promote macrophage apoptosis,which can provide new ideas and techniques for the development of new tuberculosis prevention vaccines.

关 键 词：结核病 耻垢分枝杆菌 信号肽 BID 细胞凋亡 

分 类 号：R378.911[医药卫生—病原生物学]