结核分枝杆菌DdlA多克隆抗体的制备与鉴定  

Preparation and identification of a polyclonal antibody against DdlA from Mycobacterium tuberculosis

作　　者：彭冰洁 孙雪滢 齐明花 李盛[1] 徐跃飞[1] 丁文勇[1] 张文利[1] PENG Bing-jie;SUN Xue-ying;QI Ming-hua;LI Sheng;XU Yue-fei;DING Wen-yong;ZHANG Wen-li(Department of Biochemistry,Dalian Medical University,Dalian,Liaoning 116044,China)

机构地区：[1]大连医科大学基础医学院,辽宁大连116044

出　　处：《中国病原生物学杂志》2020年第7期749-754,共6页Journal of Pathogen Biology

基　　金：国家自然科学基金项目(No.81272429);辽宁省自然科学基金(No.2019-MS-088)。

摘　　要：目的制备并鉴定参与结核分枝杆菌(Mycobacterium tuberculosis,MTB)细胞壁中肽聚糖合成与成熟的D-丙氨酰-D-丙氨酸连接酶(D-Alanyl-D-Alanine ligase,DdlA)多克隆抗体,并检测其效价。方法在NCBT数据中检索获取Rv2981c基因(ddlA)序列信息并构建其表达载体pColdⅡ-Tb-ddlA,在大肠埃希菌表达体系中用异丙基-β-D-硫代半乳糖苷(Isopropyl-beta-D-thiogalactopyranoside,IPTG)诱导DdlA蛋白表达,以琼脂糖树脂(Ni-NTA)亲和层析纯化后免疫BALB/c雌鼠,制备抗DdlA多克隆抗体。通过ELISA检测其效价,Western blot分析抗体特异性。结果成功获得可溶性表达分子质量约为40 ku的DdlA蛋白,免疫小鼠后制备的抗DdlA多克隆抗体ELISA效价为1∶2000;Western blot检测该抗体能识别分枝杆菌内源性的DdlA蛋白。结论成功制备了抗DdlA蛋白多克隆抗体。该抗体具有特异性且效价高,为研究其在结核分枝杆菌致病中的作用和筛选靶向DdlA抑制剂奠定了基础。Objective To prepare and identify a polyclonal antibody against the D-alanyl-D-alanine ligase A(DdlA)protein,which is involved in synthesis and maturation of peptidoglycan in the cell wall of Mycobacterium tuberculosis.Methods The sequence of the Rv2981 c gene(ddlA)was obtained from the NCBI website.The ddlA gene was amplified using high-fidelity PCR and ligated with pColdII to construct the expression plasmid pColdⅡ-Tb-ddlA,which was introduced into E.coli BL21(DE3)to express the fusion protein DdlA.The expressed DdlA was purified using Ni-NTA affinity chromatography followed by preparation of a polyclonal antibody against DdlA by immunizing BALB/c female mice.Antiserum was obtained by collecting blood from the eyeballs of the immunized mice.The titer of the polyclonal antibody was detected using ELISA,and its specificity was identified using Western blotting.Results The soluble fusion protein DdlA was obtained in E.coli using pColdⅡ-Tb-ddlA,which was constructed as an expression vector.Then,the polyclonal antibody against DdlA was prepared using the expressed DdlA as an antigen.The ELISA assay indicated that the titer of the prepared polyclonal antibody was 1:2000 and Western blotting indicated that the prepared antibody was able to recognize endogenous DdlA from Mycobacterium.Conclusion A polyclonal antibody against DdlA has been successfully prepared in this study,and the prepared antibody displays a high level of specificity and a high titer against DdlA from Mycobacterium.Thus,this study has provided a foundation for the study of the role of DdlA in the pathogenesis of Mycobacterium tuberculosis and the screening of inhibitors targeting ddlA.

关 键 词：结核分枝杆菌 D-丙氨酰-D-丙氨酸连接酶 原核表达 多克隆抗体 

分 类 号：R378.911[医药卫生—病原生物学]