[1]张青,黄健,黄美容,等.淋病奈瑟菌NGO2105蛋白Passenger结构域的克隆表达、多克隆抗体制备及定位分析[J].中国皮肤性病学杂志,2020,(03):262-267.[doi:10.13735/j.cjdv.1001-7089.201906114]
　ZHANG Qing,HUANG Jian,HUANG Meirong,et al.Cloning Expression,Polyclonal Antibody Preparation and Localization Analysis of NGO2105 Protein Passenger Domain of Neisseria Gonorrhoeae[J].The Chinese Journal of Dermatovenereology,2020,(03):262-267.[doi:10.13735/j.cjdv.1001-7089.201906114]
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淋病奈瑟菌NGO2105蛋白Passenger结构域的克隆表达、多克隆抗体制备及定位分析
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《中国皮肤性病学杂志》[ISSN:1001-7089/CN:61-1197/R]

卷:
期数:
2020年03期
页码:
262-267
栏目:
论著
出版日期:
2020-03-01

文章信息/Info

Title:
Cloning Expression,Polyclonal Antibody Preparation and Localization Analysis of NGO2105 Protein Passenger Domain of Neisseria Gonorrhoeae
文章编号:
1001-7089（2020)03-0262-06
作者:
张青1; 黄健1; 黄美容2; 张涛1; 陈洁1; 闵迅1
1.遵义医科大学附属医院医学检验科，贵州 遵义563003；2. 遵义医科大学附属医院输血科，贵州 遵义563003
Author(s):
ZHANG Qing1; HUANG Jian1; HUANG Meirong2; ZHANG Tao1; CHEN Jie1; MIN Xun1
(1.Department of Clincal Laboratory,Affiliated Hospital of Zunyi Medical University,Zunyi 563003,China;2.Department of Blood Transfusion,Affiliated Hospital of Zunyi Medical University,Zunyi 563003,China)
关键词:
淋病奈瑟菌; NGO2105蛋白; Passenger结构域; 原核表达; 多克隆抗血清; 定位分析
Keywords:
Neisseria gonorrhoeae; NGO2105 protein; Passenger domain; Prokaryotic expression; Polyclonal antiserum; Localization analysis
分类号:
R 759.2
DOI:
10.13735/j.cjdv.1001-7089.201906114
文献标志码:
A
摘要:
目的拟原核表达淋病奈瑟菌NGO2105蛋白的Passenger结构域并制备多克隆抗血清，初步分析其保守性及亚细胞定位。方法PCR扩增Passenger结构域编码基因并克隆入pCold TF原核表达质粒中，重组质粒转化大肠杆菌E.coil DH5α，通过PCR和测序鉴定后，再将重组质粒转化大肠杆菌BL21（DE3）菌中诱导表达目的蛋白，纯化目的蛋白后免疫BALB/c小鼠以制备多克隆抗体。Western blot分析NGO2105蛋白在临床分离菌株中的保守性。采用流式细胞技术分析淋病奈瑟菌中NGO2105蛋白Passenger结构域细胞定位。结果成功表达出可溶形式的NGO2105蛋白Passenger结构域，重组蛋白免疫小鼠后可获得效价达到5.12×105的多克隆抗血清，Western blot分析显示Passenger抗血清能与不同临床分离菌株中的NGO2105蛋白特异性反应，流式细胞技术分析显示Passenger结构域定位于淋病奈瑟菌菌体表面。结论成功获得可溶形式表达的Passenger蛋白及其多克隆抗体，NGO2105蛋白在临床分离的淋病奈瑟菌中有较好的保守性。亚细胞定位分析提示NGO2105蛋白为一种膜蛋白，其Passenger结构域定位于菌体表面，本研究可为进一步研究淋病奈瑟菌NGO2105蛋白的结构与功能奠定实验基础。
Abstract:
Objective To express the Passenger domain of Neisseria gonorrhoeae(NG) NGO2105 protein in prokaryotic cells and prepare polyclonal antiserum,and analyze its conservatism and subcellular localization. Methods Gene of the Passenger domain was amplified by PCR and cloned into the prokaryotic expression plasmid pCold TF,and then the recombinant plasmid was transformed into E.coil DH5α.After identification by PCR and sequencing,the recombinant plasmid was transformed into E.coil BL21 (DE3) to induce the expression of the target protein.BALB/c mice were immunized with purified target protein to prepare polyclonal antibody.Western blot was used to analyze the conservation of NGO2105 protein in clinical isolates.Flow cytometry was used to analyze the cellular localization of the Passenger domain of NGO2105 protein in Neisseria gonorrhoeae. Results The soluble form of the Passenger domain of NGO2105 protein was successfully expressed.The titer of anti-Passenger polyclonal antibodies was 5.12×105 detected by ELISA.Western blot analysis showed that Passenger antiserum reacted specifically to NGO2105 protein in different clinical isolates.Flow cytometry analysis showed that Passenger domain was located on the surface of Neisseria gonorrhoeae. Conclusion The soluble Passenger protein and its polyclonal antibody has been successfully obtained.NGO2105 protein is conserved in clinical isolates of Neisseria gonorrhoeae.Subcellular localization analysis suggests that NGO2105 protein is a kind of membrane protein,and its Passenger domain is located on the surface of bacteria.This study can lay an experimental foundation for further study on the structure and function of NGO2105 protein in Neisseria gonorrhoeae.

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备注/Memo

备注/Memo:
［基金项目］国家自然科学基金（81760358，31800130）
［通信作者］闵迅，E-mail:2815400619@qq.com
［Corresponding author］MIN Xun,E-mail:2815400619@qq.com
更新日期/Last Update: 2020-03-30