肠炎沙门菌SipA多克隆抗体的制备及非编码小RNA RyhB-1/2对其表达调控的分析

doi:10.16431/j.cnki.1671-7236.2021.11.026

中国畜牧兽医 ›› 2021, Vol. 48 ›› Issue (11): 4162-4169.doi: 10.16431/j.cnki.1671-7236.2021.11.026

肠炎沙门菌SipA多克隆抗体的制备及非编码小RNA RyhB-1/2对其表达调控的分析

孟霞^1,2, 何梦萍^1,2, 韦骅栩^1,2, 张珂^1,2, 王亨^1,2, 朱国强^1,2

1. 扬州大学兽医学院, 扬州 225009;
2. 江苏高校动物重要疫病与人兽共患病防控协同创新中心, 扬州 225009

收稿日期:2021-03-25 出版日期:2021-11-20 发布日期:2021-11-01
通讯作者: 孟霞 E-mail:mengxia_1@126.com
作者简介:孟霞(1980-),女,山东泰安人,博士,副教授,研究方向:病原菌致病机理,E-mail:mengxia_1@126.com;何梦萍(1996-),女,河南信阳人,硕士生,研究方向:病原菌致病机理,E-mail:MX120190738@yzu.edu.cn
基金资助:
国家自然科学基金（31972651、31101826）；江苏省高校自然科学研究项目（14KJB230002）；兽医生物技术国家重点实验室开放课题（SKLVBF201509）；江苏高校优势学科建设工程资助项目（PAPD）；扬州市自然科学基金青年科技人才项目（YZ2014019）

Preparation of Polyclonal Antibody of SipA in Salmonella Enteritidis and Analysis of Its Expression Regulation by Non-coding Small RNA RyhB-1/2

MENG Xia^1,2, HE Mengping^1,2, WEI Huaxu^1,2, ZHANG Ke^1,2, WANG Heng^1,2, ZHU Guoqiang^1,2

1. College of Veterinary Medicine, Yangzhou University, Yangzhou 225009, China;
2. Jiangsu Co-innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses, Yangzhou 225009, China

Received:2021-03-25 Online:2021-11-20 Published:2021-11-01

摘要/Abstract

摘要： 本研究旨在通过pET原核表达系统表达沙门菌毒力蛋白SipA，制备抗SipA蛋白的多克隆抗体，以分析肠炎沙门菌SE50336非编码小RNA RyhB-1、RyhB-2对SipA的调控作用。利用pET原核表达系统构建pET28a-sipA重组质粒，转化大肠杆菌BL21（DE3）感受态细胞，优化并获得最佳蛋白诱导条件后对蛋白进行诱导表达；通过10% SDS-PAGE分析重组蛋白表达形式；利用镍柱亲和层析法纯化重组蛋白；将纯化的SipA蛋白与弗式佐剂混合免疫BALB/c小鼠，获得SipA蛋白的特异性多克隆抗体，分析制备血清的特异性；利用Western blotting技术检测野生株SE50336、RyhB单缺失株SE50336ΔryhB-1、SE50336ΔryhB-2，以及RyhB双缺失株SE50336ΔryhB-1ΔryhB-2中SipA蛋白的表达水平，分析RyhB-1和RyhB-2对SipA蛋白的调控作用。结果显示，试验成功构建了重组质粒pET28a-sipA，转化大肠杆菌BL21（DE3）感受态细胞后成功表达SipA蛋白；SDS-PAGE结果显示，重组蛋白主要以包涵体形式表达，大小为75 ku；经亲和层析纯化后，SipA蛋白纯度较高；Western blotting结果显示，制备的多克隆抗体可特异性检测肠炎沙门菌SipA重组蛋白；与野生株相比，RyhB单缺失株SE50336ΔryhB-1和SE50336ΔryhB-2中SipA蛋白表达量下降，双缺失株下降则更明显，表明RyhB-1和RyhB-2均可上调SipA蛋白的表达。SipA重组蛋白的纯化及SipA多克隆抗体的制备为进一步研究RyhB-1和RyhB-2对SipA蛋白的调控作用机制奠定基础。

关键词: 肠炎沙门菌; SipA; RyhB; 原核表达; 多克隆抗体

Abstract: The purpose of this study was to express Salmonella virulence protein SipA through pET prokaryotic expression system and prepare polyclonal antibodies against SipA protein, so as to analyze the regulatory effect of noncoding small RNAs RyhB-1 and RyhB-2 of Salmonella Enteritidis SE50336 on SipA. pET28a-sipA recombinant plasmid was constructed by pET prokaryotic expression system and transformed into E. coli BL21(DE3) competent cells. After optimizing and obtaining the best protein induction conditions, the protein was induced and expressed. The expression of recombinant protein was analyzed by 10% SDS-PAGE. The recombinant protein was purified by nickel affinity chromatography. BALB/c mice were immunized with purified SipA protein and Freund adjuvant to obtain specific polyclonal antibody against SipA protein, and the specificity of the prepared serum was analyzed. Western blotting technique was used to detect wild strain SE50336 and RyhB single deletion strains SE50336ΔryhB-1, SE50336ΔryhB-2 and RyhB double deletion strain SE50336ΔryhB-1ΔryhB-2 so as to analyze the regulatory effects of RyhB-1 and RyhB-2 on SipA protein. The results showed that the recombinant plasmid pET28a-SipA was successfully constructed and transformed into E. coli BL21(DE3) competent cells. SDS-PAGE showed that the recombinant protein was mainly expressed in the form of inclusion body, with a size of 75 ku. After purification by affinity chromatography, the purity of SipA protein was high. The results of Western blotting showed that the polyclonal antibody could specifically detect the SipA recombinant protein of Salmonella Enteritidis. Compared with wild strain, the expression of SipA protein in RyhB single deletion strain SE50336ΔryhB-1 and SE50336ΔryhB-2 decreased, especially in double deletion strains, indicating that RyhB-1 and RyhB-2 could upregulate the expression of SipA protein. The purification of SipA recombinant protein and the preparation of SipA polyclonal antibody laid a foundation for further study on the regulatory mechanism of RyhB-1 and RyhB-2 on SipA protein.

Key words: Salmonella Enteritidis; SipA; RyhB; prokaryotic expression; polyclonal antibodies

中图分类号:

S852.61⁺2

孟霞, 何梦萍, 韦骅栩, 张珂, 王亨, 朱国强. 肠炎沙门菌SipA多克隆抗体的制备及非编码小RNA RyhB-1/2对其表达调控的分析[J]. 中国畜牧兽医, 2021, 48(11): 4162-4169.

MENG Xia, HE Mengping, WEI Huaxu, ZHANG Ke, WANG Heng, ZHU Guoqiang. Preparation of Polyclonal Antibody of SipA in Salmonella Enteritidis and Analysis of Its Expression Regulation by Non-coding Small RNA RyhB-1/2[J]. China Animal Husbandry and Veterinary Medicine, 2021, 48(11): 4162-4169.

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肠炎沙门菌SipA多克隆抗体的制备及非编码小RNA RyhB-1/2对其表达调控的分析

Preparation of Polyclonal Antibody of SipA in Salmonella Enteritidis and Analysis of Its Expression Regulation by Non-coding Small RNA RyhB-1/2

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