猪丹毒丝菌SpaA基因原核表达及表达蛋白质的免疫原性分析

doi:10.11843/j.issn.0366-6964.2017.03.012

畜牧兽医学报 ›› 2017, Vol. 48 ›› Issue (3): 492-500.doi: 10.11843/j.issn.0366-6964.2017.03.012

猪丹毒丝菌SpaA基因原核表达及表达蛋白质的免疫原性分析

姚焱彬¹，陆萍¹，杨志鹏¹，魏建忠¹，孙裴¹，李郁^1,2*

(1.安徽农业大学动物科技学院，合肥 230036；2.安徽省畜禽产业共性技术研究院，合肥 230036)

收稿日期:2016-09-12 出版日期:2017-03-23 发布日期:2017-03-23
通讯作者: 李郁, E-mail: liyouer@163.com
作者简介:姚焱彬(1992-)，男，江苏启东人，硕士生，主要从事动物传染病学研究,Tel：0513-83683368,E-mail：1147481655@qq.com
基金资助:
国家星火计划重点项目(2014GA710002)；安徽省质量工程项目(2013sxzx008)；安徽省生猪产业体系基金

Prokaryotic Expression and Immunogenicity Analysis of SpaA Gene in Erysipelothrix rhusiopathiae

YAO Yan-bin¹，LU Ping¹， YANG Zhi-peng¹， WEI Jian-zhong¹，SUN Pei¹，LI Yu^1,2*

(1. College of Animal Science and Technology, Anhui Agricultural University, Hefei 230036, China； 2. Anhui Institute of Generic Industrial Science and Technology in Livestock and Poultry, Hefei 230036, China)

Received:2016-09-12 Online:2017-03-23 Published:2017-03-23

摘要/Abstract

摘要：

旨在克隆表达猪丹毒丝菌SpaA蛋白，分析其免疫原性，为猪丹毒新型疫苗的研发奠定基础。运用PCR扩增SpaA基因，连接至pGEX-6P-1载体，将阳性重组质粒转化大肠杆菌Rosetta (DE3)，IPTG诱导表达，SDS-PAGE分析表达产物，GST亲和层析纯化融合蛋白质，Western blot鉴定；纯化SpaA蛋白免疫小鼠，间接ELISA检测血清中IgG和Th1、Th2相关细胞因子；不同致病力猪丹毒丝菌攻击免疫小鼠，观察病理组织变化及其免疫保护率。结果显示，猪丹毒丝菌SpaA基因在大肠杆菌中实现表达，获得大小为75 ku的纯化目的蛋白质。该蛋白质能特异性识别猪丹毒丝菌抗血清，可诱导小鼠产生较高水平的特异性抗体IgG，并使TNF-β、IFN-γ、IL-5、IL-10含量显著升高，对攻毒菌的免疫保护率均为100%，病理组织变化与攻毒对照组之间差异明显。可见成功获得的重组SpaA蛋白具有良好的免疫原性，能激发机体的体液免疫和细胞免疫，产生较强的免疫保护力，可以作为研发猪丹毒亚单位疫苗的候选抗原。

Abstract:

The aim of this study was to clone and express the SpaA gene of Erysipelothrix rhusiopathiae, and analyze its immunogenicity, thus provide the basis for the development of a new swine erysipelas vaccine. The SpaA gene was amplified using PCR and then connected to the pGEX-6P-1 vector. The positive recombinant plasmid was transformed into Rosetta (DE3) of E. coli and SpaA protein was expressed by induction of IPTG. The expression product was analyzed using SDS-PAGE. The fusion protein was purified by GST affinity chromatography and identified by western blot. The mice were immunized with purified SpaA protein, and then the IgG antibody and cytokine levels of Th1, Th2 were measured with an indirect ELISA. All immunized mice were challenged by Erysipelothrix rhusiopathiae with different pathogenicity, and the pathological changes and the protective rate were observed. The results indicated that the SpaA gene of Erysipelothrix rhusiopathiae was successfully expressed in E. coli, and the molecular weight of purified target protein was 75 kD, which can specific recognize antiserum of Erysipelothrix rhusiopathiae. SpaA can induce high levels of specific IgG antibody in mice, and the levels of TNF-β, IFN-γ, IL-5, IL-10 were significantly increased. The immune challenge-protection rate was 100%. There were obvious differences between pathological changes in control group and experimental group. The experiment suggests that SpaA recombinant protein has good antigenicity, which can stimulate the humoral and celluar immunity, and produce powerful immune protection. Therefore, it can be used as a candidate antigen for developing subunit vaccine of swine erysipelas.

中图分类号:

S855.12

姚焱彬，陆萍，杨志鹏，魏建忠，孙裴，李郁. 猪丹毒丝菌SpaA基因原核表达及表达蛋白质的免疫原性分析[J]. 畜牧兽医学报, 2017, 48(3): 492-500.

YAO Yan-bin，LU Ping， YANG Zhi-peng， WEI Jian-zhong，SUN Pei，LI Yu. Prokaryotic Expression and Immunogenicity Analysis of SpaA Gene in Erysipelothrix rhusiopathiae[J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2017, 48(3): 492-500.

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猪丹毒丝菌SpaA基因原核表达及表达蛋白质的免疫原性分析

Prokaryotic Expression and Immunogenicity Analysis of SpaA Gene in Erysipelothrix rhusiopathiae

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