猪圆环病毒2型ORF2基因重组植物乳杆菌的构建及其免疫原性分析

doi:10.16431/j.cnki.1671-7236.2017.08.027

《中国畜牧兽医》 ›› 2017, Vol. 44 ›› Issue (8): 2408-2416.doi: 10.16431/j.cnki.1671-7236.2017.08.027

猪圆环病毒2型ORF2基因重组植物乳杆菌的构建及其免疫原性分析

倪能能¹, 王新珂¹, 赖强¹, 焦茂兴², 黄毓茂¹

1. 华南农业大学兽医学院, 广东省动物源性人畜共患病预防与控制重点实验室, 国家生猪种业科学技术研究中心, 广州 510642;
2. 宜春学院, 宜春 336000

收稿日期:2017-02-08 出版日期:2017-08-20 发布日期:2017-08-18
通讯作者: 黄毓茂 E-mail:ymaohuang@scau.edu.cn
作者简介:倪能能(1990-),女,江苏南通人,硕士生,研究方向:预防兽医学,E-mail:1791755789@qq.com
基金资助:
广东省省级科技计划项目（2013B020202002）

Construction and Immunogenicity Analysis of Recombinant Lactobacillus plantarum Expressing ORF2 Gene of Porcine Circovirus Type 2

NI Neng-neng¹, WANG Xin-ke¹, LAI Qiang¹, JIAO Mao-xing², HUANG Yu-mao¹

1. National Engineering Research Center for Breeding Swine Industry, Key Laboratory of Zoonosis Prevention and Control of Guangdong Province, College of Veterinary Medicine, South China Agricultural University, Guangzhou 510642, China;
2. Yichun University, Yichun 336000, China

Received:2017-02-08 Online:2017-08-20 Published:2017-08-18

摘要/Abstract

摘要：

为研制一种能有效控制猪圆环病毒2型（porcine circovirus type 2，PCV2）的新型疫苗，本试验将PCV2的ORF2基因克隆至植物乳杆菌表达载体pSCPSP超强启动子SCP的下游，构建重组表达质粒pSCPSP-Cap，用电击转化法转化克隆至表达宿主菌植物乳杆菌，获得一株重组植物乳杆菌。以SDS-PAGE和Western blotting法检测24 h上清液中表达的蛋白；将8周龄的SPF级BALB/c小鼠随机分组，每组8只，分别设置对照组、空载体组、灌胃组、饮水组、灭活疫苗组，试验14、28、42和56 d后对小鼠采血，分离血清，利用PCV2 ELISA抗体检测试剂盒检测小鼠血清中PCV2的抗体水平。SDS-PAGE、Western blotting结果显示，PCV2衣壳蛋白在植物乳杆菌中成功分泌表达，表达的蛋白分子质量为28 ku，且具有良好的反应原性，重组植物乳杆菌免疫小鼠诱导机体产生PCV2特异性抗体显著高于其他组（P<0.05）。结果表明，PCV2的ORF2基因在植物乳杆菌中成功表达，且具有良好的生物活性，可为PCV2口服疫苗的开发研究提供理论参考。

关键词: 猪圆环病毒2型; Cap蛋白; 植物乳杆菌; 表达; 口服疫苗

Abstract:

To develop an effective vaccine against porcine circovirus type 2 (PCV2), ORF2 gene was cloned into a Lactobacillus plantarum (L.plantarum) pSCPSP under the control of a SCP promoter to construct recombinant plasmid pSCPSP-Cap that was transformed into LP1 by electroporation. Extracellular expression of the Cap protein was confirmed by SDS-PAGE and Western blotting analysis. Afterwards, 8-week-old SPF BALB/c mice were randomly divided into control group, carrier group, lavage group, group of drinking water and inactived vaccine group. After 14,28,42 and 56 d, mice serums were collected to evaluate the level of specific antibody by SDS-PAGE and Western blotting. Eventually, SDS-PAGE and Western blotting results indicated that the Cap protein of PCV2 could be efficiently expressed in L. plantarum, expression of protein molecular weight was 28 ku and with good immunogenicity. In addition, higher levels of specific antibody in the serum of mice was observed by oral administration with L. plantarum expressing the PCV2 Cap antigen (P<0.05). In conclusion, the ORF2 gene of PCV2 could be successfully expressed in recombinant L. plantarum and expressed protein with good biological activity, which would lay the foundation for the development of PCV2 oral vaccination.

Key words: porcine circovirus type 2(PCV2); Cap protein; Lactobacillus plantarum; expression; oral vaccination

中图分类号:

S852.65

倪能能, 王新珂, 赖强, 焦茂兴, 黄毓茂. 猪圆环病毒2型ORF2基因重组植物乳杆菌的构建及其免疫原性分析[J]. 《中国畜牧兽医》, 2017, 44(8): 2408-2416.

NI Neng-neng, WANG Xin-ke, LAI Qiang, JIAO Mao-xing, HUANG Yu-mao. Construction and Immunogenicity Analysis of Recombinant Lactobacillus plantarum Expressing ORF2 Gene of Porcine Circovirus Type 2[J]. , 2017, 44(8): 2408-2416.

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猪圆环病毒2型ORF2基因重组植物乳杆菌的构建及其免疫原性分析

Construction and Immunogenicity Analysis of Recombinant Lactobacillus plantarum Expressing ORF2 Gene of Porcine Circovirus Type 2

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