弓形虫截短SAG2基因的克隆表达与免疫活性分析

doi:10.11843/j.issn.0366-6964.2015.12.018

畜牧兽医学报 ›› 2015, Vol. 46 ›› Issue (12): 2258-2263.doi: 10.11843/j.issn.0366-6964.2015.12.018

弓形虫截短SAG2基因的克隆表达与免疫活性分析

赵东岳^*，陈金锋，韦剑辉

（福建师范大学南方生物医学研究中心，福州 350117）

收稿日期:2014-10-11 出版日期:2015-12-23 发布日期:2015-12-23
通讯作者: 赵东岳，Tel：0591-22868832，E-mail：mountainous@163.com
作者简介:赵东岳（1985-），山东高青人，硕士，实验师，主要从事动物病原与分子生物学研究，E-mail:296158826@qq.com
基金资助:
国家卫生和计划生育委员会科研基金（WKJ-FJ-28）

Prokaryotic Soluble Expression of Truncated SAG2 Protein of Toxoplasma gondii and Its Antigenic Activity

ZHAO Dong-yue^*，CHEN Jin-feng，WEI Jian-hui

（Southern Biomedical Research Center at Fujian Normal University，Fuzhou 350117，China）

Received:2014-10-11 Online:2015-12-23 Published:2015-12-23

摘要/Abstract

摘要：

构建截短的弓形虫表面抗原2（SAG2）原核表达系统，并探讨其抗原活性。PCR扩增去掉信号肽和C-末端的SAG2基因片段，插入原核表达载体pGEX-4T-3后转化到大肠杆菌DH5α，并用IPTG诱导。SDS-PAGE和Western blot鉴定重组SAG2t的表达及其免疫反应原性。每升菌液约纯化出4 mg rSAG2t蛋白；Western blot显示，ME49株感染的鼠血清和rSAG2t免疫的鼠血清均可强烈识别表达的截短SAG2蛋白。原核表达体系实现了截短的SAG2蛋白的可溶性表达，并具有良好的完全抗原活性。

Abstract:

To express SAG2 gene of Toxoplasma gondii by prokaryotic expression system and to identify its antigenicity，truncated SAG2 without the highly hydrophobic signal peptide and C-terminus were amplified by PCR，and recombinant prokaryotic expression plasmid (pGEX-4T-3-SAG2t) with SAG2t protein gene was constructed.Then the recombinant plasmids were transferred into E.coli DH5α，and the bacteria were induced by IPTG.The expression and immune-reactivity of SAG2t protein were detected by SDS-PAGE and Western blotting respectively.The yield of purified rSAG2t was more than 4 mg per liter of culture medium.Western blot showed that rSAG2t can be strongly recognized by mice serum infected with T.gondii ME49 strain and mice serum immunized by rSAG2t .The result showed that the soluble expression of SAG2t protein with favorable complete antigenic activity was implemented by prokaryotic expression system.

中图分类号:

S852.729

赵东岳，陈金锋，韦剑辉. 弓形虫截短SAG2基因的克隆表达与免疫活性分析[J]. 畜牧兽医学报, 2015, 46(12): 2258-2263.

ZHAO Dong-yue，CHEN Jin-feng，WEI Jian-hui. Prokaryotic Soluble Expression of Truncated SAG2 Protein of Toxoplasma gondii and Its Antigenic Activity[J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2015, 46(12): 2258-2263.

0
/ / 推荐

导出引用管理器 EndNote|Reference Manager|ProCite|BibTeX|RefWorks

链接本文: https://www.xmsyxb.com/CN/10.11843/j.issn.0366-6964.2015.12.018

https://www.xmsyxb.com/CN/Y2015/V46/I12/2258

参考文献

［1］GUTIERREZ J，O’DONOVAN J，WILLIAMS E，et al.Detection and quantification of Toxoplasma gondii in ovine maternal and foetal tissues from experimentally infected pregnant ewes using real-time PCR［J］.Vet Parasitol，2010，172(1-2)：8-15.
［2］PUTIGNANI L，MANCINELLI L，DEL CHIERICO F，et al.Investigation of Toxoplasma gondii presence in farmed shellfish by nested-PCR and real-time PCR fluorescent amplicon generation assay (FLAG)［J］.Exp Parasitol，2011，127(2)：409-417.
［3］LYONS R E，LYONS K，MCLEOD R，et al.Construction and validation of a polycompetitor construct (SWITCH) for use in competitive RT-PCR to assess tachyzoite-bradyzoite interconversion in Toxoplasma gondii［J］.Parasitology，2001，123(5)：433-439.
［4］YANG C D，CHANG G N，CHAO D.Protective immunity against Toxoplasma gondii in mice induced by a chimeric protein rSAG1/2［J］. Parasitol Res，2004，92(1)：58-64.
［5］MACHADO A V，CAETANO B C，BARBOSA R P，et al.Prime and boost immunization with influenza and adenovirus encoding the Toxoplasma gondii surface antigen 2 (SAG2) induces strong protective immunity［J］.Vaccine，2010，28(18)：3247-3256.
［6］TOMAVO S，MARTINAGE A，DUBREMETZ J F.Phosphorylation of Toxoplasma gondii major surface antigens［J］. Parasitol Res，1992，78(7)：541-544.
［7］龙彩虹，吴少庭，翁亚彪，等.弓形虫表面抗原 SAG2 基因片段的克隆与原核表达［J］.中国人兽共患病杂志，2003，19（2）：42-45.
LONG C H，WU S T，WENG Y B，et al.Cloning and prokaryotic expression of SAG2 gene of Toxoplasma gondii ［J］. Chinese Journal of Zoonoses，2003，19(2)：42-45.(in Chinese)
［8］雷明军，吴少庭，戴五星，等.弓形虫 RH 株膜表面抗原 2 全长基因的高效表达与抗原性分析［J］.中国寄生虫学与寄生虫病杂志，2004，22（4）：231-234.
LEI M J，WU S T，DAI W X，et al.High efficiency expression and antigenicity analysis of the SAG2 gene from Toxoplasma gondii RH strain［J］. Chinese Journal of Parasitology and Parasitic Diseases，2004，22(4)：231-234.(in Chinese)
［9］黄燕，徐梅倩，崔立，等.刚地弓形虫抗原基因 SAG2 的克隆、表达和纯化［J］.中国兽医寄生虫病，2007，15(6)：8-12.
HUANG Y，XU M Q，CUI L，et al.Cloning，expression and purification of SAG2 gene of Toxoplasma gondii ［J］.Chinese Journal of Veterinary Parasitology，2007，15(6)：8-12.(in Chinese)
［10］聂福旭，蒋蔚，王权，等.弓形虫膜表面抗原 SAG2 蛋白的高效表达及免疫活性分析［J］.生物技术通报，2010（4）：173-178.
NIE F X，JIANG W，WANG Q，et al.Expression and immunological activity analysis on the surface protein SAG2 from Toxoplasma gondii strain in RH［J］.Biotechnology Bulletin，2010（4）：173-178.(in Chinese)
［11］LI S，GALVAN G，ARAUJO F G，et al.Serodiagnosis of recently acquired Toxoplasma gondii infection using an enzyme-linked immunosorbent assay with a combination of recombinant antigens［J］.Clin Diagn Lab Immunol，2000，7(5)：781-787.
［12］LAU Y L，SHAMILAH H，FONG M Y.Characterisation of a truncated Toxoplasma gondii surface antigen 2 (SAG2) secreted by the methylotrophic yeast Pichia pastoris［J］.Trop Biomed，2006，23(2)：186-193.
［13］LAU Y L，FONG M Y.Toxoplasma gondii：serological characterization and immunogenicity of recombinant surface antigen 2 (SAG2) expressed in the yeast Pichia pastoris［J］.Exp Parasitol，2008，119(3)：373-378.

[1]	郑晓楠, 李婷婷, 王金磊, 郑文斌, 朱兴全. 弓形虫致密颗粒蛋白的生物学功能研究进展[J]. 畜牧兽医学报, 2022, 53(10): 3345-3357.
[2]	王沛, 王萌, 李婷婷, 郑晓楠, 梁勤立, 陈小庆. 弓形虫4个假定蛋白基因缺失株的构建及其基本生物功能学研究[J]. 畜牧兽医学报, 2022, 53(10): 3598-3608.
[3]	郑雨昕, 张义伟, 姜宁. 恶性疟原虫ApiAP2蛋白质家族研究进展[J]. 畜牧兽医学报, 2022, 53(5): 1354-1363.
[4]	刘林科, 王朋林, 菅忆晨, 李世杰, 王占铭, 王荣军, 张龙现, 宁长申, 菅复春. 我国部分地区羊芽囊原虫流行病学调查[J]. 畜牧兽医学报, 2021, 52(4): 1061-1068.
[5]	高俊莹, 张东超, 李璇, 王华琳, 姜宁. 弓形虫表面抗原SAG1黏附宿主细胞表面硫化肝素的特性研究[J]. 畜牧兽医学报, 2019, 50(9): 1874-1881.
[6]	叶强，王萌，张念章，殷宏，张德林. 弓形虫强免疫原性排泄分泌抗原组分的鉴定[J]. 畜牧兽医学报, 2015, 46(11): 2063-2068.

弓形虫截短SAG2基因的克隆表达与免疫活性分析

Prokaryotic Soluble Expression of Truncated SAG2 Protein of Toxoplasma gondii and Its Antigenic Activity

RichHTML

PDF

可视化

摘要/Abstract

引用本文

使用本文

参考文献

相关文章 6

编辑推荐

Metrics

本文评价