摘要
目的原核表达大肠埃希菌不耐热肠毒素B亚基(heat-labile enterotoxin B subunit,LTB),并制备其多克隆抗体。方法利用PCR技术扩增LTB基因,并将其连接至pET30a(+)原核表达载体,构建重组原核表达质粒pET30a(+)-LTB-8×His,转化大肠埃希菌BL21(DE3),IPTG诱导表达,并对目的蛋白表达条件进行优化。利用His镍柱亲和层析法纯化目的蛋白,免疫大耳白家兔制备兔源抗LTB多克隆抗体。结果重组原核表达质粒pET30a(+)-LTB-8×His经双酶切及测序鉴定证明构建正确;表达的重组LTB蛋白相对分子质量约为13000,以包涵体形式表达,最佳诱导条件为:用0.4 mmol/L IPTG于30℃诱导4 h;纯化后蛋白浓度为3.7 mg/m L;制备的多克隆抗体可用于LTB的Western blot及IFA检测。结论利用大肠埃希菌表达系统成功表达了重组LTB蛋白,并制备了具有良好反应原性和特异性的多克隆抗体,为产肠毒素型大肠埃希菌(enterotoxigenic Escherichia coli,ETEC)疾病的诊断及LTB黏膜免疫佐剂特性的研究奠定了基础。
Objective To express the heat-labile enterotoxin B(LTB)subunit of Escherichia coli in prokaryotic cells and prepare its polyclonal antibody.Methods LTB gene was amplified by PCR and inserted into prokaryotic expression vector pET30a(+).The constructed recombinant plasmid pET30a(+)-LTB-8×His was transformed to E.coli BL21(DE3)and induced by IPTG.The condition for expression of the target protein was optimized.The target protein was purified by His nickel ion affinity chromatography,with which white rabbits were immunized to prepare polyclonal antibody against LTB.Results Both restriction analysis and sequencing proved that recombinant plasmid pET30a(+)-LTB-8×His was constructed correctly.The expressed recombinant LTB protein,with a relative molecular mass of about13000,existed in a form of inclusion body.The optimal condition for induction was treatment with 0.4 mmol/L IPTG at30℃for 4 h.The concentration of purified protein was 3.7 mg/mL.The prepared polyclonal antibody could be used for Western blot and IFA of LTB.Conclusion Recombinant LTB protein was successfully expressed by using E.coli expression system,and polyclonal antibody with good reactogenicity and specificity was prepared,which laid a foundation of diagnosis of enterotoxigenic Escherichia coli(ETEC)diseases and study on the properties of LTB mucosal immune adjuvant.
作者
龚志远
张梦瑶
金宏丽
黄培
张海丽
李媛媛
王化磊
GONG Zhi-yuan;ZHANG Meng-yao;JIN Hong-li;HUANG Pei;ZHANG Hai-li;LI Yuan-yuan;WANG Hua-lei(College of Veterinary Medicine,Key Laboratory of Zoonosis Research,Ministry of Education,Jilin University,Changchun 130062,Jilin Province,China)
出处
《中国生物制品学杂志》
CAS
CSCD
北大核心
2022年第4期407-412,417,共7页
Chinese Journal of Biologicals
基金
国家重点研发计划(2021YFD1801404)。
作者简介
通信作者:王化磊,E-mail:wanghualei@jlu.edu.cn。
