摘要
目的制备2型猪链球菌(Streptococcus suis 2)酪氨酸激酶Cps2C重组蛋白及其抗血清,为进一步研究Cps2C的作用奠定基础。方法采用在线工具对cps2C基因进行生物信息学分析。设计cps2C基因引物,以05ZYH33菌株基因组DNA为模板PCR扩增cps2C基因,构建pET32a:cps2C重组质粒;重组质粒转入感受态E.coli BL21后,IPTG诱导表达Cps2C重组蛋白,经Ni2+亲和层析纯化后用His单克隆抗体进行鉴定。用纯化的重组蛋白免疫BALB/c小鼠,获得多克隆抗血清,采用间接ELISA法测定抗体效价。结果成功构建pET32a:cps2C表达质粒,获得阳性E.coli BL21Cps2C重组蛋白表达菌株,SDS-PAGE显示纯化的目的蛋白条带单一,相对分子质量为43×103,与预期一致;Western blot检测Cps2C重组蛋白可以与His单克隆抗体、感染05ZYH33全菌的猪恢复期血清反应。制备的多克隆抗血清效价为1∶819 200,且能与重组蛋白反应。结论原核表达了具有抗原性的Cps2C重组蛋白,制备的鼠源多抗血清能识别Cps2C蛋白,为研究Cps2C蛋白在2型猪链球菌中的作用奠定了基础。
Objective To express the recombinant protein Cps2 Cof Streptococcus suis 2 in a prokaryotic expression system and prepare an antiserum to it. Methods The cps2C gene was analyzed bioinformatically using online tools.A pair of primers specifically targeting the cps2C gene were designed.The cps2C gene was amplified from the 05 ZYH33 strain genomic DNA using the primers and then cloned into the expression vector pET32a.The recombinant plasmid was transformed into E.coli BL21 for overexpression of Cps2C protein.After its expression was induced with IPTG,the recombinant protein was purified using Ni2+NTA affinity chromatography and analyzed using SDS-PAGE.Western blot analysis was performed with anti-His tag monoclonal antibody and convalescent-phase serum from piglets infected with the virulent Streptococcus suis 2 strain.BALB/c mice were immunized with the purified recombinant protein,and a polyclonal antiserum was prepared.The antibody titer was determined using indirect ELISA. Results The expression plasmid pET32 a:cps2C was successfully constructed,and a E.coli BL21 strain expressing recombinant Cps2C protein was obtained.SDS-PAGE indicated that the recombinant Cps2C protein was present in both the supernatant and the precipitate of the bacterial lysate.The purified protein produced a single band with a relative molecular weight of 43×103,which was consistent with the estimated molecular mass of the recombinant Cps2C protein.Western blotting indicated that the recombinant Cps2C protein reacted with anti-His tag monoclonal antibody and convalescent-phase serum from pigs clinically infected with the Streptococcus suis 2 virulent strain 05 ZYH33.The obtained polyclonal antiserum specifically recognized the Cps2C protein at a titer of 1:819,200. Conclusion The recombinant protein Cps2 Cwas successfully expressed in a prokaryotic expression system,and mouse-derived polyclonal antiserum was obtained.This study has laid the foundation for further study of the role of the Cps2C protein in the pathogenicity of Streptococcus suis 2.
作者
张会芳
吴倩倩
王悄悄
王晶
胡丹
郑峰
汪春晖
潘秀珍
曹祥荣
ZHANG Hui-fang;WU Qian-qian;WANG Qiaoqiao;WANG Jing;HU Dan;ZHENG Feng;PAN Xui-zhen;CAO Xiang-rong(College of Life Sciences,Nanjing Normal University,Nanjing,China 210023;Hua Dong Research Institute for Medicine and Biotechnics,Nanjing,China 210002;The Wuxi Maternity and Child Health Care Hospital Affiliated with Nanjing Medical University,Wuxi 214000)
出处
《中国病原生物学杂志》
CSCD
北大核心
2020年第2期136-140,共5页
Journal of Pathogen Biology
基金
国家自然科学基金项目(No.81571965,81701635)
江苏省自然科学基金项目(No.BK20151019)。
关键词
2型猪链球菌
原核表达
酪氨酸激酶
多抗血清
Streptococcus suis 2
prokaryotic expression
Cps2C
polyclonal antiserum
