一种肺炎链球菌重组蛋白PsaA-PspA23原核表达载体的构建及表达  

Construction and expression of a prokaryotic expression vector for recombinant protein PsaA-PspA23 of Streptococcus pneumoniae

作　　者：甘忠桥 孟祥玉 李博 陈晓瑞 张悦 袁剑文 梁丹虹 吴永革 谷铁军 GAN Zhong-qiao;MENG Xiang-yu;LI Bo;CHEN Xiao-rui;ZHANG Yue;YUAN Jian-wen;LIANG Dan-hong;WU Yong-ge;GU Tie-jun(National Engineering Laboratory for AIDS Vaccine,Changchun 130012,Jilin Province,China)

机构地区：[1]吉林大学艾滋病疫苗国家工程实验室,吉林长春130012

出　　处：《中国生物制品学杂志》2018年第9期955-959,964,共6页Chinese Journal of Biologicals

摘　　要：目的优化肺炎疫苗重组蛋白基因Psa A-Psp A23的同源区序列,阻止该基因的组氨酸标签(His-tag)融合表达,对突变后的目的基因进行表达及鉴定。方法以肺炎疫苗重组蛋白基因Psa A-Psp A23为模板,设计2对具有重叠区的特异性引物,并分别对该重组基因进行扩增,得到Psa A-Psp A2和Psp A3的CDR区(亚类决定区)基因片段。利用重叠延伸PCR(Overlap PCR)技术,将Psa A-Psp A2基因片段和Psp A3的CDR区基因片段连接,得到同源区优化后的基因片段。利用PCR技术扩增同源区优化后的基因片段,使该基因3′末端引入终止密码子,阻止Psa APsp A23基因表达载体上His-tag的融合表达。将得到的片段同源区优化且无His-tag融合表达的目的基因连接至p ET20b载体后,转入大肠埃希菌(E.coli)中进行表达,表达产物进行Western blot分析。结果重组表达质粒p ET20b-Psa A-Psp A23经双酶切和测序鉴定,证明构建正确;转化E.coli中,37℃,IPTG终浓度1 mmol/L诱导5 h,获得可溶性表达的重组蛋白,且无His-tag。结论 Psa A-Psp A23蛋白基因同源区优化及His-tag成功去除,并在原核表达系统E.coli中可溶性表达,可为Psa A-Psp A23蛋白疫苗的进一步研究奠定基础。Objective To optimize the homologous region sequence of recombinant protein Psa A-Psp A23 gene of pneumonia vaccine,prevent the histidine fusion tag expression of the gene,and express and identify the target gene after mutation. Methods Two pairs of specific primers with overlapping regions were designed using the recombinant protein Psa A-Psp A23 gene of pneumonia vaccine as a template,with which the Psa A-Psp A2 gene fragment and the CDR regions（subclass-determining region）of Psp A3 gene fragment were amplified,and linked by Overlapp PCR technique to obtain an optimized homologous gene fragment. Stop codon was introduced into the 3′ end of the gene to prevent the expression of histidine fusion tag on expression vector for Psa A-Psp A23 gene. The target gene with homologous region optimized and without histidine fusion tag expression was inserted into p ET20 b vector and transferred into E. coli for expression,and the expressed protein was analyzed by Western blot. Results Restrictin analysis and sequencing proved that recombinant plasmid p ET20 b-Psa A-Psp A23 was constructed correctly. Soluble recombinant protein without histidine fusion tag was expressed in E. coli after induction with 1 mmol/L IPTG at 37 ℃ for 5 h. Conclusion Psa A-Psp A23 protein gene with homologous region optimized and histidine tag deleted was expressed in a soluble form in E. coli,which laid a foundation of further study on Psa A-Psp A23 protein vaccine.

关 键 词：组氨酸标签 肺炎链球菌表面蛋白A 同源区序列 重叠延伸PCR 

分 类 号：R378.14[医药卫生—病原生物学] Q789[医药卫生—基础医学]