摘要
为利用大肠埃希菌表达猪链球菌Ide通用截短蛋白,通过对Gen Bank公布的Ide S蛋白家族基因同源性分析,使用Primer 5.0软件设计一对特异性引物,以猪链球菌2型强毒株JZLQ全基因组为模板,采用PCR方法扩增Ide基因截短片段,经Bam HⅠ和HindⅢ双酶切后将目的片段分别克隆到p ET28a、p ET32a和p ET-sumo表达载体上,分别转化宿主菌E.coli BL21(DE3)。通过优化诱导温度及诱导剂IPTG浓度进行表达并对表达产物进行SDS-PAGE分析。结果显示,PCR扩增片段大小约为1200 bp,经双酶切和测序验证构建正确;三种重组表达载体转化大肠埃希菌后均有目的蛋白表达,但不同表达条件下目的蛋白表达量存在差异,其中p ET28a-Tr Ide重组表达载体用25℃诱导、IPTG终浓度为1 mmol/L时可实现重组蛋白的高效可溶性表达,为IdeSsuis蛋白在猪链球菌通用疫苗研发方面的研究奠定了基础。
To express the truncated Immunoglobulin M-degrading enzyme （TrIde） of Streptococcus suis serotype 2, sequence homology of the IdeS family was analyzed and one pair of specific primers was designed using software Primer 5.0. The target sequence fragment was amplified by PCR using the genome DNA of strain JZLQ as a template. The amplified target DNA named TrIde was cut by Bam H Ⅰ and Hin d Ⅲ and then was inserted into vector pET28a、pET32a and pET-sumo, respectively, to generate pET28a-TrIde、pET32a-TrIde and pET-sumo-TrIde recombinant vector. These recombinant vectors were then transformed into E.coli BL21（DE3） for over expression. The E.coli cells containing different recombinant vectors were induced in different temperature conditions with different IPTG concentration for 8 hours, respectively. These specimens were analyzed by SDS-PAGE. Results showed that a fragment about 1200 bp in length was amplified from the genome of JZLQ strain and was confirmed right by double enzyme digestion and sequencing methods. SDS-PAGE analysis indicated that TrIde protein can be expressed by all of these recombinant vector but pET28a-TrIde performed better at 25 ℃ with 1 mmol/L IPTG. This research served as a basis for the study of the vaccine development of S.suis 2.
作者
曲光刚
王长江
李书光
李茂峰
武曰星
金婷婷
韩文瑜
沈志强
QU Guang-gang;WANG Chang-jiang;LI Shu-guang;LI Mao-feng;WU Yue-xing;JIN Ting-ting;HAN Wen-yu;SHEN Zhi-qiang(Post-doctoral Research Center of Shandong Binzhou Animal Science＆ Veterinary Medicine Academy,Binzhou,Shandong,256600,China;Post-doctoral Research Mobile Station of Jilin University,Changchun 130062,China;Shandong Lvdu Biotechnology Co.,Ltd.Binzhou,Shandong 256600,China;Shandong Binzhou Animal Science ＆ Veterinary Medicine Academy,Binzhou,Shandong 256600,China;Yangxin County Comprehensive Inspection and Testing Center,Binzhou,Shandong 256600,China)
出处
《中国兽药杂志》
2018年第6期7-12,共6页
Chinese Journal of Veterinary Drug
