猪链球菌Ide通用截短蛋白原核表达及纯化研究

Prokaryotic Expression and Purification of Truncated Ide General Protein of Streptococcus suis

关键词:  猪链球菌2型  IgM裂解酶  原核表达  SDS-PAGE分析

Key words:  Streptococcus suis 2  Immunoglobulin M-degrading enzyme  prokaryotic expression  SDS-PAGE analysis

摘要:

利用大肠埃希菌表达猪链球菌Ide通用截短蛋白。通过对GenBank公布的IdeS蛋白家族基因同源性分析,利用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BamH I和Hind III双酶切后将目的片段分别克隆到pET28a、pET32a和pET-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1200bp,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pET28a-TrIde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Ide_Ssuis蛋白在猪链球菌通用疫苗研发方面的研究奠定了基础。

Abstract:

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 BamH I and Hind 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 1200bp 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.