| 罗非鱼无乳链球菌EsxA基因克隆与原核表达 |
| |
| 引用本文： | 马艳平,梁志凌,马江耀,刘振兴,郝乐,柯浩.罗非鱼无乳链球菌EsxA基因克隆与原核表达[J].动物医学进展,2016(4):35-39. |
| |
| 作者姓名： | 马艳平 梁志凌 马江耀 刘振兴 郝乐 柯浩 |
| |
| 作者单位： | 广东省农业科学院动物卫生研究所/广东省兽医公共卫生公共实验室,广东广州,510640 |
| |
| 基金项目： | 广东省海洋渔业局科技与产业发展专项(A201401C06)；广州市农业科技攻关项目(GZCQC1202FG04002-01)；广州市科技攻关项目(2014J4100229) |
| |
| 摘 要： | Ⅶ型分泌系统(T7SS)是近年来发现的分泌系统,分泌两种胞外蛋白,EsxA基因编码的ESAT6蛋白和EsxB基因编码的CFP-10蛋白。分泌蛋白具有良好的免疫原性,促进细菌从巨噬细胞吞噬体逃逸、影响巨噬细胞凋亡及裂解细胞等生物学功能,与致病性密切相关。以罗非鱼源无乳链球菌为模板,克隆到294bp的EsxA基因,将目的序列克隆到pMD18-T载体,经测序,目的序列与无乳链球菌EsxA基因同源率在99%以上。EsxA基因克隆到pET32a载体中,重组载体在28℃、0.5mmol/L IPTG诱导条件下表达量最大,可溶性表达。将纯化的ESAT6蛋白免疫Balb/c鼠,成功制备ESAT6蛋白鼠多克隆抗体,经Western blot,ESAT6蛋白具有良好的反应原性。研究结果为进一步进行无乳链球菌ESAT6蛋白的免疫学功能研究奠定了基础。
|
| 关 键 词： | Ⅶ型分泌系统 无乳链球菌 EsxA 基因 克隆与表达 |
Clonging and Protokaryon Expression of EsxA Gene of Streptococcus agalactiae in Tilapia |
| |
| Abstract: | New discovered type Ⅶ secretion system (T7SS)secrets two extracellular proteins including Es-xA coded ESAT6 and EsxB coded CFP-10,which is related to pathogenicity closely.ESAT6 and CFP-10 proteins have diverse functions such as promoting bacteria from phagosome of macrophage,impacting mac-rophage apoptosis and lysis.The two secreted proteins in T7SS of Streptococcus agalactiae form the ho-mologous dimmers,collectively known as ESAT6 protein,may be bacterial adhesion protein showing im-portant for host-cell attachment and invasion in S.agalactiae through bioinformatics analysis.But ESAT6 protein whether has the virulence and its pathogenesis is not clear,therefore it is necessary to study on it. We cloned 294 bp sequence from tilapia Streptococcus agalactiae .And the sequence was cloned into pMD18-T vector,the homology is 99% with tilapia Streptococcus agalactiae EsxA gene.EsxA gene was cloned into pET32a vector.The best solouble expression was under 0.5 mmol/L IPTG,28℃.In addition, the purified protein was used to immunize Balb/c mice to prepare mouse anti-ESAT6 protein antibody.ES-AT6 protein had sastisfied reactogenicity through Western blot test.These results encouraged further re-search for the immunological function of S.agalactiae ESAT6 protein. |
| |
| Keywords: | type Ⅶ secretion system Streptococcus agalactiae EsxA gene clonging and expression |
| 本文献已被 CNKI 万方数据 等数据库收录！ |
|
