日本血吸虫TGF-β受体Ⅰ基因胞外段的克隆与表达  

Cloning and expression of extracelluar fragment of TGF-β receptor I gene of Schistosoma japonicum

作　　者：高志岩[1] 李孜[1] 黎小妍[2] 马长玲[1] 陈姗[1] 黄俊[1] 

机构地区：[1]广州医学院病原生物学与免疫学教研室,广东广州510182 [2]中山大学附属第三医院药剂科,广东广州510630

出　　处：《热带医学杂志》2011年第1期4-7,共4页Journal of Tropical Medicine

基　　金：广东省自然科学基金(093008);国家自然科学基金(30901353)

摘　　要：目的获得日本血吸虫TGF-β受体I胞外段(SjTβRIout)的序列,行原核克隆和表达,并分析其蛋白的免疫学功能。方法应用cDNA末端快速扩增技术(rapid amplification of cDNA ends,RACE)方法获得SjTβRI基因cDNA部分序列,用RT-PCR方法扩增SjTβRI胞外段(SjTβRIout)基因,构建pET28a(+)重组原核表达载体,并测序鉴定。将质粒转入大肠埃希菌,IPTG诱导表达,并使用Ni离子亲和层析并透析处理获得纯化重组蛋白。使用纯化蛋白与弗氏佐剂共同免疫SD大鼠获得抗血清,使用ELISA方法检测血清中SjTβRIout特异抗体IgG含量。结果成功构建SjTβRIout原核表达载体。测序鉴定显示SjTβRIout包含399bp,编码133个氨基酸,并且与曼氏血吸虫TGF-β受体I的同源性较高。重组蛋白经SDS-PAGE分析,显示其相对分子质量约为20000,与目的基因编码蛋白的预测分子量相符。经重组蛋白免疫的SD大鼠产生高效价(>1:100000000)的抗原特异性IgG抗体。结论首次获得pET28a(+)-SjTβRIout的重组蛋白,并且证明其具有良好的免疫原性。Objective To clone and express the extracelluar region of TGF-β receptor-I in Schistosoma japonicum（SjTβRIout）,and to identify its immunologic functions.Methods Part of the coding region of the SjTβRI gene was obtained by rapid amplification of cDNA ends（RACE）.The extracelluar fragment of SjTβRI gene was amplified by RT-PCR,cloned into the prokaryotic expression vector pET28a（＋）,and then confirmed by sequencing method.The plasmid was then transfected into E.coli and the expression was induced by IPTG.The recombinant protein was purified using Ni-ion affinity chromatography.SD rats were immunized with the purified protein and Freund＇s adjuvant.ELISA was used to detect the production of the SjTβRIout specific antibody.Results The pET28a（＋）-SjTβRIout recombinant expression plasmid was successfully constructed.Sequence analysis showed that the SjTβRIout gene had 399 bp（encoding 133 amino acids） and the gene was highly homologous with Schistosoma mansoni TGF-β receptor I.SjTβRIout recombinant protein with a molecular weight of 20 kDa was expressed in E.coli.The recombinant protein immunized SD rats produced a high titer（ 1：100 000 000） of antigen-specific IgG antibody.Conclusion The pET28a（＋）-SjTβRIout recombinant protein was proved to be immunogenic.

关 键 词：日本血吸虫 SjTβRI 克隆 基因表达 

分 类 号：R383.24[医药卫生—医学寄生虫学]