弓形虫多表位基因的构建及其在大肠杆菌系统中的表达鉴定

目的　构建弓形虫和破伤风毒素多个表位的编码基因 ,将其在大肠杆菌系统进行表达 ,评价重组抗原的特异反应性。方法　从弓形虫保护性抗原及通用免疫增强佐剂破伤风毒素中选取含有T和 (或 )B细胞表位的抗原片段 ,通过软件分析在各片段之间插入适当数目、起间隔作用的氨基酸 ,以保持个片段的空间构象独立性 ,从而确定氨基酸顺序。根据氨基酸序列选取适当的密码子构成弓形虫多表位基因。将该基因合成并鉴定后 ,亚克隆入原核表达载体 ,在大肠杆菌中表达并分析表达产物的表达形式和抗原活性。结果　成功构建了长度为 36 0bp的弓形虫多表位基因。该基因在原核表达系统中经诱导 ,得到了 14 4kDa的包涵体形式的表达产物。免疫印迹实验表明该产物具有强特异性抗原活性。结论　弓形虫多表位基因在原核中的表达产物在弓形虫病疫苗及诊断中有潜在的应用价值

Construction of the gene encoding multiepitope of Toxoplasma gondii and its expression in E.coli

Aim To construct the gene encoding multiple epitopes of Toxoplasma gondii antigens and tetanus toxin,express the gene in E coli and evaluate the specific immunoactivity of the recombinant antigen Methods Peptide fragments containing T cell and(or) B cell epitopes were chosed from Toxoplasma gondii and tetanus toxin antigens With the help of relevant protein-analyzing software,appropriate spacer aminos were inserted to maintain the relatively independent conformation of the constituent fragments,such the final amino sequence were determined Proper codons were chosed according to the amino sequence and necessary enzyme sites were considered as well to form the DNA sequence,namely the gene encoding multiple epitopes The gene were synthesized and subcloned into E coli expression system The form and antigenicity of the expression products were analysed Results The 360bp-long-gene encoding multiple epitopes of Toxoplasma gondii and tetanus toxin were successfully constructed In E coli expression system,the product of 14 4kD is obtained It is expressed in the form of inclusion Results of immunolblotting shows strong antigenincity of the product Conclusions The product of the gene encoding multiple epitopes of Toxoplasma gondii and tetanus toxin expressed in E coli is of potential value in prepartion of vaccine and diagnosis kit of toxoplasmosis