摘要
目的构建携带天花粉蛋白基因的高效表达载体,对比原核表达的重组天花粉蛋白和天然蛋白的免疫原性。方法通过RT-PCR技术扩增无N端和C端序列的TCS基因,测序鉴定后克隆入表达载体pET-29b中,IPTG诱导表达。用金属螯合层析法纯化重组蛋白。薄层扫描及Bradford法检测纯化后重组蛋白的纯度和含量。间接ELISA法检测重组蛋白和天然蛋白产生的多克隆抗体效价。Western blotting检测重组蛋白与抗天然蛋白抗体之间的免疫反应性。结果1.双酶切鉴定结果表明,pET-29b-TCS成功构建;2.30℃,1 mmol/L的IPTG诱导4 h,重组蛋白的表达量最高(占细菌总蛋白量的36%),主要以可溶性非包涵体形式存在于胞浆中;3.His-tag特异性结合树脂纯化后,获得了纯度达95%的重组蛋白,纯化后的蛋白含量为1.2 g/L;4.间接ELISA法检测天然蛋白和重组蛋白产生的抗体效价分别为1∶4 960和1∶5 120;5.Western blotting测纯化的重组蛋白与抗天然蛋白的多克隆抗体之间有特异反应性。结论本实验成功构建了高效表达载体pET-29b-TCS,携带的天花粉蛋白基因无N端和C端编码序列。重组蛋白的抗体效价显著低于天然蛋白,表明天然蛋白的糖基化与其免疫原性之间有一定的关系。
Objective The TCS gene without N-and C-terminal sequences was cloned into pET-29b in order to get the recombinant trichosanthin （rTCS） at the relatively high level in E. coli, and the rTCS immunogenicity was compared with natural TCS （nTCS）. Methods TCS gene without the N-and C-terminal coding sequences was amplified by RT-PCR, and then inserted into pET-29b. The soluble rTCS was induced with IPTG and purified through metal chelate affinity chromatography, the purity and content were showed by the thin-layer scan analysis and bradford assay. After the rabbits were immunized with purified rTCS and nTCS, respectively, the titer and specificity of polyclonal antibody were detected by indirect ELISA assay and Western blotting. Results 1. The vector pET-29b-TCS was constructed successfully after it was confirmed with the same restriction enzymes. 2. The rTCS attained the maximum in the presence of 1mmol/L IPTG for4 h at 30℃ . 3 .The soluble rTCS was expressed at relatively high level （36% of total protein in E. coli） and the yield of rTCS with a purity of 95% was 1.2 g in 1 L bacterium. 4. The polyclonal antisera titer of nTCS was significantly higher than that of rTCS （1：40 960 and 1：5 120 respectively）. 5. The specific reaction between rTCS and anti-nTCS antibody was detected by Western blotting. Conclusion The high level expressing vector pET-29b-TCS, carrying TCS gene withoug the N-and C-terminal coding sequences, was constructed successfully. The immunogenieity of rTCS was remarkably lower than that of nTCS, which indicates that glyeosylation of nTCS has a relationship with the antigenieity.
出处
《解剖学报》
CAS
CSCD
北大核心
2006年第2期172-176,共5页
Acta Anatomica Sinica
基金
浙江省科技厅资助项目(2003C30057)
浙江省教育厅资助项目(20040226)
浙江省中医药管理局科研基金资助项目
