重组DT/bFGF融合蛋白基因表达载体的构建及表达产物的纯化

Construction of Expression Vector for Recombinant DT/bFGF Fusion Protein and Purification of Expressed Product

目的构建重组DT/bFGF融合蛋白表达载体,制备高纯度的DLF融合毒素。方法PCR扩增DT389和bFGF的编码DNA序列,经酶切和连接,克隆至表达载体pET-30a,转化E.coliBL21(DE3),鉴定阳性克隆菌落,经IPTG诱导表达融合蛋白DLF,镍离子螯合层析纯化,用Westernblot分析其抗原性。结果测序表明,插入片段可编码含545个氨基酸残基的融合蛋白DLF。SDS-PAGE分析表明,该融合蛋白可在大肠杆菌中表达,其表达量约占菌体总蛋白的20%,表达形式主要为包涵体。纯化后纯度达90%以上。Westernblot证实,该融合蛋白同时具有DT和bFGF两种抗原性。结论已成功构建DT/bF-GF表达载体,并获得较纯的DLF融合毒素。

Objective To construct the expression vector for DT/bFGF fusion protein and prepare highly purified DLF fusion toxin. Methods The DNA sequences encoding DT389 and bFGF were amplified by PCR and linked together by restriction endonuclease digestion and ligation ,then cloned into expression vector pET-30a. The constructed plasmid pET-30a/DLF was transformed to E. coli BL21 （ DE3 ） ,and the positive clones were screened. The fusion protein was expressed by IPTG induction and purified by nickel ion affinity chromatography,then identified by Western blot. Results Sequence analysis showed that the inserted DNA fragment encoded fusion protein DLF consisting of 545 amino acid residues. SDS-PAGE showed that the fusion protein in a form of inclusion body was expressed in E. coli and contained about 20% of total somatic protein. The purity of the fusion protein reached more than 90% after purification and showed antigenicities of both DT and bFGF as proved by Western blot. Conclusion The expression vector of DT/bFGF fusion protein was successfully constructed, and highly purified DLF fusion toxin was obtained.