天蚕素B和表皮生长因子融合蛋白的原核表达、纯化与发酵

目的原核表达天蚕素B和表皮生长因子融合蛋白,并进行纯化和发酵。方法全基因合成带有凝血酶切割位点的天蚕素B和表皮生长因子融合基因,克隆入pET22b(+)-X表达载体中,构建重组质粒pET22b-CecropinB-EGF,分别转化大肠杆菌BL21(DE3)和Rosetta-gami2(DE3),经IPTG诱导后,进行SDS-PAGE及Westernblot分析。镍离子亲和层析纯化融合蛋白并复性后,对其促表皮生长活性进行测定,最后在5L发酵罐中进行发酵。结果酶切及测序结果显示,融合蛋白基因已克隆入pET22b(+)-X表达载体中;SDS-PAGE分析显示,天蚕素B和表皮生长因子融合蛋白在Rosetta-gami2(DE3)宿主菌中得到了有效表达,表达量约占全菌总蛋白的30%;Westernblot分析显示,表达产物可与表皮生长因子单克隆抗体特异结合;融合蛋白的表达形式为包涵体;纯化、复性后的融合蛋白具有促BALB/c3T3细胞生长的活性,在5L发酵罐中进行发酵,1次可收获菌体约60g。结论已成功地在大肠杆菌Rosetta-gami2(DE3)中表达了天蚕素B和表皮生长因子融合蛋白,为研究具有抗菌和促伤口愈合的双功能药物奠定了基础。

Prokaryotic Expression,Purification and Fermentation of Cecropin B and Epidermal Growth Factor Fusion Protein

Objective To express cecropin B and epidermal growth factor(EGF)fusion protein in prokaryotic cells, and purify and ferment the expressed product. Methods The full-length of cecropin B and EGF fusion gene, with the cleavage site of thrombin, was synthesized and cloned into expression vector pET22b(+)-X. The constructed recombinant plasmid pET22b-Cecropin B-EGF was transformed to E. coli BL21(DE3) and Rosetta-gami2(DE3) for expression under induction of IPTG. The expressed products were identified by SDS-PAGE and Western blot, then purified by nickel ion affinity chromatography, re-naturalized, deter-mined for activity of promoting epidermal growth and, finally, fermented in a 5 L fermentor. Results Both restriction analysis and sequencing proved that cecropin B and EGF fusion gene was cloned into expression vector pET22b (+)-X. SDS-PAGE showed effec-tive expression of cecropin B and EGF fusion protein in E. coli Rosetta-gami2(DE3). The expressed product contained about 30% of total somatic protein, and bound specifically to McAb against EGF as proved by Western blot. The fusion protein in a form of inclu-sion body, after purification and re-naturalization, promoted the growth of 3T3 cells of BALB / c mice significantly. A portion of 60 g of bacteria was harvested from a single fermentation in 5 L fermentor. Conclusion Cecropin B and EGF fusion protein was success-fully expressed in E. coli Rosetta-gami2(DE3), which laid a foundation of developing bifunctional drug killing bacteria and promoting the healing of wound.