重组牛乳过敏原β-乳球蛋白的可溶性表达及其特性分析

目的 实现β-乳球蛋白(β-lactoglobulin, BLG)的可溶性表达,并比较重组BLG和天然BLG在结构和免疫原性两个方面的差异性。方法 将pET-30a-BLG重组质粒转入大肠杆菌BL21(DE3)感受态细胞中,用异丙基-β-D-硫代半乳糖苷(isopropyl-β-D-thiogalactopyranoside,IPTG)进行体外诱导表达,并对表达条件进行优化,采用十二烷基硫酸钠-聚丙烯酰胺电泳方法分析重组蛋白的可溶性。利用荧光光谱和圆二色谱比较重组BLG空间结构变化,同时采用竞争抑制酶联免疫吸附实验评价重组BLG致敏性能力。结果 在Tris-HCl溶液体系中重组BLG的可溶性表达可达到50%以上,可溶性表达的最优条件为:在Tris-HCl溶液体系中,诱导温度为37℃, IPTG终浓度1.0 mmol/L,诱导4 h。重组BLG表面疏水性下降,可溶性重组BLG的α-螺旋结构减少,经除盐处理后致敏性下降,包涵体重组BLG无规则卷曲结构减少、致敏性上升。结论 本研究成功从上清液中纯化得到可溶性重组BLG,且可溶性表达获得的BLG空间结构更接近天然蛋白。

Soluble expression and characteristic analysis of recombinant β-lactoglobulin

Objective In order to achieve the soluble expression of β-lactoglobulin (BLG), and compare the differences in structure and immunogenicity between recombinant BLG and natural BLG. Methods In this study, recombinant plasmid pET-30a-BLG was transferred into Escherichia coli BL21(DE3) competent cells and induced by isopropyl-β-D-thiogalactopyranoside (IPTG) in vitro. Then optimized expression conditions and the solubility of the recombinant protein was analyzed by electrophoresis of sodium dodecyl sulfate and polyacrylamid. The spatial structure of recombinant BLG was ccompared by fluorescence spectrum and circular dichroism chromatography, and the sensitization ability of recombinant BLG was evaluated by competitive-enzyme linked immunosorbent assay. Results The soluble expression of recombinant BLG in Tris-HCl solution can reach more than 50%, and the optimal conditions for soluble expression are as follows: in Tris-HCl solution system, the induction temperature is 37℃, the final concentration of IPTG is 1.0 mmol/L, and the induction time is 4 h. The surface hydrophobicity of recombinant BLG decreased, and the α-helix structure of soluble recombinant BLG decreased, while the sensitization of BLG decreased after desalting treatment.. However, the random curly structure of BLG decreased and the sensitization increased in the inclusion bodies. Conclusion In this study, soluble recombinant BLG was successfully purified from supernatant, and the spatial structure of BLG obtained by soluble expression was closer to that of natural protein.