温度响应的黄曲霉毒素B_(1)纳米抗体重组表达、复性及生物活性

Recombinant Expression,Renaturation and Biological Activity of Temperature-Responsive Nanobodies against Aflatoxin B_(1)

将不同长度类弹性蛋白多肽(elastin-like polypeptide,ELP)与纳米抗体融合表达,获得具有温度响应能力的抗黄曲霉毒素B_(1)(aflatoxin B_(1),AFB_(1))纳米抗体。采用递归定向连接克隆得到重组表达载体pET30a-G8-ELP20、pET30a-G8-ELP40、pET30a-G8-ELP60、pET30a-G8-ELP80,分别转化大肠杆菌BL21(DE3)菌株,表达后经十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(sodium dodecyl sulfate-polyacrylamide gel electrophoresis,SDS-PAGE)分析显示4种融合蛋白均以不溶性包涵体形式存在于菌体沉淀中,对其进行变性处理后,分别采用稀释复性、可逆相变循环(inverse transition cycling,ITC)纯化、透析复性、柱上复性4种方式对包涵体蛋白进行复性。SDS-PAGE分析4种复性方式分别获得的4种融合蛋白,结果显示其纯度差异不显著,但ITC纯化蛋白得率最高,分别为83%、90.7%、89.5%、88.3%;间接竞争酶联免疫吸附实验测定复性后融合蛋白活性,结果表明由稀释复性法获得的G8-ELP80重组蛋白IC50最低(4.35 ng/mL);浊度分析法测得融合不同长度ELP标签纳米抗体的相变温度分别为45、38、32、28℃;圆二色谱分析显示4种融合蛋白二级结构均以β-折叠、β-转角为主,与预估结果相符。本研究将ELP标签与抗AFB1纳米抗体融合表达,实现了纳米抗体的温度刺激响应性,系统比较了不同复性方式对蛋白特性的影响,为后续AFB1检测分析提供了基础。

In this study,temperature-responsive anti-aflatoxin B_(1)(AFB_(1))nanoantibodies were obtained by fusion expression of elastin-like polypeptides(ELPs)of different lengths with nanoantibodies.The recombinant expression vectors pET30a-G8-ELP20,pET30a-G8-ELP40,pET30a-G8-ELP60 and pET30a-G8-ELP80 were synthesized by recursive directional ligation(RDL)and transformed into Escherichia coli BL21(DE3)cells,separately.Sodium dodecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE)analysis showed that the four fusion proteins were expressed in the form of inclusion bodies in the bacterial precipitate.The inclusion bodies were dissolved in denaturing buffer and refolded by dilution,inverse transition cycling(ITC)purification,dialysis or column refolding.SDS-PAGE analysis showed that the purity of the four proteins obtained achieved using the four refolding methods was similar.After refolding by ITC,the highest yield of 83%,90.7%,89.5%,and 88.3% was obtained for the four fusion proteins,respectively.The half maximal inhibitory concentration(IC_(50))of G8-ELP80 obtained by dilution refolding was the lowest(4.35 ng/mL),as determined by indirect competitive enzyme-linked immunosorbent assay(ELISA).The phase transition temperatures(Tt)of nanoantibodies tagged with the different ELPs,as determined by the turbidity method,were 45,38,32 and 28℃,respectively.Circular dichroism(CD)spectroscopy showed that the main secondary structures of the four fusion proteins were β-sheet and β-turn.This study provides a basis for subsequent AFB1 detection and analysis.