卵形鲳鲹RelB蛋白原核表达及其多克隆抗体制备

段家文　乔瑞峰　雷坤　杨萌　陆专灵　韦友传

摘要：【目的】制備卵形鲳鲹（Trachinotus ovatus）RelB蛋白（TroRelB）多克隆抗体，为深入研究TroRelB蛋白的结构、功能及蛋白—蛋白相互作用打下基础。【方法】将TroRelB基因与pET-32a表达载体连接构建pET-32a-TroRelB重组质粒，转化大肠杆菌（Escherichia coli）BL21（DE3）感受态细胞，采用异丙基-β-D-硫代半乳糖苷（IPTG）进行诱导表达，以Ni-IDA琼脂糖纯化树脂柱纯化的TroRelB重组蛋白免疫Balb/C小鼠制备多克隆抗体，应用ELISA和Western blotting分别检测抗体效价及特异性。【结果】以构建的pET-32a-TroRelB重组质粒转化BL21（DE3）感受态细胞，经IPTG诱导能成功表达获得TroRelB重组蛋白，其相对分子量为84.0 kD，且主要以包涵体的形式进行表达。经Ni-IDA琼脂糖纯化树脂柱纯化透析的TroRelB重组蛋白浓度为0.498 mg/mL，能被抗His标签抗体识别。以纯化TroRelB重组蛋白免疫Balb/C小鼠获得抗TroRelB血清（多克隆抗体），ELISA检测结果显示其抗体效价为1∶256000;Western blotting检测结果显示，5和15 ng的TroRelB重组蛋白均可特异性结合TroRelB多克隆抗体，而阴性对照（免疫前血清）未出现预期条带。【结论】原核系统诱导表达的TroRelB重组蛋白主要以包涵体的形式进行表达，且携带有His标签，以其免疫Balb/C小鼠制备获得的TroRelB多克隆抗体具有较强的特异性和较高的抗体效价，可用于深入研究TroRelB蛋白的生物学功能及揭示卵形鲳鲹的免疫机制。

关键词： 卵形鲳鲹;RelB蛋白;原核表达;重组蛋白;多克隆抗体

中图分类号： S965.331                        文献标志码： A 文章编号：2095-1191（2021）01-0238-07

Abstract：【Objective】The polyclonal antibody against Trachinotus ovatus RelB protein（TroRelB） was prepared tolay the foundation for further study on the structure， function and protein-protein interaction of TroRelB protein. 【Me-thod】The recombinant plasmid pET-32a-TroRelB was constructed by linking TroRelB gene to expression vector pET-32a then transformed into Escherichia coli BL21（DE3） competent cells. The protein expression was induced by isopropyl-β-D-thiogalactoside（IPTG）. The recombinant TroRelB protein purified with a Ni-IDA agarose resin column was applied to immunize Balb/C mice to prepare polyclonal antibody. The titer and specificity of this antibody were detected by ELISA and Western blotting assay respectively. 【Result】The BL21（DE3） competent cells were transformed by recombinant plasmid pET-32a-TroRelB and the recombinant TroRel Bprotein with a molecular weight of 84.0 kD was successfully expressed mainly in the form of inclusion body by IPTG inducing. The concentration of the recombinant TrorelB protein which could be recognized by antibody of anti-His tag was 0.498 mg/mL after purification by Ni-IDA agarose resin co-lumn. The anti-TrorelB serum（polyclonal antibody） was obtained by immunizing Balb/Cmice with the purified recombinant TrorelB protein. The ELISA result showed that the titer of this polyclonal antibody was 1∶256000， and the Western blotting result showed that both 5 and 15 ng of the recombinant TrorelB protein reacted specifically withthe polyclonal antibody while the negative control（serum before the immune） did not show the expected band. 【Conclusion】The recombinant TrorelB protein induced by the prokaryotic system is mainly expressed in the form of inclusion body and carries His label. The polyclonal antibody of TrorelB prepared by immunizing Balb/C mice with this recombinant protein possess strong specificity and high antibody titer， which can be used to further study the biological function of TrorelB protein and reveal the immune mechanism of T. ovatus.

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