卵形鯧鲹RelB蛋白原核表達及其多克隆抗體制備

段家文　喬瑞峰　雷坤　楊萌　陸專靈　韋友傳

摘要：【目的】制備卵形鯧鲹（Trachinotus ovatus）RelB蛋白（TroRelB）多克隆抗體，為深入研究TroRelB蛋白的結構、功能及蛋白—蛋白相互作用打下基礎。【方法】將TroRelB基因與pET-32a表達載體連接構建pET-32a-TroRelB重組質粒，轉化大腸桿菌（Escherichia coli）BL21（DE3）感受態細胞，采用異丙基-β-D-硫代半乳糖苷（IPTG）進行誘導表達，以Ni-IDA瓊脂糖純化樹脂柱純化的TroRelB重組蛋白免疫Balb/C小鼠制備多克隆抗體，應用ELISA和Western blotting分別檢測抗體效價及特異性。【結果】以構建的pET-32a-TroRelB重組質粒轉化BL21（DE3）感受態細胞，經IPTG誘導能成功表達獲得TroRelB重組蛋白，其相對分子量為84.0 kD，且主要以包涵體的形式進行表達。經Ni-IDA瓊脂糖純化樹脂柱純化透析的TroRelB重組蛋白濃度為0.498 mg/mL，能被抗His標簽抗體識別。以純化TroRelB重組蛋白免疫Balb/C小鼠獲得抗TroRelB血清（多克隆抗體），ELISA檢測結果顯示其抗體效價為1∶256000;Western blotting檢測結果顯示，5和15 ng的TroRelB重組蛋白均可特異性結合TroRelB多克隆抗體，而陰性對照（免疫前血清）未出現預期條帶。【結論】原核系統誘導表達的TroRelB重組蛋白主要以包涵體的形式進行表達，且攜帶有His標簽，以其免疫Balb/C小鼠制備獲得的TroRelB多克隆抗體具有較強的特異性和較高的抗體效價，可用于深入研究TroRelB蛋白的生物學功能及揭示卵形鯧鲹的免疫機制。

關鍵詞： 卵形鯧鲹;RelB蛋白;原核表達;重組蛋白;多克隆抗體

中圖分類號： S965.331? ? ? ? ? ? ? ? ? ? ? ? 文獻標志碼： A 文章編號：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.

Staal J，Beyaert R. 2018. Inflammation and NF-κB signaling in prostate cancer：Mechanisms and clinical implications[J]. Cells，7（9）：122. doi：10.3390/cells7090122.

Suzuki K，Yamamoto T，Inoue J. 1995. Molecular cloning of cDNA encoding the Xenopus homolog of mammalian RelB[J]. Nucleic Acids Research，23（22）：4664-4669.

Tan J K H，Ni K P，Le F，O'Neill H C. 2007. Hematopoiesis of immature myeloid dendritic cells in stroma-dependent spleen long-term cultures occurs independently of NF-κB/RelB function[J]. Experimental Hematology，35（10）：1580-1593.

Wang L，Zhou Z C，Guo C J，Rao X Y，Xiao J，Weng S P，Yin Z X，Yu X Q，He J G. 2009. The alpha inhibitor of NF-kappaB（IkappaBalpha） from the mandarin fish binds with p65 NF-kappaB[J]. Fish & Shellfish Immunology，26（3）：473-482.

Wang X Z，Liu S K，Yang Y J，Fu Q，Abebe A，Liu Z J. 2017. Identification of NF-κB related genes in channel catfish and their expression profiles in mucosal tissues after columnaris bacterial infection[J]. Developmental and Comparative Immunology，70：27-38.

Xu Y，Josson S，Fang F，Oberley T D，Clair D K S，Wan X S，Sun Y L，Bakthavatchalu V，Muthuswamy A，Clair W H S. 2009. RelB enhances prostate cancer growth：Implications for the role of the nuclear factor-kappaB alternative pathway in tumorigenicity[J]. Cancer Research，69（8）：3267-3271.

Zhao C，Xiu Y，Ashton J，Xing L P，Morita Y，Jordan C T，Boyce B F. 2012. Noncanonical NF-κB signaling regulates hematopoietic stem cell self-renewal and microenvironment interactions[J]. Stem Cells，30（4）：709-718.

Zuklys S，Balciunaite G，Agarwal A，Fasler-Kan E，Palmer E，Holl?nder G A. 2000. Normal thymic architecture and negative selection are associated with Aire expression，the gene defective in the autoimmune-polyendocrinopathy-candidiasis- ectodermal dystrophy（APECED）[J]. Journal of Immunology，165（4）：1976-1983.

（責任編輯 蘭宗寶）