CTA1-DD蛋白在枯草芽胞杆菌中的分泌表达  

Secretion and expression of CTA1-DD protein in Bacillus subtilis

作　　者：张元鹏[1,2] 侯立婷[1,2] 杜露平[1,2] 于晓明[1,2] 李兰[1,2] 杨利[1,2] 张浩明[1,2] 王义伟[1,2] 乔绪稳[1,2] 程海卫[1,2] 秦竹[1,2,3,4,5] 陈瑾 郑其升[1,2,3,4,5] ZHANG Yuanpeng;HOU Liting;DU Luping;YU Xiaoming;LI Lan;YANG Li;ZHANG Haoming;WANG Yiwei;QIAO Xuwen;CHENG Haiwei;QIN Zhu;CHEN Jin;ZHENG Qisheng(Institute of Veterinary Immunology&Engineering,Jiangsu Academy of Agricultural Sciences,Nanjing 210014,China;National Research Center of Engineering and Technology for Veterinary Biologicals,Jiangsu Academy of Agricultural Sciences,Nanjing 210014,China;Jiangsu Co-innovation Center for the Prevention and Control of Important Animal Infectious Disease and Zoonosis,Yangzhou University,Yangzhou 225009,China;Jiangsu Key Laboratory for Food Quality and Safety-State Key Laboratory Cultivation Base,Ministry of Science and Technology,Nanjing 210014,China;GuoTai(Taizhou)Center of Technology Innovation for Veterinary Biologicals,Taizhou 225300,China)

机构地区：[1]江苏省农业科学院动物免疫工程研究所,江苏南京210014 [2]江苏省农业科学院国家兽用生物制品工程技术研究中心,江苏南京210014 [3]江苏省动物重要疫病与人兽共患病防控协同创新中心,江苏扬州225009 [4]江苏省食品质量安全重点实验室-省部共建国家重点实验室培育基地,江苏南京210014 [5]兽用生物制品(泰州)国泰技术创新中心,江苏泰州1225300

出　　处：《畜牧与兽医》2023年第9期42-46,共5页Animal Husbandry & Veterinary Medicine

基　　金：“十四五”重点研发专项(2022YFD1800800);江苏省农业自主创新项目[CX(21)3135]。

摘　　要：有研究证实,霍乱毒素(CT)A1亚基与葡萄球菌G蛋白的D肽偶联构建的融合蛋白CTA1-DD具有良好的佐剂效果。为了获得重组CTA1-DD蛋白在枯草芽胞杆菌中的表达,将CTA1-DD基因连接到表达载体pHT43中,构建重组枯草杆菌表达载体pHT43-CTA1-DD。将重组载体转化到枯草芽胞杆菌WB600中,用IPTG诱导重组菌表达,发酵液离心后取上清液,通过SDS-PAGE和Western blot方法分析蛋白表达情况。发酵上清液经Ni-IDA琼脂糖树脂纯化,与流感病毒HA抗原混合后共同滴鼻免疫小鼠后采集小鼠血清和支气管肺泡灌洗液检测IgG和IgA的抗体水平。结果显示:重组CTA1-DD蛋白在枯草芽胞杆菌中能够分泌表达,在发酵罐中发酵36 h情况下蛋白表达量最高,目的蛋白大小在42 kDa左右,蛋白产量最高能到700μg/mL,Western blot检测目的蛋白正确表达。重组蛋白经过Ni-IDA琼脂亲和层析纯化后与HA抗原混和滴鼻免疫小鼠,ELISA结果显示CTA1-DD蛋白组针对HA抗原能够诱导更高的血清IgG和黏膜IgA抗体水平,说明枯草芽胞杆菌表达的CTA1-DD蛋白具有佐剂活性。试验结果为进一步研究CTA1-DD蛋白在黏膜佐剂的大规模生产应用奠定了基础。The fusion protein CTA1-DD construscted by coupling cholera toxin(CT)subunit A1 with D-peptide of staphylococcal G protein has been previously demonstrated to have good adjuvant effect.In order to verify the expression of recombinant CTA1-DD protein inBacillus subtilis,the CTA1-DD gene was inserted into p HT43 to construct a recombinant Bacillus subtilis expression vector p HT43-CTA1-DD.The recombinant vector was transformed into Bacillus subtilis WB600,and the expression of the recombinant protein was induced by IPTG.The protein in the supernatant was collected by centrifugation and analyzed by SDS-PAGE and Western blot.The CTA1-DD protein in the supernatant was purified by Ni-IDA agarose-agar resin.Then,mice were immunized intra-nasally with HA plus CTA1-DD,and serum samples and mucosal bronchial veolar lavages were collected for detection of Ig G and Ig A antibody levels.The results showed that the recombinant CTA1-DD protein was secreted and expressed in Bacillus subtilis.Under the condition of fermentation for 36 h,the protein expression level was the highest,and that the protein yield was up to 700μg/m L.The correct expression of the target protein which was about 42 k Da was detected by Western blot.The recombinant protein was purified by Ni-IDA affinity chromatography,and was mixed with HA antigen,and the mice were immunized with nasal drops.The ELISA results showed that CTA1-DD induced higher serum Ig G antibody and mucosal Ig A antibody levels,indicating that the CTA1-DD protein expressed by Bacillus subtilis possessed adjuvant activity.The results of this study indicated the possibility of CTA1-DD protein in large-scale production and application of mucosal adjuvant.

关 键 词：CTA1-DD蛋白 枯草芽胞杆菌 表达 黏膜免疫 

分 类 号：Q786[生物学—分子生物学]