CTB-FimA融合蛋白原核表达纯化及其免疫效果  

Prokaryotic expression,purification and immune effect of CTB-FimA fusion protein

作　　者：梁梦歌 李智涛[1] 韩海芳 王烁 陈义祥 高社干[1,2] LIANG Mengge;LI Zhitao;HAN Haifang;WANG Shuo;CHEN Yixiang;GAO Shegan(School of Basic Medicine and Forensic Medicine,Henan University of Science and Technology,Luoyang 471003,China;Henan Key Laboratory of Cancer Epigenetics,Cancer Institute,The First Affiliated Hospital,College of Clinical Medicine of Henan University of Science and Technology,Luoyang 471003,China)

机构地区：[1]河南科技大学基础医学与法医学院,河南洛阳471003 [2]河南科技大学临床医学院,河南科技大学第一附属医院,河南科技大学肿瘤研究所,河南省肿瘤表观遗传重点实验室,河南洛阳471003

出　　处：《生物技术》2024年第1期12-19,共8页Biotechnology

基　　金：国家自然科学基金项目(81972571);洛阳市社会发展专项医疗卫生重点项目(2101038A)。

摘　　要：[目的]构建牙龈卟啉单胞菌(Porphyromonas gingivalis,Pg)菌毛蛋白(FimA)与霍乱毒素B亚基(CTB)重组质粒pET-32a/CTB-FimA,表达纯化CTB-FimA融合蛋白并对其黏膜免疫效果进行研究。[方法]根据GenBank查找FimA基因和CTB基因,利用(Gly4Ser)3连接肽序列构建pET-32a/CTB-FimA原核表达质粒,转化至大肠杆菌BL21(DE3),确定IPTG诱导浓度及温度,SDS-PAGE分析目的蛋白的可溶性;采用亲和层析和凝胶过滤层析纯化目的蛋白CTB-FimA;用纯化的目的蛋白作为免疫原鼻腔滴注免疫BALB/c小鼠观察其黏膜免疫效果。[结果]经双酶切和测序鉴定pET-32a/CTB-FimA重组表达质粒构建成功;IPTG最适诱导浓度为0.5 mmol/L,最适诱导温度为23℃,重组蛋白CTB-FimA主要以可溶形式表达;Expasy软件ProtParam工具预测重组蛋白分子量约为72.08 kDa,亲水性总平均值为-0.313;每升发酵液表达CTB-FimA蛋白约100 mg;免疫BALB/c小鼠产生sIgA抗体效价显著高于对照组。[结论]成功构建pET-32a/CTB-FimA重组蛋白表达质粒,获得可溶性CTB-FimA重组蛋白,鼻腔滴注免疫BALB/c小鼠能够产生高效价的sIgA抗体,为防治口腔牙龈卟啉单胞菌感染疫苗的研制奠定基础。[Objective]To construct a recombinant plasmid pET-32a/CTB-FimA containing Porphyromonas gingivalis(Pg)fimbrial protein(FimA)and cholera toxin B subunit(CTB),to express and purify the fusion protein CTB-FimA and study its mucosal immune effect.[Method]The(Gly_4Ser)_3 linker peptide sequence was used to construct the pET-32a/CTB-FimA prokaryotic expression plasmid,which was transformed into E.coli BL21(DE3).The induction concentration and temperature of IPTG were determined,and the soluble protein was analyzed by SDS-PAGE.The target protein CTB-FimA was purified by affinity chromatography and gel filtration chromatography.BALB/c mice were immunized with the purified target protein by intranasal instillation to observe the mucosal immune effect.[Result]The recombinant expression plasmid pET-32a/CTB-FimA was successfully constructed and confirmed by restriction enzyme digestion and sequencing.The optimal concentration of IPTG was 0.5 mmol/L,and the induction temperature was 23℃.The recombinant protein CTB-FimA was mainly expressed in soluble form.The molecular weight was 72.08 kDa predicated by the ProtParam tool of Expasy software,and the mean hydrophilicity was-0.313.Approximately 100 mg CTB-FimA protein was expressed per liter of fermentation broth.sIgA antibody titers in immunized BALB/c mice were significantly higher than those in the control group.[Conclusion]The recombinant expression plasmid pET-32a/CTB-FimA was successfully constructed,and the soluble CTB-FimA recombinant protein was obtained.High-titer sIgA antibody was produced in BALB/c mice were immunized with CTB-fimA through nasal drip,which laid the foundation for the development of a vaccine against oral P.gingivalis infection.

关 键 词：牙龈卟啉单胞菌 CTB-FimA重组蛋白 诱导表达 亲和层析 凝胶过滤层析 鼻腔黏膜免疫 

分 类 号：R735.1[医药卫生—肿瘤]