幽门螺旋杆菌脂蛋白Lpp20重组抗原的可溶性表达及其免疫学性质分析  

Soluble expression,purification and immunological properties of recombinant lipoprotein Lpp20 from H.pylori

作　　者：李元 齐召雄 张富蕊 李欣 张国林 刘宏鹏 郭乐 刘昆梅[2] LI Yuan;QI Zhao-xiong;ZHANG Fu-rui;LI Xin;ZHANG Guo-lin;LIU Hong-peng;GUO Le;LIU Kun-mei(Provincial Key Laboratory of Clinical Pathogenic Microbiology,School of Clinical Medicine,Ningxia Medical University,Yinchuan 750004,China;Breeding Base of State Key Laboratory for Craniocerebral Diseases,Ningxia Medical University,Yinchuan 750021,China;Suzhou Pharmaceutical Testing and Research Center,Suzhou 215000,China)

机构地区：[1]宁夏医科大学临床医学院,宁夏临床病原微生物重点实验室,宁夏银川750021 [2]宁夏医科大学,颅脑疾病国家重点实验室培育基地,宁夏银川750021 [3]苏州市药品检验检测研究中心,江苏苏州215000

出　　处：《生物技术》2022年第2期141-145,152,共6页Biotechnology

基　　金：宁夏高等学校科学研究项目(NGY2020043);国家自然科学基金项目(32070930,82160497);宁夏重点研发计划项目(2020BFG02012);自治区级大学生创新创业训练计划项目(S202110752034);中科院“西部之光”人才培养计划项目,江苏省市场监督管理局科技计划项目(KJ207561)。

摘　　要：[目的]利用原核表达系统表达幽门螺旋杆菌脂蛋白Lpp20(rLpp20),分离纯化获得重组融合蛋白rLpp20,利用其制备抗rLpp20多克隆抗体,并分析其抗体的特异性。[方法]利用生物信息学软件分析rLpp20的抗原结构;通过拼接引物PCR法,获得幽门螺旋杆菌Lpp20基因序列,将其插入到质粒pCzn1中,构建重组质粒pCzn1-rLpp20,转入大肠杆菌Arctic Express中;经IPTG诱导表达rLpp20后,超声波裂菌,用Ni-IDA柱亲和纯化获得目的蛋白rLpp20。然后,利用Western Blot鉴定rLpp20与anti-H.pylori和anti-His tag的免疫反应性;最后,脂蛋白重组抗原rLpp20与弗氏佐剂混合,免疫BALB/c小鼠,制备anti-Lpp20多克隆抗血清,利用ELISA鉴定anti-Lpp20的抗体特异性。[结果]利用生物信息学软件证实rLpp20具有较好的抗原性;重组质粒pCZN1-rLpp20经双酶切和基因测序鉴定,证实rLpp20序列与理论值一致;基因工程菌株pCzn1-rLpp20/Arctic Express可以以可溶形式表达rLpp20,经Ni-IDA亲和层析柱纯化,纯度约达98.2%。Western Blot结果证实:重组抗原rLpp20可特异性与anti-H.pylori和anti-His tag结合。ELISA结果证实:所制备的anti-Lpp20抗血清具有良好的抗体特异性,可特异性识别rLpp20和H.pylori裂解物。[结论]利用E.coli Arctic Express实现了脂蛋白重组抗原rLpp20的可溶性表达,经Ni-IDA柱亲和纯化获得了高纯度rLpp20;脂蛋白重组抗原rLpp20具有良好的免疫反应性和特异性,为进一步研制幽门螺旋杆菌疫苗及相关诊断试剂盒奠定了良好的实验基础。[Objective]After expression and purification of recombinant lipoprotein(rLpp20) from Helicobacter pylori,the anti-Lpp20 polyclonal antibody was prepared,and the immunological properties of rLpp20 were analyzed.[Method] The structure of rLpp20 was analyzed by bioinformatics software,the pCzn1-rLpp20 plasmids were obtained by inserting the Lpp20 gene into the plasmid pCzn1,and then transferred into E.coli Arctic Express.After IPTG induction,the rLpp20 protein was expressed and purified by Ni-IDA affinity chromatography.Western Blot was used to identify the immunoreactivity of rLpp20 by incubation with anti-His tag or anti-H.pylori antibodies.After BALB/c mice were immunized with rLpp20 plus Freund′s adjuvant,the anti-Lpp20 polyclonal antibodies were prepared,and then analyzed by ELISA.[Result] Bioinformatics software predicted that the rLpp20 protein has good antigenic properties.The experiments of double enzyme digestion and gene sequencing confirmed that the pCzn1-rLpp20 plasmid was fully consistent with the theoretical sequence.The rLpp20 protein can be expressed in soluble form in recombinant strains pCzn1-rLpp20/Arctic Express.The purity of the rLpp20 protein was about 95.8% after purification by Ni-IDA affinity chromatography.Western Blot results showed that the rLpp20 protein could react with anti-His tag and anti-H.pylori antibodies.In addition,ELISA results confirmed that anti-Lpp20 polyclonal antibodies could bind to rLpp20 and H.pylori lysates,suggesting anti-Lpp20 polyclonal antibodies had high antibody specificity.[Conclusion] The recombinant lipoprotein rLpp20 was expressed as a soluble way in pCZN1-rLpp20/Arctic Express,and obtained with high purity after purification.The rLpp20 protein had good antigenicity and specificity,which laid a good experimental foundation for further development of H.pylori vaccine and related diagnostic reagents.

关 键 词：幽门螺旋杆菌 脂蛋白Lpp20 多克隆抗体 特异 

分 类 号：Q784[生物学—分子生物学] Q786