产气荚膜梭菌Alpha毒素突变体的表达及单克隆抗体制备  

Expression and Preparation of Monoclonal Antibodies of Clostridium perfringens Alpha-Toxin Mutant

作　　者：韩凤烨 刘莹[2] 潘晨帆 张乾义[2] 陈小云[2] 朱真[2] 印春生[2] 温永俊[1] 王凤雪 杜吉革 HAN Fengye;LIU Ying;PAN Chenfan;ZHANG Qianyi;CHEN Xiaoyun;ZHU Zhen;YIN Chunsheng;WEN Yongjun;WANG Fengxue;DU Jige(Key Laboratory of Animal Diseases Clinical Diagnosis and Treatment Technology of Ministry of Agriculture and Rural Affairs,College of Veterinary Medicine,Inner Mongolia Agricultural University,Hohhot 010000,China;China Institute of Veterinary Drug Control,Beijing 100081,China)

机构地区：[1]内蒙古农业大学兽医学院,农业农村部动物疾病临床诊疗技术重点实验室,呼和浩特010000 [2]中国兽医药品监察所,北京100081

出　　处：《中国畜牧兽医》2025年第1期442-450,共9页China Animal Husbandry & Veterinary Medicine

基　　金：国家自然科学基金地区项目(32260894);内蒙古自治区教育厅“高校青年科技英才”项目(NJYT22043);内蒙古农业大学青年教师科研能力提升专项(BR220113);“十四五”国家重点研究计划项目课题:动物疫病综合防控关键技术研究与应用(2022YFD1800704)。

摘　　要：[目的]获得产气荚膜梭菌Alpha毒素(Clostridium perfringens alpha-toxin, CPA)的重组突变体,评价其毒力和抗原性,进而制备针对CPA的单克隆抗体,并评价单克隆抗体特性。[方法]通过人工合成含6个氨基酸突变(第56和130位的天冬氨酸突变为甘氨酸、第275、307和331位的酪氨酸突变为苯丙氨酸、第336位的天冬氨酸突变为天冬酰胺)的CPA基因片段,并将其克隆至pET-30a(+)载体,转化大肠杆菌BL21(DE3)感受态细胞进行诱导表达与纯化,获得重组蛋白rCPA_(m6),并检测其毒力与免疫原性。将rCPA_(m6)作为包被抗原建立CPA抗体的间接ELISA检测方法,并用灭活的天然CPA作为免疫原按照常规方法免疫BALB/c小鼠,制备单克隆抗体,对其功能进行鉴定。[结果]重组蛋白rCPA_(m6)在大肠杆菌BL21(DE3)感受态中能够以可溶性和包涵体两种形式表达。毒力测定结果显示,100μg/只rCPA_(m6)攻毒后,小鼠全部存活。免疫原性分析结果显示,1×最小致死量(MLD)的天然CPA攻毒后,对照组小鼠全部死亡,10和20μg/只rCPA_(m6)免疫组小鼠存活率为100%。利用rCPA_(m6)成功建立了CPA抗体的间接ELISA检测方法,并筛选获得4株分泌抗CPA的单克隆抗体杂交瘤细胞株,分别命名为6E3、6B8、10A11、13D10,且4种细胞上清抗体效价均≥1∶3 200,其中单克隆抗体6B8细胞上清能中和天然CPA,且能与rCPA_(m6)发生反应。[结论]试验成功获得具有良好的安全性和免疫原性的重组蛋白rCPA_(m6),制备的CPA单克隆抗体6B8具有一定的中和活性及较高的特异性和敏感性。研究结果为CPA亚单位疫苗的研制提供了候选抗原,同时为CPA中毒症的治疗以及抗原/抗体检测方法的建立提供了物质基础。[Objective]The purpose of this experiment was to obtain the recombinant mutant of Clostridium perfringens alpha-toxin(CPA),evaluate its virulence and antigenicity,and then prepare monoclonal antibodies against CPA,and evaluate the characteristics of the monoclonal antibodies.[Method]The CPA gene fragment containing 6 amino acid mutations(aspartic acid mutation at positions 56 and 130 to glycine,tyrosine mutation at positions 275,307 and 331 to phenylalanine,aspartic acid mutation at position 336 to aspartic acid)was synthesized.The synthetic fragment was cloned into pET-30a(+)vector,and transformed into Escherichia coli BL21(DE3)competent cells for induction expression and purification,and the recombinant protein rCPA m6 was obtained.The toxicity and immunogenicity were detected.The indirect ELISA assay based on rCPA m6 for the detection of CPA antibody was established.BALB/c mice were immunized with inactivated natural CPA as immunogen,and the monoclonal antibody was prepared and its function was identified.[Result]Recombinant protein rCPA m6 could be expressed in both soluble and inclusion form in Escherichia coli BL21(DE3)competent cells.The results of toxicity test showed that all mice survived after 100μg/mice rCPA m6 challenge.The results of immunogenicity analysis showed that after 1×minimum lethal dose(MLD)of natural CPA,all mice died in control group,and the survival rate of mice in 10 and 20μg/mice rCPA m6 immune groups was 100%.An indirect ELISA method for the detection of CPA antibody was successfully established using rCPA m6,and 4 hybridoma cell lines secreting anti-CPA monoclonal antibody were screened,named 6E3,6B8,10A11 and 13D10,respectively.The titers of the 4 cell supernatants were all≥1∶3200,among which the monoclonal antibody 6B8 cell supernatants could neutralize the natural CPA and react with rCPA m6.[Conclusion]The recombinant protein rCPA m6 with good safety and immunogenicity was successfully obtained,and the CPA monoclonal antibody 6B8 had certain neutralizing activity as well a

关 键 词：产气荚膜梭菌Alpha毒素(CPA) 突变体 原核表达 单克隆抗体 

分 类 号：S852.61[农业科学—基础兽医学]