摘要
【目的】旨在克隆兔豆状囊尾蚴(Cysticercus pisiformis)半胱氨酸蛋白酶抑制剂(cysteine protease inhibitor)Stefin基因(CpStefin),原核表达并纯化兔豆状囊尾蚴半胱氨酸蛋白酶抑制剂CpStefin蛋白,制备多克隆抗体并测定其效价。【方法】根据GenBank登录的猪带绦虫半胱氨酸蛋白酶抑制剂Stefin基因序列设计引物,以提取的兔豆状囊尾蚴总RNA为模板,运用RT-PCR方法克隆兔豆状囊尾蚴Stefin基因,构建克隆载体pMD-19TCpStefin,分析Cpstefin蛋白氨基酸序列的二级结构、理化性质、结构域,并分析其与其它绦虫Stefin基因的亲缘关系,构建系统进化树。以克隆载体pMD-19T-CpStefin为模板扩增兔豆状囊尾蚴Stefin基因,回收纯化的扩增产物连接到原核表达载体pGEX-4T-1,构建重组质粒pGEX-4T-CpStefin,双酶切、测序结果与预期结果一致。重组质粒pGEX-4T-CpStefin转化BL21(DE3)大肠杆菌感受态细胞,IPTG诱导表达获得重组蛋白。应用亲和层析法纯化可溶性CpStefin重组蛋白,进行SDS-PAGE鉴定。重组蛋白免疫家兔制备多克隆抗体,运用ELISA方法测定其效价。【结果】首次克隆兔豆状囊尾蚴半胱氨酸蛋白酶抑制剂CpStefin基因,基因大小约为290 bp,成功构建克隆载体pMD-19T-CpStefin;菌液PCR和双酶切鉴定结果表明重组质粒pGEX-4T-CpStefin构建正确,通过IPTG诱导表达获得分子质量大小约为35 kU的可溶性融合蛋白,亲和层析法纯化获得纯度较高的CpStefin重组蛋白,制备出的多克隆抗体效价达到1∶25600。【结论】本研究首次克隆出豆状囊尾蚴半胱氨酸蛋白酶抑制剂CpStefin基因,运用原核表达系统可溶性表达豆状囊尾蚴半胱氨酸蛋白酶抑制剂CpStefin蛋白,纯化获得纯度较高的豆状囊尾蚴半胱氨酸蛋白酶抑制剂CpStefin重组蛋白,制备的多克隆抗体效价达到1∶25600。
【Objective】This study aimed to clone the cysteine protease inhibitor stefin gene from Cysti⁃cercus pisiformis(CpStefin)of Taenia pisiformis,purify the recombinant proteins using a prokaryotic ex⁃pression system,and generate polyclonal antibodies with subsequent titer detection.【Method】A pair of primers was designed based on the gene sequence of the cysteine protease inhibitor stefin gene from Taenia solium.The stefin gene from C.pisiformis was amplified via RT-PCR using C.pisiformis RNA as a tem⁃plate.Subsequently,the plasmid pMD-19T-CpStefin was constructed,and secondary structure analysis,physicochemical property assessment,domain identification of the Cpstefin protein amino acid sequence,and construction of a phylogenetic tree comparing Cpstefin gene with other tapeworm stefin genes were con⁃ducted.The CpStefin gene was then ligated into a prokaryotic vector to obtain the recombinant plasmid pGEX-4T-CpStefin,which was confirmed by sequencing and double enzyme.The sequencing results matched the expected outcomes.The recombinant plasmid pGEX-4T-CpStefin was transformed into BL21(DE3)E.coli,and the recombinant fusion protein was induced by IPTG and purified using affinity chroma⁃tography.The purified CpStefin recombinant protein was used to immunize New Zealand white rabbits to produce polyclonal antibodies against soluble CpStefin,and the antibody titers were determined via ELISA assay.【Result】The CpStefin gene was successfully cloned with a size of 290 bp,and the plasmid pMD-19T-CpStefin was successfully constructed.The recombinant plasmid pGEX-4T-CpStefin was also suc⁃cessfully generated based on PCR and enzyme digestion results.The CpStefin protein was expressed as a soluble 35 kU fusion protein,and the CpStefin protein was purified.Rabbit antiserum was prepared using the purified soluble CpStefin recombinant expression protein as an antigen.The titers of polyclonal antibod⁃ies against CpStefin reached 1∶25600 as detected by ELISA.【Conclusions】The CpStefin gene of Cysticer⁃cus pisiformis was successfully cloned and recombinant plasmid pGEX-4T-CpStefin was successfully con⁃structed.The recombinant CpStefin protein was obtained by prokaryotic expression system,and the recom⁃binant CpStefin protein was successfully purified with high-purity.The polyclonal antibodies with titer of 1∶25600 against the recombinant protein CpStefin of Cysticercosis pisiformis was successfully prepared.
作者
王泽祥
张国强
王由森
罗永禄
薛萍
白玉婷
柳春霞
WANG Zexiang;ZHANG Guoqiang;WANG Yousen;LUO Yonglu;XUE Ping;BAI Yuting;LIU Chunxia(Colllege of Veterinary Medicine,Gansu Agricultural University,Lanzhou 730070,China;Animal Disease Prevention and Control Center of Yongdeng County,Lanzhou 730300,China;Animal Disease Prevention and Control Center of Pingliang City,Pingliang 744000,China)
出处
《甘肃农业大学学报》
CAS
CSCD
2023年第4期21-29,共9页
Journal of Gansu Agricultural University
基金
甘肃省自然科学基金项目(21JR7RA823)
国家自然科学基金地区科学基金项目(32160843)
甘肃农业大学国家级大学生创新创业训练计划项目(202010733011)。
关键词
豆状囊尾蚴
半胱氨酸蛋白酶抑制剂
原核表达
纯化
多克隆抗体
Cysticercus pisiformis
cysteine protease inhibitor
prokaryotic expression
purification
poly⁃clonal antibodies
作者简介
第一作者:王泽祥,讲师,博士,研究方向为兽医寄生虫学。E-mail:wangzx@gsau.edu.cn。
