抗B组柯萨奇病毒3型3C蛋白多克隆抗体的制备及初步应用  

The generation and characterization of the polyclonal antibody against Coxsackievirus B33C protease

作　　者：牛孙敏 张箭[1] 王立鑫 王瑶[1] 赵朔暄 邵恩泽 沃晓嫚[1] 赵文然[1] Niu Sunmin;Zhang Jian;Wang Lixin;Wang Yao;Zhao Shuoxuan;Wo Xiaoman;Zhao Wenran(Department of Cell Biology,Harbin Medical University,Harbin 150081,China)

机构地区：[1]哈尔滨医科大学细胞生物学教研室,哈尔滨150081

出　　处：《国际免疫学杂志》2023年第3期233-239,共7页International Journal of Immunology

基　　金：国家自然科学基金(81971920)。

摘　　要：目的制备抗B组柯萨奇病毒(Coxsackievirus,CVB)3C蛋白的多克隆抗体。方法通过聚合酶链式反应从pcDNA3.1(+)-EGFP-3C中扩增编码3C的DNA片段,构建pET28a(+)-3C-6His表达载体,转化至大肠埃希菌(Escherichia coli,E.Coli)BL21(DE3)以表达3C重组蛋白。优化表达条件及菌体蛋白的提取方法,经超声破碎制备菌体总蛋白,经十二烷基硫酸钠聚丙烯酰胺凝胶电泳(sodium dodecyl sulfate polyacrylamide gel electrophoresis,SDS-PAGE)后,用氯化钾进行胶染色,切胶回收相对分子质量为26000的蛋白,即纯化的3C重组蛋白(3C-6His的相对分子质量为26000)。用纯化的1 mg 3C重组蛋白加等量弗氏佐剂免疫新西兰大白兔,共免疫5次,每次间隔1周,免疫结束后1周取兔血清,检测抗体的特异性。结果在相对分子质量为26000的位置检测到了3C-6His融合蛋白的表达,成功构建了高效表达带His标签的3C重组蛋白载体。重组3C蛋白原核优化表达条件为37℃培养细菌6 h,加0.05 mmol/L异丙基β-D-硫代半乳糖苷(isopropy-β-D-thiogalactoside,IPTG)进行诱导。经SDS-PAGE分离菌体总蛋白,得到了纯化的3C重组蛋白。获得的兔免疫血清可以结合E.Coli及HeLa细胞表达的3C蛋白,还能识别感染CVB3或肠道病毒A71的HeLa细胞中表达的3C蛋白。结论获得了抗CVB33C蛋白酶的多克隆抗体,这一抗体可特异结合肠道病毒的3C蛋白酶,为进一步研究3C蛋白酶在肠道病毒致病机制中的作用奠定了基础。Objective To generate and characterize the polyclonal antibody against the 3C protease of Coxsackievirus B3(CVB3).Methods The DNA of 3C was amplified from pcDNA3.1(+)-EGFP-3C by polymerase chain reaction.The pET28a(+)-3C-6His plasmid,which expresses His-tagged 3C,was constructed and used to transform Escherichia coli(E.Coli)BL21(DE3).The expression conditions and the extraction protocol for the recombinant 3C(3C-6His)protein were optimized.After ultrasonic treatment,the recombinant 3C expressed in the bacteria was separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis(SDS-PAGE).The gel was stained by potassium chloride and sliced to collect the proteins with the relative molecular weight of 26000,which corresponds to the recombinant 3C-6His(26000).New Zealand white rabbits were immunized with 1 mg of the purified 3C recombinant protein in combination with equal amount of Freunds adjuvant for a total of 5 times,with an interval of one week.One week after the final immunization,the sera of the rabbits were collected to test the characterization of the antibody.Results The expression of 3C-6His fusion protein was detected at a relative molecular weight of 26000.The plasmid expressing the His-tagged recombinant 3C protein was constructed,and the recombinant 3C was expressed with high quality.The optimal expression condition was established,in which E.Coli were cultured at 37℃for 6 h with the addition of 0.05 mmol/L sopropylβ-D-thiogalactoside(IPTG).High quantity of recombinant 3C protein was obtained after SDS-PAGE separation.The obtained rabbit antisera can bind 3C protein expressed in E.Coli and HeLa cells.The anti-3C antiserum can also recognize the 3C protease expressed in HeLa cells infected with CVB3 or enterovirus A71.Conclusion A polyclonal antibody against CVB33C protease was obtained,which can specifically bind to the 3C protease of enterovirus.This result maybe provided the basis for the further study about the role played by 3C protease in the pathogenesis of enteroviruses.

关 键 词：B组柯萨奇病毒 3C蛋白 多克隆抗体 

分 类 号：R392[医药卫生—免疫学]