猪Ⅰ型补体受体与C3b活性片段相互结合的体外检测  

Detection of Interaction Between Porcine Type Ⅰ Complement Receptor and C3b Active Fragment in Vitro

作　　者：孙雨晨 贾瑞璞 范阔海[1] 孙娜[1] 孙耀贵[1] 孙盼盼 李宏全[1] 尹伟[1] SUN YuChen;JIA RuiPu;FAN KuoHai;SUN Na;SUN YaoGui;SUN PanPan;LI HongQuan;YIN Wei(College of Veterinary Medicine,Shanxi Agricultural University,Taigu 030801,Shanxi)

机构地区：[1]山西农业大学动物医学学院,山西太谷030801

出　　处：《中国农业科学》2021年第19期4243-4254,共12页Scientia Agricultura Sinica

基　　金：国家自然科学基金项目(31640082);国家自然科学基金青年科学基金项目(31702221);山西省研究生优秀创新项目(2019SY216)。

摘　　要：【目的】检测猪红细胞类补体受体Ⅰ型(Complement receptor 1-like,CR1-like)与C3b活性片段能否发生结合,以期为阐明猪红细胞发挥免疫粘附功能的分子机理提供科学数据。【方法】利用前期已构建的CR1-like_(3-6)、CR1-like_(8-11)功能域片段的重组质粒建立酵母双杂交检测体系,运用酵母共转化的方法将诱饵质粒(重组pGBKT7-CR1-like)与捕获质粒(重组pGADT7-C3b)共同转入Y2HGold酵母细胞中,分别利用一缺平板SD/-Leu、SD/-Trp和二缺平板SD/-Leu/-Trp(DD0)严格筛选共转化成功的酵母细胞,再根据报告因子是否表达来鉴别转化子在SD/-Leu/-Trp/X-α-Ga1 (DDO/X)、SD/-Leu/-Trp/X-α-Ga1/Aba (DDO/X/A)二缺培养板上的生长情况,并结合菌落的颜色变化现象综合判定CR1-like活性片段与补体C3b在酵母细胞中是否发生相互结合;然后运用免疫沉淀技术分离酵母细胞中CR1-like与C3b结合复合物,并对该复合物的特异性进行Western blot鉴定。【结果】试验成功将pGBKT7-CR1-like与pGADT7-C3b基因共转入Y2HGold酵母细胞。共转化的酵母克隆在SD/-Leu、SD/-Trp、DDO平板上能够正常生长,在DDO/X、DDO/X/A平板上正常生长且菌落呈现蓝色,由此表明,试验中酵母双杂交系统建立成功,并通过试验获得了阳性酵母克隆。共同转化了 pGBKT7-CR1-like和pGADT7-C3b质粒的酵母菌落PCR反向鉴定结果显示,在共转化的酵母菌中含有目的基因CR1-like_(3-6)和CR1-like_(8-11),共转化组的质粒酶切后出现C3b基因片段,与设计大小一致,说明重组质粒成功共转化入酵母细胞中。免疫沉淀试验中应用pGBKT7载体的标签抗体c-Myc沉淀酵母细胞中的融合蛋白,以c-Myc为一抗进行Western blot检测发现,单独转化了pGBKT7-CR1-like_(3-6)和pGBKT7-CR1-like_(8-11)的融合蛋白在 50 kD 处出现特异性条带;共转化 pGBKT7-CR1-like_(3-6)+pGADT7-C3b和共转化pGBKT7-CR1-like(8-11)+pGADT7-C3b的酵母融合蛋白在83kD处出现特异性条带;以HA单克【Objective】In order to provide scientific data for elucidating the molecular mechanism of porcine erythrocyte immune adhesion function,it was investigated whether CR1-like(Complement receptor 1-like,CR1-like)of porcine erythrocyte could bind to the C3b or not.【Method】In this study,the recombinant plasmids of CR1-like_(3-6)and CR1-like_(8-11)functional domain fragments were constructed first,which were used to establish a yeast two-hybrid detection system.The bait plasmid(recombinant pGBKT7-CR1-like)and capture plasmid(recombinant pGADT7-C3b)were co-transformed into Y2HGold yeast cells.The single deficient SD/-Leu,SD/-Trp and double-deficient SD/-Leu/-Trp(DDO)media were used to strictly screen the co-transformed yeast cells.Then,according to the expression of report factor,the growth of transformants were identified on the double-deficient medium SD/-Leu/-Trp/X-α-Gal(DDO/X)or SD/-Leu/-Trp/X-α-Gal/Aba(DDO/X/A)combined with the color change phenomenon of the colony to comprehensively determine whether CR1-like active fragments and complement C3b bind to each other in yeast cells or not.The CR1-like-C3b binding complex in yeast cells was then separated by immunoprecipitation,and the specificity of the complex was identified by Western blot.【Result】The co-transformed yeast clones showed normal growth on SD/-Leu,SD/-Trp,DDO and DDO/X,DDO/X/A media with blue color colonies,and this indicated that positive yeast colonies were successfully obtained.The results of PCR reverse identification showed that the co-transformed yeast contained the target genes CR1-like_(3-6)and CR1-like_(8-11).The C3b gene fragment appeared after the plasmid was digested,indicating that the recombinant plasmid pGBKT7-CR1-like and pGADT7-C3b were successfully co-transformed into yeast cells.In the immunoprecipitation test,the tag antibody c-Myc of the pGBKT7 vector was used to precipitate the fusion protein in yeast cells.Western blot detection with c-Myc as the primary antibody revealed that the fusion protein transformed pGBKT7-CR1

关 键 词：CR1-like C3B 酵母双杂交 免疫粘附 

分 类 号：S852.4[农业科学—基础兽医学]