猪SIgA分泌片的表达及其多克隆抗体的制备  

PROKARYOTIC EXPRESSION AND PREPARATION OF POLYCLONAL ANTIBODIES AGAINST SECRETORY COMPONENT OF PORCINE SIGA

作　　者：朱利塞 贾爱卿 王贵平 王娟 李菲 邓胜朝 钱雪桥 丁壮 ZHU Li-sai;JIA Ai-qing;WANG Gui-ping;WANG Juan;Li Fei;DENG Sheng-chao;QIAN Xue-qiao;DING Zhuang(Key Laboratory of Utilization for Microbiological Resources in Breeding Industries,Ministry of Agriculture and Rural Affairs,Haid Central Research Institute,Animal Husbandry and Fisheries Research Center of Guangdong Haid Group Co.,Ltd.,Guangzhou 511400,China;Guangdong Haid Institute of Animal Husbandry&Vterinary,Guangzhou 511400,China;College of Veterinary Medicine,Jilin University,Changchun 130062,China)

机构地区：[1]广东海大集团股份有限公司畜牧水产研究中心、海大中央研究院农业农村部微生态资源养殖利用重点实验室,广州511400 [2]广东海大畜牧兽医研究院,广东511400 [3]吉林大学动物医学学院,长春130062

出　　处：《中国动物传染病学报》2020年第3期68-72,共5页Chinese Journal of Animal Infectious Diseases

基　　金：博士后科研(基金配套)项目经费(4000826);广东海大畜牧兽医研究院基于畜禽重要疫病诊断技术院士工作站(2017B090904029);猪重要疫病快速诊断试剂的研发及产业化(201804020006)。

摘　　要：SIgA是评价黏膜免疫的重要指标,分泌片(SC)是SIgA的特有成分,为了更方便的获得大量的SIgA的分泌片,本研究应用RT-PCR方法扩增出SC基因片段,将SC基因片段克隆到pGEX-4T-1原核表达载体上,经酶切和测序验证获得pGEX-4T-1-SC重组质粒;将获得的pGEX-4T-1-SC转化至大肠杆菌BL21(DE3)感受态细胞中,经IPTG诱导表达重组蛋白rSC.重组蛋白rSC经鉴定和纯化后,按常规免疫程序制备多克隆抗体;利用Western blot对抗重组蛋白rSC的多克隆抗体进行鉴定.结果显示,该抗体能特异性识别猪初乳中的SIgA免疫球蛋白,而与猪初乳中的IgG不发生反应.因此,抗SC重组蛋白的多克隆抗体可以作为抗SIgA免疫球蛋白的特异性抗体,为进一步建立SIgA的检测方法及黏膜免疫的研究奠定基础.Secretory component(SC)is a special part of secretary IgA and represents an important assessment index of mucosal immune level.In order to obtain the recombinant plasmid pGEX-4T-1-SC,the SC gene was cloned into prokaryotic expression vector pGEX-4T-1 using RT-PCR.After confirmation of th recombinant plasmid by enzymatic digestion and DNA sequencing,the pGEX-4T-1-SC plasmid was transformed into E.coli BL21(DE3)for expression with IPTG.Subsequently,rabbit polyclonal antibodies against the recombinant SC were prepared and examined in Western blot.The results demonstrated that the recombinant SC and native SIgA reacted with rabbit polyclonal antibodies against the recombinant SC.In conclusion,the preparations of the recombinant SC protein and polyclonal antibodies provided the tools to develop the detection method of SIgA antibodies in sow colostrum and study mucosal immunity.

关 键 词：SIGA 分泌片 多克隆抗体 黏膜免疫 

分 类 号：S852.42[农业科学—基础兽医学]