草鱼C5a、C5aR和FⅡ多克隆抗体的制备与表达特性  

Preparation and expression characteristics of polyclonal antibodies against C5a,C5aR and FⅡin grass carp(Ctenopharyngodon idella)

作　　者：苏杭 肖调义[1] 许宝红[1] 刘巧林[1] 吕丽刚 刘益 SU Hang;XIAO Tiaoyi;XU Baohong;LIU Qiaolin;Lü Ligang;LIU Yi(Hunan Engineering Technology Research Center of Featured Aquatic Resources Utilization,Hunan Agricultural University,Changsha 410128,China)

机构地区：[1]湖南农业大学,湖南省特色水产资源利用工程技术研究中心,湖南长沙410128

出　　处：《水产学报》2022年第5期785-796,共12页Journal of Fisheries of China

基　　金：国家自然科学基金(U20A2063);湖南省自然科学基金青年项目(2019JJ50265)。

摘　　要：为研究草鱼补体蛋白5a(complement component 5a,C5a)、补体蛋白5a受体(C5a receptor,C5aR)和凝血因子Ⅱ(blood coagulation factorⅡ,FⅡ)在感染草鱼呼肠孤病毒(grass carp reovirus,GCRV)时的蛋白表达和相互作用,针对草鱼C5a和FⅡ蛋白构建了原核表达体系、针对草鱼C5aR蛋白构建C5aR-KLH偶联物,纯化蛋白,免疫日本大耳白兔制备3种蛋白的多克隆抗体。用蛋白质印迹(Western blot)、免疫共沉淀(Co-IP)和拉下(pull down)实验检测3种蛋白表达与互作关系,Western blot结果显示,C5a和C5aR在健康草鱼的肝脏、脾脏、肾脏、头肾、肠、鳃和肌肉中均有蛋白表达,FⅡ在肝脏、脾脏和肠中表达,而在肾脏、头肾、鳃和肌肉中不表达;在感染GCRV的草鱼肝脏组织中C5a、C5aR和FⅡ蛋白均随病程进展呈现上升趋势。Co-IP结果显示,在GCRV处理后,C5a、C5aR和FⅡ蛋白具有相互作用关系。pull down结果显示,C5a pull down共鉴定得到C3、RIG-I等28种候选蛋白,C5aR pull down共鉴定得到转醛醇酶、巨球蛋白等24种候选蛋白。该研究为深入探讨C5a、C5aR和FⅡ互作调控关系以及进一步探索补体和凝血级联系统中3个关联蛋白响应GCRV感染的作用机制奠定了基础。Complement and coagulation cascade are important components of innate immunity,in which complement protein 5a(complement component 5a,C5a),complement protein 5a receptor(C5a receptor,C5aR)and coagulation factor II(blood coagulation factor II,FII)play a key role in dealing with virus infection.In order to study their protein expression and interaction in Ctenopharyngodon idella infected with grass carp reovirus(GCRV),the prokaryotic expression system of C5a and FII protein was constructed,the C5aR-KLH conjugate against C5aR protein was constructed,and the protein was purified.Japanese big-eared rabbits were immunized to prepare polyclonal antibodies against the three kinds of proteins.The expression and interaction of the three proteins were detected by western blot,Co-immunoprecipitation(Co-IP)and pulldown test.Western blot results showed that C5a and C5aR proteins were expressed in liver,spleen,kidney,head kidney,intestine,gill and muscle of C.idella.FII protein was expressed in liver,spleen and intestine,but not in kidney,head kidney,gill and muscle.Expression of C5a,C5aR and FII proteins in liver tissues of C.idella infected with GCRV showed an upward trend with the progression of the disease.Co-IP results showed that C5a,C5aR and FII proteins interacted with each other after GCRV treatment.Pull down results showed that 28 candidate proteins such as C3 and RIG-I were identified by C5a pull down,and 24 candidate proteins such as transaldolase and macroglobulin were identified by C5aR pull down.This study provides a basis for further exploring the interaction and regulation of C5a,C5aR and FII,and for further exploring the mechanism of the three associated proteins in the complement and coagulation cascade system responding to GCRV infection.

关 键 词：草鱼 C5A C5AR FⅡ 多克隆抗体 草鱼呼肠孤病毒(GCRV) 表达特性 

分 类 号：S941[农业科学—水产养殖]