猪FcRn与IgG抗体Fc段CH2结构域的细胞定位与互作  

Cellular Localization and Interaction Between CH2 Domain of IgG Fc Segment and Porcine(Sus scrofa) FcRn

作　　者：许发芝[1] 朱正萱 丁小玲[1] XU Fa-Zhi;ZHU Zheng-Xuan;DING Xiao-Ling(Anbui Province Key Laboratory of Veterinary Pathobiology and Disease Control,Anbui Agricultural University,Hefei 230036.China)

机构地区：[1]安徽农业大学兽医病理生物学与疫病防控安徽省重点实验室,合肥230036

出　　处：《农业生物技术学报》2021年第3期521-528,共8页Journal of Agricultural Biotechnology

基　　金：安徽省高校自然科学研究重点项目(KJ2015A078);安徽省自然科学基金(2008085MC91);高校优秀青年人才项目(gxyq2018005)。

摘　　要：新生儿Fc受体(neonatal Fc receptor, FcRn)能够特异性识别并跨越黏膜屏障转运IgG。为了确定猪(Sus scrofa) FcRn与IgG抗体Fc段CH2结构域的互作关系,本研究通过逆转录PCR技术克隆获得猪IgG抗体Fc段CH2基因片段,并将其亚克隆至原核表达载体pEGX-4T-1,经异丙基硫代半乳糖苷(isopropylβ-D-thiogalactoside, IPTG)诱导融合蛋白表达并通过谷胱甘肽巯基转移酶(glutathione S-transferase, GST)亲和柱纯化。同时将IgG CH2基因片段亚克隆至真核表达载体pEGFP-C1,通过共转染确定IgG CH2与FcRn在非洲绿猴(Chlorocebus sabaeus)肾细胞(COS-7)中的细胞共定位,免疫共沉淀验证IgG CH2与FcRn的相互作用;进一步通过ELISA方法检测CH2与FcRn在不同pH值下的结合力。结果表明,克隆获得猪IgG CH2基因全长330 bp,编码110个氨基酸。诱导表达的融合蛋白GST-CH2相对分子量为38.4 k D,该蛋白在诱导菌体中以可溶性和包涵体两种形式存在。激光共聚焦观察显示,IgG CH2与FcRn共定位于细胞质中,具有共聚集现象。免疫共沉淀显示,通过标签蛋白GFP和Flag,在所有的共沉淀条带中,均能检测到对应的目的条带,说明IgG CH2能够结合FcRn。ELISA结果表明,IgG CH2与FcRn的结合呈现pH值依赖性。综上所述,本研究确定了FcRn与IgG抗体Fc段CH2结构域存在细胞共定位与互作关系,二者的结合具有pH依赖性,这为构建新的基于Fc的小型抗体融合蛋白提供了材料。Neonatal Fc receptor(FcRn) can specifically recognize and transport IgG across the mucosal barrier. In order to determine the interaction between porcine(Sus scrofa) FcRn and IgG Fc segment CH2 domain, the porcine IgG CH2 gene was cloned by the reverse transcription(RT-PCR) and further subcloned into prokaryotic expression vector PEGX-4 T-1. The fusion protein was induced by isopropyl β-Dthiogalactoside(IPTG) and purified by glutathione S-transferase(GST) Affinity Column. In addition, IgG CH2 gene was subcloned to eukaryotic expression vector pEGFP-C1 and Co-transfection was carried out to determine whether the IgG CH2 and FcRn co-located in African green monkey(Chlorocebus sabaeus) kidney cells(COS-7). The interaction of IgG CH2 and FcRn was verified by coimmunoprecipitation. Furthermore,the binding ability of IgG CH2 and FcRn under different pH condition was detected by ELISA. The results showed the amplified porcine IgG CH2 gene was 330 bp, encoding 110 amino acids. The relative molecular weight of fusion protein GST-CH2 was 38.4 kD. The GST-CH2 in the induced bacteria existed in the form of soluble protein and inclusion body. The soluble GST-CH2 protein will possess the better ability of combining the FcRn. Laser confocal microscope observation showed that the IgG CH2 and FcRn were located in the cytoplasm and had an aggregation phenomenon. The coimmunoprecipitation showed that the corresponding target bands could be detected in all the coprecipitation strips through labeled protein GFP and Flag, which indicated IgG CH2 could bind FcRn. By ELISA, it was found that the IgG CH2 and FcRn had the binding ability when pH value was 6.0, but they were not combined at pH 7.4, suggesting that the combination of IgG CH2 and FcRn was dependent on pH. In conclusion, the results showed that FcRn and CH2 domain of IgG Fc segment had a co-localization and interaction, their combination was pH-dependent, which provides materials for the construction of Fc-based small-size antibody fusion protein.

关 键 词：猪 IgG CH2 新生儿Fc受体(FcRn) 细胞定位 互作关系 

分 类 号：S831.2[农业科学—畜牧学]