肠道免疫相关的猪富含半胱氨酸肠蛋白2三维建模、分子特征及mRNA表达的组织分布

doi:10.11843/j.issn.0366-6964.2021.011.021

畜牧兽医学报 ›› 2021, Vol. 52 ›› Issue (11): 3194-3207.doi: 10.11843/j.issn.0366-6964.2021.011.021

肠道免疫相关的猪富含半胱氨酸肠蛋白2三维建模、分子特征及mRNA表达的组织分布

李美娣¹, 赵锃珏², 刘汉清³, 傅嘉莉¹, 张玲华^2*, 武力^1*

1. 广东华农高科生物药业有限公司, 广州 510642;
2. 华南农业大学生命科学学院广东省农业生物蛋白质功能与调控重点实验室, 广州 510642;
3. 华南农业大学兽医学院, 广州 510642

收稿日期:2021-03-01 出版日期:2021-11-23 发布日期:2021-11-24
通讯作者: 张玲华,主要从事生物化学与分子生物学研究,E-mail:lhzhang@scau.edu.cn;武力,主要从事新兽药研究与推广,E-mail:1187131629@qq.com
作者简介:李美娣(1981-),女,广东梅州人,博士,高级兽医师,主要从事新兽药研究与推广,E-mail:limeidi@163.com;赵锃珏(2000-),男,湖南湘潭人,本科生,主要从事微生物与免疫研究,E-mail:zj_zhao@stu.scau.edu.cn。
基金资助:
广东省自然基金项目（2018A030313625）

Porcine Cysteine-rich Intestinal Protein 2 Involved in the Gut Immune: Three-dimensional Modeling, Molecular Characterization and Tissue Distribution of mRNA Expression

LI Meidi¹, ZHAO Zengjue², LIU Hanqing³, FU Jiali¹, ZHANG Linghua^2*, WU Li^1*

1. Guangdong Huanong Hi-Tech Biological Pharmaceutical Co. Ltd., Guangzhou 510642, China;
2. Guangdong Provincial Key Laboratory of Protein Function and Regulation in Agricultural Organisms, College of Life Sciences, South China Agricultural University, Guangzhou 510642, China;
3. College of Veterinary Medicine, South China Agricultural University, Guangzhou 510642, China

Received:2021-03-01 Online:2021-11-23 Published:2021-11-24

摘要/Abstract

摘要： 本研究利用广泛用于肠道感染机制研究的IPEC-J2细胞系探究poCRIP2在猪胃肠道炎症中的作用。在识别poCRIP2基因全长序列的基础上研究poCRIP2的有关特征，同时对其组织表达模式进行分析，并建立有效的poCRIP2三维结构模型以解释其功能，了解poCRIP2在细菌感染过程中的免疫作用及其与NF-κB通路的关系。采用逆转录聚合酶链反应（RT-PCR）技术从猪心获得poCRIP2的全长序列；利用Primer Premier5.0和NCBI Primer BLAST程序设计了特异性引物对，采用保家基因β-肌动蛋白（β-actin）对结果进行归一化处理，测试CRIP2基因在猪体内的组织分布；采用生物信息学方法对poCRIP2的特性进行分析，构建poCRIP2蛋白模型；利用ROBETTA服务器，以高度同源的小鼠LIM-homeodomain protein islet 1（Isl1）（PDB：4 JCJ）为模板，模拟poCRIP2的三维结构；用PROCHECK和ProSA对模型进行了分析；应用Procheck对蛋白质折叠进行立体化学和拓扑分析，并应用ProSA对蛋白质折叠进行评价；使用PyMOL程序对建模结果进行评估，分析poCRIP2的保守结构域；设计肠道感染试验，分析poCRIP2在肠道免疫中的功能。结果表明，从猪心获得的poCRIP2 cDNA全长序列为1 118 bp，poCRIP2蛋白与人和小鼠的同源性分别为94.23%和93.75%，它还含有两个保守区（LIM-TLP和LIM-CRP）。poCRIP2的组织表达模式分析表明，poCRIP2在所有组织中均有表达，其中在小肠、肺、胃、脾和肌肉等中表达较低。poCRIP2在革兰阴性菌作用下的表达水平比对照组高1~2倍，但NF-κB通路在同一作用下被激活，无抑制作用，poCRIP2可能不与NF-κB通路相互作用，而与其它通路共同作用于肠道免疫。本研究成功获得了猪CRIP2基因的全长序列，poCRIP2的组织表达模式表明，poCRIP2与人CRIP2相似，建立了可靠的三维结构模型，基于该模型推测poCRIP2中包含的功能主要由β折叠和α-螺旋结构实现，革兰阴性菌感染肠道后poCRIP2能被显著上调，poCRIP2应不依赖于NF-κB通路，在肠道免疫中起其他作用。

关键词: poCRIP2, 长白猪, IPEC-J2, 感染

Abstract: This study uses the IPEC-J2 cell line, which is widely used in studying the mechanism of intestinal infection, to explore the role of poCRIP2 in porcine gastrointestinal inflammation. The full-length sequence of poCRIP2 gene was identified and the relevant characteristics and its tissue expression pattern were analyzed. An effective three-dimensional structure model of poCRIP2 was established to explain its function, and to understand the immune function of poCRIP2 in the process of bacterial infection and its relationship with NF-κB pathway. Using reverse transcription polymerase chain reaction (RT-PCR) technology to obtain the full-length sequence of poCRIP2 from pig heart; using Primer Premier5.0 and NCBI Primer BLAST program to design a specific primer pair, using the protection gene β-actin normalizes the results to test the tissue distribution of the CRIP2 gene in pigs; using bioinformatics methods to analyze the characteristics of poCRIP2 to construct the poCRIP2 protein model; using the ROBETTA server and the high homology mouse LIM-homeodomain protein islet 1 (Isl1) (PDB: 4 JCJ) to simulate the three-dimensional structure of poCRIP2; the model was analyzed by PROCHECK and ProSA; the stereochemical and topological analysis of protein folding was performed by Procheck, and applied ProSA to evaluate protein folding; PyMOL program was used to evaluate the modeling results and analyzes the conserved domains of poCRIP2; designs an intestinal infection test to analyze the function of poCRIP2 in intestinal immunity. The results showed that the full-length sequence of poCRIP2 cDNA obtained from porcine heart was 1 118 bp. The homology of poCRIP2 protein to human and mouse were 94.23% and 93.75%, respectively. It also contained two conserved regions (LIM-TLP and LIM-CRP). Tissue expression pattern analysis of poCRIP2 showed that poCRIP2 was expressed in all tissues, but less in the small intestine, lung, stomach, spleen and muscle and other tissues. The expression level of poCRIP2 under the action of Gram-negative bacteria is 1~2 times higher than that of the control group, but the NF-κB pathway is activated under the same action without inhibition. PoCRIP2 may not interact with the NF-κB pathway, but may have combined effect with other pathways on intestinal immunity. This study has successfully obtained the full-length sequence of the pig CRIP2 gene. The tissue expression pattern of poCRIP2 shows that poCRIP2 is similar to human CRIP2. A reliable 3D structural model has been established. Based on this model, it is speculated that the functions contained in poCRIP2 are mainly composed of β folding and α-helical structure are realized, and poCRIP2 can be significantly upregulated after Gram-negative bacteria infect the intestine. PoCRIP2 may not depend on the NF-κB pathway and play other roles in intestinal immunity.

Key words: poCRIP2, Landrace, IPEC-J2, infection

中图分类号:

S852.4
Q51

李美娣, 赵锃珏, 刘汉清, 傅嘉莉, 张玲华, 武力. 肠道免疫相关的猪富含半胱氨酸肠蛋白2三维建模、分子特征及mRNA表达的组织分布[J]. 畜牧兽医学报, 2021, 52(11): 3194-3207.

LI Meidi, ZHAO Zengjue, LIU Hanqing, FU Jiali, ZHANG Linghua, WU Li. Porcine Cysteine-rich Intestinal Protein 2 Involved in the Gut Immune: Three-dimensional Modeling, Molecular Characterization and Tissue Distribution of mRNA Expression[J]. Acta Veterinaria et Zootechnica Sinica, 2021, 52(11): 3194-3207.

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肠道免疫相关的猪富含半胱氨酸肠蛋白2三维建模、分子特征及mRNA表达的组织分布

Porcine Cysteine-rich Intestinal Protein 2 Involved in the Gut Immune: Three-dimensional Modeling, Molecular Characterization and Tissue Distribution of mRNA Expression

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