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

Abstract

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

CLC Number:

S852.4
Q51

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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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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