牛卵泡CART受体的筛选及其表达特性分析

doi:10.11843/j.issn.0366-6964.2020.03.010

摘要/Abstract

摘要： 旨在筛选可卡因-苯丙胺调节转录肽（cocaine and amphetamine regulated transcript peptide，CART）的受体，明确其受体在优势卵泡（dominant follicles，DF）和从属卵泡（subordinate follicles，SF）的表达特性。本研究使用琼脂糖Protein A/G磁珠免疫共沉淀（co-immunoprecipitation，Co-IP）鉴定CART及其相互作用蛋白；利用HMMTOP V2.0分析其跨膜次数并获得G蛋白偶联受体（G-protein-coupled receptors，GPCRs）；运用SWISS-MODEL和PDB数据库对CART和筛选出的GPCRs同源建模，获得模型分子的PDBQT文件，并通过评分函数对构建模型质量和每个氨基酸残基的模型质量进行评价；将CART和待分析受体PDBQT文件输入ZDOCK进行分子对接，获得复合体立体空间模型和评分函数值；利用qRT-PCR和免疫组织化学技术对筛选出的靶蛋白趋化因子样受体1（chemokine-like receptor 1，CMKLR1）在牛DF和SF中的表达及定位进行分析。Co-IP获得的111个蛋白质组分中包含10个膜蛋白，分别为A2M、C5、CMKLR1、COX2、DDOST、HEATR5A、B3AT、ADT2、RPN2、SLC4A1；其中CMKLR1具有7次跨膜的α螺旋结构，属于GPCRs；利用SWISS-MODEL建模技术构建CART和CMKLR1的分子模型，ZDOCK分子对接后获得复合体立体空间模型，其中评分函数值最高为1 977.34。qRT-PCR分析表明，CMKLR1 mRNA在SF中的表达量显著高于DF（P<0.05）；免疫组织化学分析结果表明，CMKLR1存在于牛DF和SF颗粒层、膜层，在SF颗粒层和膜层细胞显色强度均高于DF，这与qRT-PCR的分析结果相一致。结果显示，蛋白同源建模和分子对接技术应用于受体筛选是可行的。CMKLR1作为神经肽CART的候选受体，在SF的表达量显著高于DF，该研究对CART受体的鉴定及深入阐明CART调控牛卵泡发育的作用机理具有重要意义。

关键词: 卵泡, CART, 受体, 同源建模, 分子对接

Abstract: The aim of this study was to screen CART receptor and clarify its expression characteristics in dominant follicles (DF) and subordinate follicles (SF). CART and proteins associated with CART were identified by using immunomagnetic Protein A/G Co-IP; The membrane proteins were predicted, transmembrane times were analyzed, GPCRs were obtained by HMMTOP V2.0; CART and screened GPCRs were modeled homologously by using SWISS-MODEL and PDB database, PDBQT file of model molecules was obtained, respectively. The quality of constructed model and each amino acid residue were evaluated by scoring function; Inputting CART and PDBQT file of receptors to be analyzed in ZDOCK interface for molecular docking, respectively, complex three-dimensional space model and score function value were obtained; Expression and localization of CMKLR1 in bovine DF and SF were analyzed by qRT-PCR and immunohistochemistry. One hundred and eleven proteins were obtained by Co-IP, which contained 10 membrane proteins (A2M, C5, CMKLR1, COX2, DDOST, HEATR5A, B3AT, ADT2, RPN2, SLC4A1); CMKLR1 had 7 transmembrane helix structures, which belonged to GPCRs; Molecular models of CART and CMKLR1 were constructed using SWISS-MODEL technology, complex three-dimensional space model was obtained by ZDOCK molecular docking, and the highest score function value was 1 977.34. qRT-PCR analysis showed that expression level of CMKLR1 mRNA in SF was significantly higher than that in DF (P<0.05); Immunohistochemical analysis showed that CMKLR1 was expressed in GCs and membranelayer cells layer of DF and SF, and specific color intensity showed that expression levels of CMKLR1 in SF GCs and membrane cells were higher than those in DF, which were consistent with qRT-PCR results. The results show that protein homology modeling and molecular docking techniques are feasible for receptor screening. CMKLR1, as a candidate receptor for neuropeptide CART, has a significantly higher expression level in SF than DF. This study is of great significance for the identification of CART receptors and for elucidating the mechanism of CART regulating bovine follicular development.

Key words: follicle, CART, receptor, homology modeling, molecular docking

中图分类号:

S823.3

侯淑宁, 郝庆玲, 景炅婕, 王锴, 成俊丽, 吕丽华, 李鹏飞. 牛卵泡CART受体的筛选及其表达特性分析[J]. 畜牧兽医学报, 2020, 51(3): 505-513.

HOU Shuning, HAO Qingling, JING Jiongjie, WANG Kai, CHENG Junli, Lü Lihua, LI Pengfei. Screening and Expression Analysis of CART Receptor in Bovine Follicle[J]. Acta Veterinaria et Zootechnica Sinica, 2020, 51(3): 505-513.

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