牛卵泡CART受体的筛选及其表达特性分析

Screening and Expression Analysis of CART Receptor in Bovine Follicle

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.