羊口疮病毒129蛋白互作宿主蛋白的筛选与C1QBP基因的克隆分析

doi:10.16431/j.cnki.1671-7236.2023.01.001

Abstract

Abstract: 【Objective】 The aim of the present study was to screen and validate the interacting protein of Orf virus 129(ORFV129) using yeast two-hybrid experiment with the cDNA library from goat fetal turbinate cells (GFTCs).【Method】 The Smart^TM technology was used to construct the GFTCs cDNA library.A bait vector pGBKT7-129 was constructed, which was then transformed into Y2HGold yeast competent cells, to verify the activity of self-activating.The toxicity effect induced by pGBKT7-129 transfection was also evaluated by measuring the growth curve of the bacterial.Using ORFV129 as the bait vector, the host protein that interact with ORFV129 were screened and the positive colonies were identified by PCR method and sequencing.The GO databases was used for function annotation and pathway analysis, following which a Cytoscape v 3.8.0 software was used for protein-protein interaction visualization.The coding domain sequence of Complement C1q binding protein (C1QBP) gene was cloned by RT-PCR method from goat spleen tissue, which was then ligated to pcDNA3.1(+) to construct a eukaryotic expression vector pcDNA3.1-C1QBP, and it was transfected into GFTCs for subcellular localization analysis.【Result】 The GFTCs cDNA library was constructed successfully with a capacity of 6.0×10⁶ CFU/mL.The bait plasmid pGBKT7-129 was successfully constructed with no self-activation ability and no toxicity to yeast cells.A total of 14 cellular proteins interacted with ORFV129 were selected from yeast two-hybrid experiment and the positive clones were confirmed by PCR and sequencing.The CDS region of goat C1QBP gene was successfully cloned, with a length of 837 bp, encoding 279 amino acids.The phylogenetic tree showed that there was the closest genetic relationship between Capra hircus and Ovis aries.C1QBP protein was an unstable hydrophilic protein without signal peptide structure and transmembrane domain, mainly including three kinds of phosphorylation sites, including 18 serine, 4 threonine and 3 tyrosine sites.The secondary structure of C1QBP protein was composed of alpha helix (33.81%), random coil (46.40%), extended chain (16.91%) and beta turn (2.88%), and the tertiary structure was consistent with the secondary structure.Indirect immunofluorescence test showed that C1QBP protein was scattered in the cytoplasm.【Conclusion】 The host intracellular protein C1QBP gene that interacts with ORFV129 protein and plays a role in the natural immune response was screened.The indirect immunofluorescence test verified that C1QBP was located in the cytoplasm.It was speculated that ORFV129 interacted with C1QBP to induce inflammation, laying a foundation for further verifying the process of ORFV129 protein mediated ORFV inhibiting the immune response of the body.

Key words: goat fetal turbinate cells (GFTCs); Orf virus 129 (ORFV129); yeast two-hybrid; interacting protein; C1QBP

CLC Number:

S855.3

DAN Yixin, XIANG Hua, ZHANG Huanrong, YANG Lu, REN Yupeng, XU Songwei, HE Honghong, ZHU Jiangjiang. Screening of Host Proteins Interacting with ORFV129 Protein and Cloning and Analysis of C1QBP Gene[J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(1): 1-14.

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Screening of Host Proteins Interacting with ORFV129 Protein and Cloning and Analysis of C1QBP Gene

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