中国畜牧兽医2025，Vol.52Issue(2)：534-544,11.DOI:10.16431/j.cnki.1671-7236.2025.02.004
马山黑山羊MAPK8基因克隆、生物信息学分析及真核表达载体构建
Cloning,Bioinformatics Analysis and Eukaryotic Expression Vector Construction of MAPK8 Gene in Mashan Black Goats
摘要
Abstract
[Objective]This study was aimed to clone the mitogen-activated protein kinase 8(MAPK8)gene sequence in Mashan Black goats,conduct bioinformatic analysis,and construct its eukaryotic expression vector,so as to provide experimental materials for research on goat mammary gland development and the role of MAPK8 gene in transdifferentiation processes.[Method]Primers were designed based on MAPK8 gene sequence in Capra hircus(GenBank accession No.:XM_018042429.1).The MAPK8 gene CDS sequence was amplified from Mashan Black goats using PCR and cloned.Sequence alignment and phylogenetic tree construction were performed using DNAStar and Mega 11.0 software.Bioinformatic analysis was conducted using tools such as ProtParam and ProtScale.The eukaryotic expression vector was constructed,and lentiviral packaging was performed to collect viral supernatant,which infected the fibroblasts in Mashan Black goats.The expression of MAPK8 gene were detected using Real-time quantitative PCR in virus-infected and control groups.[Result]The sequence of MAPK8 gene CDS in Mashan Black goats was 1 155 bp in length,encoding a protein of 384 amino acids with a molecular weight of 43.98 ku.The molecular formula was C1968H3114N528O569S22,and the isoelectric point(pI)was 7.13.Similarity alignment results revealed that the amino acid sequence similarity of MAPK8 gene between Mashan Black goats and different species was higher than 85％,indicating that the amino acid sequence of MAPK8 gene was relatively conserved.The phylogenetic tree indicated that the MAPK8 amino acid sequence in Mashan Black goats was most closely related to that of Bos taurus and least related to that of Danio rerio.MAPK8 protein in Mashan Black goats was hydrophilic and did not belong to transmembrane proteins.MAPK8 protein was primarily distributed in cytoplasm(52.2％),mitochondria(26.1％),nucleus(17.4％)and cell membrane(4.3％),and contained a serine/threonine protein kinases,catalytic domain(amino acids 26-321).The secondary structure of MAPK8 protein in Mashan Black goats included random coil,alpha helix,beta turn and extended chain.Protein interaction predictions suggested that MAPK8 protein might interact with MAPK9,JUN,TP53,FOS,JUNB,MAP2K4,MAP2K7,MAPK8IP1 and NFATC3 proteins.The lentiviral eukaryotic expression vector of MAPK8 gene pCDH-CMV-MAPK8-mcherry-Puro was successfully constructed,and transfection into Mashan Black goat fibroblasts resulted in the production of a red fluorescence signal.The expression of MAPK8 gene in experimental group was extremely significantly higher than that in control group(P＜0.01).[Conclusion]In this study,the CDS sequence of MAPK8 gene in Mashan Black goats was successfully cloned,and the pCDH-CMV-MAPK8-mcherry-Puro eukaryotic expression vector was constructed,providing important foundational data for future research on the function and application of MAPK8 gene in goats.关键词
马山黑山羊/MAPK8基因/生物信息学/真核表达Key words
Mashan Black goats/MAPK8 gene/bioinformatics/eukaryotic expression分类
农业科技引用本文复制引用
雷志刚,潘宏,张丹丹,刘权辉,孙哲,邓珊,黄奔..马山黑山羊MAPK8基因克隆、生物信息学分析及真核表达载体构建[J].中国畜牧兽医,2025,52(2):534-544,11.基金项目
国家自然科学基金(32160171) （32160171）
