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

doi:10.16431/j.cnki.1671-7236.2025.02.004

摘要/Abstract

摘要： 【目的】克隆马山黑山羊丝裂原活化蛋白激酶8(mitogen-activated protein kinase 8,MAPK8)基因序列，并对该基因进行生物信息学分析，构建其真核表达载体，为山羊乳腺发育及MAPK8基因在转分化过程中的研究提供试验材料。【方法】参考山羊MAPK8基因序列(GenBank登录号：XM_018042429.1)设计引物，利用PCR扩增马山黑山羊MAPK8基因CDS序列并克隆；分别使用DNAStar和Mega 11.0软件进行序列比对及系统进化树构建，并利用ProtParam、ProtScale等软件进行生物信息学分析；构建真核表达载体并进行慢病毒包装后收集病毒液上清，感染马山黑山羊成纤维细胞，并通过实时荧光定量PCR检测感染病毒组和对照组MAPK8基因表达水平。【结果】马山黑山羊MAPK8基因CDS区全长1 155 bp，编码384个氨基酸，蛋白分子质量为43.98 ku，分子式为C₁₉₆₈H₃₁₁₄N₅₂₈O₅₆₉S₂₂，等电点(pI)为7.13。相似性比对结果显示，马山黑山羊与不同物种间MAPK8基因氨基酸序列相似性均高于85%，表明MAPK8氨基酸序列较为保守。系统进化树显示，马山黑山羊MAPK8基因氨基酸序列与牛的亲缘关系最近，与斑马鱼的亲缘关系最远。马山黑山羊MAPK8蛋白为亲水性，不属于跨膜蛋白，主要分布在细胞质(52.2%)、线粒体(26.1%)、细胞核(17.4%)和细胞膜(4.3%)中，含有丝氨酸/苏氨酸蛋白激酶催化结构域(第26―321位氨基酸处)。MAPK8蛋白二级结构包括无规则卷曲、α-螺旋、β-转角和延伸链。互作蛋白预测显示，MAPK8蛋白可能与MAPK9、JUN、TP53、FOS、JUNB、MAP2K4、MAP2K7、MAPK8IP1和NFATC3蛋白存在互作。试验成功构建MAPK8基因慢病毒真核表达载体pCDH-CMV-MAPK8-mchery-Puro，转染马山黑山羊成纤维细胞后产生红色荧光信号，试验组细胞内MAPK8基因表达量极显著高于对照组(P＜0.01)。【结论】本研究成功克隆了马山黑山羊MAPK8基因CDS序列，并构建了pCDH-CMV-MAPK8-mchery-Puro真核表达载体，为山羊MAPK8基因功能及应用研究提供了基础数据。

关键词: 马山黑山羊; MAPK8基因; 生物信息学; 真核表达

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.

Key words: Mashan Black goats; MAPK8 gene; bioinformatics; eukaryotic expression

中图分类号:

S827
Q78

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.

LEI Zhigang, PAN Hong, ZHANG Dandan, LIU Quanhui, SUN Zhe, DENG Shan, HUANG Ben. Cloning, Bioinformatics Analysis and Eukaryotic Expression Vector Construction of MAPK8 Gene in Mashan Black Goats[J]. China Animal Husbandry and Veterinary Medicine, 2025, 52(2): 534-544.

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