猴源葡萄糖调节蛋白78生物信息学分析及其真核表达

doi:10.16431/j.cnki.1671-7236.2021.04.003

中国畜牧兽医 ›› 2021, Vol. 48 ›› Issue (4): 1170-1178.doi: 10.16431/j.cnki.1671-7236.2021.04.003

猴源葡萄糖调节蛋白78生物信息学分析及其真核表达

焦文强^1,2,3, 刘运超², 邢广旭², 陈鹏举⁴, 徐引弟^1,3, 王治方^1,3, 张青娴^1,3, 许峰^1,3

1. 河南省农业科学院畜牧兽医研究所, 郑州 450002;
2. 河南省农业科学院动物免疫学重点实验室, 郑州 450002;
3. 河南省畜禽繁育与营养调控重点实验室, 郑州 450002;
4. 河南省现代中兽医研究院, 郑州 450002

收稿日期:2020-09-18 出版日期:2021-04-20 发布日期:2021-04-16
通讯作者: 邢广旭 E-mail:11508559@qq.com
作者简介:焦文强(1982-),男,河南郑州人,博士,助理研究员,研究方向:分子病毒学,E-mail:wenqiangjiao@163.com
基金资助:
河南省农业科学院优秀青年科技基金（2020YQ25）；河南省科技攻关项目（212102110376）

Bioinformatics Analysis and Eukaryotic Expression of Chlorocebus sabaeus Glucose Regulatory Protein 78

JIAO Wenqiang^1,2,3, LIU Yunchao², XING Guangxu², CHEN Pengju⁴, XU Yindi^1,3, WANG Zhifang^1,3, ZHANG Qingxian^1,3, XU Feng^1,3

1. Institute of Animal Husbandry and Veterinary, Henan Academy of Agricultural Sciences, Zhengzhou 450002, China;
2. Henan Provincial Key Laboratory of Animal Immunology, Henan Academy of Agricultural Sciences, Zhengzhou 450002, China;
3. Key Laboratory of Livestock Breeding and Nutrition Regulation, Zhengzhou 450002, China;
4. Henan Institute of Modern Chinese Veterinary Medicine, Zhengzhou 450002, China

Received:2020-09-18 Online:2021-04-20 Published:2021-04-16

摘要/Abstract

摘要： 试验旨在探究葡萄糖调节蛋白78（glucose regulatory protein 78，GRP78）基因的理化性质和结构特点，阐述GRP78在猪流行性腹泻病毒复制中的分子伴侣作用和调控机制。通过RT-PCR方法扩增GRP78基因，并插入到pMD20-T Simple载体进行克隆测序，利用生物信息学方法对其氨基酸序列、跨膜结构、糖基化位点、磷酸化位点、三级结构等进行预测和分析。将GRP78基因插入pCDNA 3.1中，然后转染Vero-E6细胞。Western blotting检测Vero-E6细胞中GRP78的表达。生物信息学分析结果表明，GRP78基因全长1 965 bp，编码654个氨基酸，蛋白质分子质量为72.33 ku，理论等电点为5.07，分子式为C₃₁₈₉H₅₁₅₃N₈₆₅O₁₀₁₉S₁₃。遗传进化树分析显示，猴源GRP78基因与双峰驼、犬、猫、大猩猩、蝙蝠、狒狒、猪、马的氨基酸序列同源性为99.5%~99.8%；遗传进化分析显示，大猩猩和狒狒亲缘关系最为接近。跨膜区和信号肽预测结果显示，该蛋白存在信号肽但不存在跨膜结构。GRP78蛋白无N-糖基化修饰位点，存在6个O-糖基化位点、28个磷酸化位点，表明GRP78可能有与激酶磷酸化有关的PKC、PKA特异性蛋白激酶的结合位点，可能参与己糖代谢和单糖代谢。

关键词: GRP78基因; 生物信息学分析; 真核表达

Abstract: The present study was aimed to explore the physicochemical properties and structural characteristics of chlorocebus sabaeus glucose regulatory protein 78 (GRP78) gene,and to elucidate the molecular chaperone effect and regulatory mechanism of GRP78 gene in porcine epidemic diarrhea virus replication.GRP78 gene was amplified by RT-PCR and inserted into pMD20-T Simple vector for cloning and sequencing,and its amino acids sequence,transmembrane structure,glycosylation site,phosphorylation site,and tertiary structure were predicted and analyzed by bioinformatics methods.The GRP78 gene was inserted into pCDNA 3.1,and transfected into Vero-E6 cells.Western blot was employed to confirm the expression of GRP78 in Vero-E6 cells.Bioinformatics analysis results showed that the total length of GRP78 gene was 1 965 bp,encoding 654 amino acids.The molecular weight of GRP78 protein was 72.33 ku.The theoretical isoeletric point was 5.07,and the molecular formula was C₃₁₈₉H₅₁₅₃N₈₆₅O₁₀₁₉S₁₃.The homology of amino acid sequence of GRP78 gene in Chlorocebus sabaeus with Camelus bactrianus, Canis lupus, Felis catus, Gorilla, Myotis lucifugus, Papio anubis, Sus scrofa, Equus caballus were 99.5% to 99.8%.Genetic analysis showed that gorillas and baboons were the closest relatives.The prediction results of transmembrane region and signal peptide showed that the protein had signal peptide but no transmembrane region.The protein had 6 O-glycosylation sites and 28 phosphorylation sites but no N-glycosylation modification sites,which suggested that GRP78 might have binding sites for PKC and PKA specific protein kinases related to kinase phosphorylation,and might be involved in hexose metabolism and monosaccharide metabolism.

Key words: GRP78 gene; bioinformatics analysis; eukaryotic expression

中图分类号:

Q786

焦文强, 刘运超, 邢广旭, 陈鹏举, 徐引弟, 王治方, 张青娴, 许峰. 猴源葡萄糖调节蛋白78生物信息学分析及其真核表达[J]. 中国畜牧兽医, 2021, 48(4): 1170-1178.

JIAO Wenqiang, LIU Yunchao, XING Guangxu, CHEN Pengju, XU Yindi, WANG Zhifang, ZHANG Qingxian, XU Feng. Bioinformatics Analysis and Eukaryotic Expression of Chlorocebus sabaeus Glucose Regulatory Protein 78[J]. China Animal Husbandry and Veterinary Medicine, 2021, 48(4): 1170-1178.

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猴源葡萄糖调节蛋白78生物信息学分析及其真核表达

Bioinformatics Analysis and Eukaryotic Expression of Chlorocebus sabaeus Glucose Regulatory Protein 78

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