鸡朊样蛋白Doppel基因的克隆及其与PrPC相互关系分析

doi:10.11843/j.issn.0366-6964.2015.08.019

畜牧兽医学报 ›› 2015, Vol. 46 ›› Issue (8): 1425-1431.doi: 10.11843/j.issn.0366-6964.2015.08.019

鸡朊样蛋白Doppel基因的克隆及其与PrP^C相互关系分析

张天亮¹，吴润^1*，杨润霞¹，魏姣¹，万学瑞¹，刘霞²，刘磊¹，张小丽¹

（1.甘肃农业大学动物医学院，兰州 730070；2.甘肃农业大学生命科学技术学院，兰州 730070）

收稿日期:2014-11-25 出版日期:2015-08-23 发布日期:2015-08-23
通讯作者: 吴润，男，教授，博士生导师，E-mail：wurun@gsau.edu.cn
作者简介:张天亮（1988-），男，甘肃天水人，硕士生，主要从事鸡朊蛋白及朊样蛋白研究，E-mail：382598970@qq.com
基金资助:
国家自然科学基金资助项目（31160510）；甘肃省自然科学研究基金计划项目（1107RJZA198）

Clone of Chicken Prion-like Doppel Gene and the Analysis of Its Interrelation with PrP^C

ZHANG Tian-liang¹，WU Run^1* ，YANG Run-xia¹，WEI Jiao¹，WAN Xue-rui¹，LIU Xia²，LIU Lei¹，ZHANG Xiao-li¹

(1.College of Veterinary Medicine，Gansu Agricultural University，Lanzhou 730070，China；2.College of Life Science and Technology，Gansu Agricultural University，Lanzhou 730070，China)

Received:2014-11-25 Online:2015-08-23 Published:2015-08-23

摘要/Abstract

摘要：

克隆鸡Doppel蛋白（ChDpl）基因（PRND）并初步分析其与正常细胞型朊蛋白（PrP^C）的相互关系，为禽源Dpl结构与功能、Dpl与PrP^C互作关系机制的研究提供理论依据与分子基础；以多物种Dpl氨基酸序列为基础，通过比对找到Dpl氨基酸序列保守区，设计简并引物并依据鸡的密码子偏好性对引物进行优化，以逐步降低其简并性。用此特异性引物以聚合酶链式反应（PCR）扩增鸡PRND基因，并将其克隆到pMD-18-T载体，鉴定后将序列上传GenBank数据库。结合Dpl与PrP^C相关研究初步分析两者的相互关系；多物种Dpl氨基酸序列比对发现，第11—150位（139个氨基酸残基）为Dpl氨基酸序列保守区，以此区域设计引物并进行PCR扩增，得到约417 bp的目的条带，GenBank登录号为KP140962。综合分析PrP^C与Dpl相关研究发现，尽管Dpl与PrP^C具有相似的翻译后修饰和空间结构，但两者多表现为拮抗作用，即PrP^C能够拮抗Dpl的神经毒作用，尤其是其N-端包含八肽重复区的第23—88位残基对于保护Purkinje细胞免受Dpl诱导的神经退化作用至关重要。禽源Dpl的成功克隆不仅填补了目前研究的空白，更在分子水平为后续研究Dpl结构与功能及其与PrP^C的拮抗机制奠定了基础。

Abstract:

This study aimed to clone the chicken Doppel protein (ChDpl) gene (PRND)，preliminary analyze its relationship with the normal cellular prion protein (PrP^C)，and to provide a theoretical and molecular basis for the research of avian Dpl structure and function，and the mechanism of the interaction relationship between Dpl and PrP^C.Based on the amino acid sequence of multi-species Dpl，the conserved region of Dpl amino acid sequence was found，and the degenerate primers and optimize primers were designed with chicken codon preference to reduce the degeneracy gradually.Chicken PRND gene was amplified by polymerase chain reaction (PCR) with the specific primers，and then was inserted into pMD-18-T vector.The sequence was uploaded to the GenBank database after identified.Besides，the relationship between Dpl and PrP^C was analyzed.Through the alignments of multi-species Dpl’s amino acid sequence，we found that the section 11-150 bits (140 amino acid residues) was the conserved region of Dpl’s amino acid sequence，by designing primers with this area and PCR amplification，we obtained about 417 bp target band，its GenBank accession number was KP140962.Through comprehensively analysis of the related researches of Dpl and PrP^C，we found that although Dpl and PrP^C have a similar post-translational modifications and spatial structure，they mostly show antagonism，namely PrP^C can antagonize the neurotoxicity of Dpl，especially the 23-88 th residues of its N-terminal peptide containing octapeptide repeat region are crucial for the protection of Purkinje cells from Dpl induced neurodegeneration.The successful cloning of avian Dpl not only fills the gaps of current researches，even more at the molecular level laid a foundation for further study of the structure and function of Dpl and its antagonistic mechanism with PrP^C.

中图分类号:

S852.659.7

张天亮，吴润，杨润霞，魏姣，万学瑞，刘霞，刘磊，张小丽. 鸡朊样蛋白Doppel基因的克隆及其与PrP^C相互关系分析[J]. 畜牧兽医学报, 2015, 46(8): 1425-1431.

ZHANG Tian-liang，WU Run，YANG Run-xia，WEI Jiao，WAN Xue-rui，LIU Xia，LIU Lei，ZHANG Xiao-li. Clone of Chicken Prion-like Doppel Gene and the Analysis of Its Interrelation with PrP^C[J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2015, 46(8): 1425-1431.

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参考文献

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鸡朊样蛋白Doppel基因的克隆及其与PrP^C相互关系分析

Clone of Chicken Prion-like Doppel Gene and the Analysis of Its Interrelation with PrP^C

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鸡朊样蛋白Doppel基因的克隆及其与PrPC相互关系分析

Clone of Chicken Prion-like Doppel Gene and the Analysis of Its Interrelation with PrPC

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鸡朊样蛋白Doppel基因的克隆及其与PrP^C相互关系分析

Clone of Chicken Prion-like Doppel Gene and the Analysis of Its Interrelation with PrP^C