柔嫩艾美耳球虫保守蛋白CHP559基因克隆与真核表达

doi:10.13560/j.cnki.biotech.bull.1985.2015.07.024

生物技术通报 ›› 2015, Vol. 31 ›› Issue (7): 161-168.doi: 10.13560/j.cnki.biotech.bull.1985.2015.07.024

柔嫩艾美耳球虫保守蛋白CHP559基因克隆与真核表达

翟颀, 黄兵, 董辉, 赵其平, 朱顺海, 梁思婷, 李莎, 杨斯涵, 韩红玉

(中国农业科学院上海兽医研究所农业部动物寄生虫学重点实验室，上海 200241)

收稿日期:2014-10-24 出版日期:2015-07-16 发布日期:2015-07-16
作者简介:翟颀，男，硕士研究生，研究方向：寄生虫分子生物学；E-mail：zhaixiaoqi@126.com
基金资助:
国家自然科学基金项目(31201699)，中央级公益性科研院所基本科研业务费专项(2014JB03)

Gene Cloning and Eukaryotic Expression of a Conserved Hypothetical Protein Gene CHP559 in Eimeria tenella

Zhai Qi, Huang Bing, Dong Hui, Zhao Qiping, Zhu Shunhai, Liang Siting, Li Sha, Yang Sihan, Han Hongyu

(Key Laboratory of Animal Parasitology of Ministry of Agriculture，Shanghai Veterinary Research Institute of Chinese Academy of Agricultural Sciences，Shanghai 200241)

Received:2014-10-24 Published:2015-07-16 Online:2015-07-16

摘要/Abstract

摘要： 为构建柔嫩艾美耳球虫保守蛋白EtCHP559基因的真核表达重组质粒pcDNA3.1-flag-EtCHP559，并转染DF-1细胞进行表达。利用5' RACE技术克隆获得了柔嫩艾美耳球虫EtCHP559基因全长cDNA序列，该基因全长为1 746 bp，ORF为104-1 327 bp，编码407个氨基酸，编码蛋白的分子量约为46 kD，并利用生物信息学分析该基因编码蛋白的性质、结构等特征，发现该蛋白含有跨膜结构和信号肽。通过RT-PCR扩增得到含完整开放阅读框的基因片段，并将其与真核表达载体pcDNA3.1-flag连接，构建了真核重组表达质粒pcDNA3.1-flag-EtCHP559，所构建的真核重组表达质粒pcDNA3.1-flag-EtCHP559经过PCR和双酶切鉴定，可见一条大小约为1 224 bp的目的条带；重组质粒转染DF-1细胞后，免疫印迹检测可见大小约为47 kD的目的蛋白条带，间接免疫荧光实验显示在DF-1细胞中检测到绿色荧光。这些研究结果表明已成功获得了EtCHP559的全长序列，并成功构建了EtCHP559的真核重组表达质粒，在真核细胞DF-1中获得表达，为深入研究EtCHP559的功能奠定了基础。

关键词: 柔嫩艾美耳球虫, CHP559基因, DF-1细胞, 真核表达

Abstract: The objective of this study is to construct eukaryotic recombinant expression plasmids of a conserved hypothetical protein gene(EtCHP559)of Eimeria tenella, and study the expression of EtCHP559 gene in transfected DF-1 cells. The full-length cDNA of EtCHP559 was cloned using 5'RACE approaches. Bioinformatics analysis revealed that the gene contained 1 746 bp, of which the ORF was 1 224 bp(position 104 - 1 327 bp)encoding 407 amino acids with molecular weight of 46 kD. The deduced amino acid sequence of the protein had a transmembrane region and signal peptide. The cDNA fragment consisting of complete ORF was amplified by RT-PCR, and ligated to the eukaryotic expression vector pcDNA3. 1-flag. The recombinant plasmid pcDNA3. 1-flag-EtCHP559 was identified successfully by PCR and restriction enzyme digestion, and the target band of approximate 1 224 bp was observed. Subsequently, the recombinant plasmid was transfected into DF-1 cells, Western blot recognized target protein of around 47 kD, and immunofluorescence also showed that green fluorescence in transfected DF-1 cells was observed. These results indicated that the full-length sequence of EtCHP559 was obtained, and the recombinant plasmid of EtCHP559 was successfully constructed and expressed in eukaryotic cells, which laid a foundation for the future research on functions of EtCHP559.

Key words: Eimeria tenella, CHP559 gene, DF-1 cell, eukaryotic expression

翟颀, 黄兵, 董辉, 赵其平, 朱顺海, 梁思婷, 李莎, 杨斯涵, 韩红玉^{. 柔嫩艾美耳球虫保守蛋白CHP559基因克隆与真核表达[J]. 生物技术通报, 2015, 31(7): 161-168.}

Zhai Qi, Huang Bing, Dong Hui, Zhao Qiping, Zhu Shunhai, Liang Siting, Li Sha, Yang Sihan, Han Hongyu. Gene Cloning and Eukaryotic Expression of a Conserved Hypothetical Protein Gene CHP559 in Eimeria tenella[J]. Biotechnology Bulletin, 2015, 31(7): 161-168.

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柔嫩艾美耳球虫保守蛋白CHP559基因克隆与真核表达

Gene Cloning and Eukaryotic Expression of a Conserved Hypothetical Protein Gene CHP559 in Eimeria tenella

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