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

为构建柔嫩艾美耳球虫保守蛋白Et CHP559基因的真核表达重组质粒pc DNA3.1-flag-Et CHP559,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Et CHP559基因全长c DNA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 k D,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pc DNA3.1-flag连接,构建了真核重组表达质粒pc DNA3.1-flag-Et CHP559,所构建的真核重组表达质粒pc DNA3.1-flag-Et CHP559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 k D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Et CHP559的全长序列,并成功构建了Et CHP559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Et CHP559的功能奠定了基础。

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-I cells, Western blot recognized target protein of around 47 kD, and immunofiuorescence 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 ceils, which laid a foundation for the future research on functions of EtCHP559.