中国动物传染病学报2016，Vol.24Issue(2)：53-61,9.
柔嫩艾美耳球虫胱硫醚β合成酶基因克隆及在真核细胞中的表达
GENE CLONING AND EUKARYOTIC EXPRESSION OF CYSTATHIONINE β-SYNTHASE OF EIMERIA TENELLA
摘要
Abstract
The objective of the present study was to construct recombinant expression plasmids of cystathionine β-synthase of Eimeria tenella(Et CBS) and study the expression of Et CBS gene in transfected DF-1 cells. The full-length cDNA of Et CBS was obtained using rapid amplification of cDNA ends (RACE). The ORF of Et CBS was amplified in RT-PCR and ligated to the eukaryotic expression vector pBiFC-VC155. The recombinant plasmid pBiFC-VC155-Et CBS was confi rmed in PCR and restriction enzyme digestion. Subsequently, the recombinant plasmid was transfected into DF-1 cells. Western blot and indirect immunofluorescence assay (IFA) were used to examine the expression of recombinantEt CBS. The results showed that the full-length cDNA of Et CBS was 2423 bp in length and contained a 1887-bp ORF located between 201~2087 bp encoding 407 amino acids with molecular weight of 68 kDa. The deduced amino acidsequence of the protein had neither transmembrane region nor signal peptide. Western blot indicated that a protein with molecular weight of 68 kDa in the transfected cells was strongly recognized by the antiserum of recombinant EtCBS protein (rEt CBS). Specific green fluorescence was observed in the DF-1 cells by IFA. These results will provide important rationale to study the functions of Et CSB.关键词
柔嫩艾美耳球虫/胱硫醚β合成酶/DF-1细胞/真核表达Key words
Eimeria tenella/cystathionineβ-synthase/DF-1 cell/eukaryotic expression分类
农业科技引用本文复制引用
李聪,董辉,朱顺海,朱雪龙,王自文,赵其平,夏伟丽,门启斐,黄兵..柔嫩艾美耳球虫胱硫醚β合成酶基因克隆及在真核细胞中的表达[J].中国动物传染病学报,2016,24(2):53-61,9.基金项目
国家自然科学基金(31201699、31572266) （31201699、31572266）
农业部中央级公益性科研院所项目(2015JB10) （2015JB10）
