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当前位置: 首页 > 文章 > 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 中国动物传染病学报 2016,24 (2) 53-61
Position: Home > Articles > GENE CLONING AND EUKARYOTIC EXPRESSION OF CYSTATHIONINE β-SYNTHASE OF EIMERIA TENELLA Chinese Journal of Animal Infectious Diseases 2016,24 (2) 53-61

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

作  者：
李聪;董辉;朱顺海;朱雪龙;王自文;赵其平;夏伟丽;门启斐;黄兵
单  位：
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开放实验室
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
摘  要：
为构建柔嫩艾美耳球虫胱硫醚β合成酶(Eimeria tenella cystathionineβ-synthase,Et CBS)基因的真核重组表达质粒p Bi FC-VC155-Et CBS,利用RACE技术克隆了Et CBS基因的全长c DNA序列,对其编码的蛋白进行相关生物信息学分析。通过RT-PCR扩增含完整开放阅读框(open reading frame,ORF)的c DNA片段,将其与真核表达载体p Bi FC-VC155连接,经PCR、双酶切和测序鉴定正确后,转染DF-1细胞,用免疫印迹和间接免疫荧光技术鉴定重组蛋白表达情况。结果显示,成功获得了Et CBS基因的全长c DNA序列,该基因全长2423 bp,ORF位于第201~2087 bp之间,编码629个氨基酸,编码蛋白的分子量约为68k Da。生物信息学分析发现该基因编码的蛋白不具有信号肽,也不存在跨膜结构。Western blot可见大小约为68 k Da的重组表达蛋白条带,间接免疫荧光实验显示在转染的DF-1细胞中能检测到绿色荧光。研究结果表明,本研究已成功获得了Et CBS基因的全长c DNA序列,成功构建了Et CBS基因含完整ORF的真核重组表达质粒,并在DF-1细胞中实现了表达,为深入研究Et CBS基因的功能奠定了基础。
译  名：
GENE CLONING AND EUKARYOTIC EXPRESSION OF CYSTATHIONINE β-SYNTHASE OF EIMERIA TENELLA
作  者：
LI Cong;DONG Hui;ZHU Shun-hai;ZHU Xue-long;WANG Zi-wen;ZHAO Qi-ping;XIA Wei-li;MEN Qi-fei;HUANG Bing;HAN Hong-yu;Key Laboratory of Animal Parasitology of Ministry of Agriculture,Shanghai Veterinary Research Institute,CAAS;College of Life and Environment Sciences;Shanghai Normal Unversity;Jiangsu Coinnovation Center for Prevention and Control of Important Animal Infections Diseases and Zoonoses;
关键词：
Eimeria tenella;;cystathionine β-synthase;;DF-1 cell;;eukaryotic expression
摘  要：
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 c DNA of Et CBS was obtained using rapid amplification of c DNA ends(RACE). The ORF of Et CBS was amplified in RT-PCR and ligated to the eukaryotic expression vector p Bi FC-VC155. The recombinant plasmid p Bi FC-VC155-Et CBS was confirmed 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 recombinant Et CBS. The results showed that the full-length c DNA 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 k Da. The deduced amino acid sequence of the protein had neither transmembrane region nor signal peptide. Western blot indicated that a protein with molecular weight of 68 k Da in the transfected cells was strongly recognized by the antiserum of recombinant Et CBS protein(r Et 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.

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