利用BiFC技术研究柔嫩艾美耳球虫顶膜抗原1结构域Ⅰ与棒状体颈部蛋白2互作关系

DEMONSTRATION OF INTERACTION BETWEEN DOMAIN Ⅰ OF APICAL MEMBRANCE ANTIGEN 1 AND RHOPTRY NECK PROTEIN 2 OF EIMERIA TENELLA BY BIFC TECHNOLOGY

为了验证柔嫩艾美耳球虫(Eimeria tenella)顶膜抗原1结构域Ⅰ(apical membrance antigen 1 domain I,E tAMA1-DⅠ)与棒状体颈部蛋白2(rhoptry neck protein 2,EtRON2)的互作关系,以柔嫩艾美耳球虫子孢子cDNA为模板,PCR扩增出492 bp的E tAMA1-DⅠ片段和1395 bp的E tRON2片段,并与pGEM-T-easy载体连接构建相应重组质粒。获得的阳性重组质粒及双分子荧光互补技术的真核表达载体pBiFC-VN155和pBiFC-VC155用E c oRⅠ和B g I II进行双酶切,将E tAMA1-DⅠ、E tRON2分别与pBiFC-VN155、pBiFC-VC155连接,构建真核重组质粒pBiFC-VN155-E tAMA1-DⅠ和pBiFC-VC155-E tRON2。将2个真核重组质粒分别转染BHK细胞进行表达,经间接免疫荧光鉴定,可在BHK细胞中成功表达。将2个真核重组质粒共转染至BHK细胞中,同时将pBiFC-bJunVN155和pBiFC-bFos(deltaZIP)VC155、pBiFC-bJunVN155和pBiFC-bFos(delta)VC155共转染至细胞中分别作为阳性和阴性对照组。BiFC结果发现真核重组质粒共转染组和阳性对照组的BHK细胞均产生绿色荧光,而阴性对照组无荧光,表明E tAMA1-DⅠ与E tRON2蛋白之间存在互作关系。本研究结果为深入研究E tAMA1及E tRON2在球虫入侵过程中的功能与作用机制奠定基础。

In order to understand the interaction between domain I of apical membrance antigen 1 (Et AMA1-D Ⅰ) and rhoptry neck protein 2 (Et RON2) of Eimeria tenella (E.tenella ),a 492 bp fragment of Et AMA1-DⅠ and a 1395 bp fragment of Et RON2 were amplified by PCR using the cDNAs of E.tenella sporozoite as template.The PCR products were subcloned into pGEM-T-easy vector for constructing the recombinant plasmids and confirmed with PCR and sequencing.Both recombinant plasmids and bimolecular fluorescence complementary technology (BiFC) eukaryotic expression vectors pBiFC-VN155 and pBiFC-VC155 were double-digested with Eco R Ⅰ and Bg I II.The Et AMA1-DⅠand Et RON2 were ligated with respective pBiFC-VN155 and pBiFC-VC155 to construct recombinant eukaryotic plasmids pBiFC-VN155-Et AMA1-DⅠand pBiFC-VC155-Et RON2.Recombinant eukaryotic plasmids were transfected into BHK cells and visualized with indirect immunofluorescence assay.The result showed that these plasmids were expressed in BHK cells.The recombinant eukaryotic plasmids pBiFC-VN155-Et AMA1-DⅠand pBiFC-VC155-Et RON2 were co-transfected into BHK cells.At the same time,the plasmids pBiFC-bJunVN155 and pBiFC-bFos (deltaZIP) VC155,pBiFC-bJunVN155 and pBiFC-bFos (delta) VC155 were co-transfected into BHK cells to serve as positive and negative controls.The BiFC results showed that BHK cells co-transfected with pBiFC-VN155-Et AMA1-DⅠand pBiFC-VC155-Et RON2 as well as positive control produced green fluorescence while negative control was not,indicating there was an interaction between Et AMA1-DⅠ and EtRON2.These results would help further elucidate the function and mechanism of Et AMA1 and Et RON2 in Eimeria invasion.