【作者】 刘玉松；

【导师】 夏平安； 崔保安；

【作者基本信息】 河南农业大学 ， 预防兽医学， 2010， 硕士

【摘要】 目前研究发现PRRSV感染中存在抗体依赖性增强现象,亚中和剂量抗体能结合靶细胞表面Fc受体而增强病毒的感染。Fc受体主要有FcγRⅠ(CD64)、FcγRⅡ(CD32)、FcγRⅢ(CD16)和FcgRIV四个亚群,FcγRII(CD32)在人发现存在FcγRIIA,FcγRIIB和FcγRIIC三种亚型。FcγRIIB是抑制型受体,已在人、小鼠和牛上发现了两类主要的剪接异构体。本实验克隆并鉴定了猪FcγRIIB剪接异构体,为研究其介导PRRSV侵入细胞的机制奠定基础。根据GenBank中猪IgGIIB类Fc受体(swFcγRⅡB)(DQ026064)cDNA基因序列,利用Primer软件设计合成了一对特异性引物,应用RT-PCR技术,从猪外周血白细胞cDNA中扩增swFcγRⅡB基因,将扩增的基因片段插入到pTG19-T载体中,进行序列测定和分析。结果表明,克隆的swFcγRⅡB基因序列(FJ608551)长度为951bp,编码316个氨基酸,包括45个氨基酸的N-端信号肽序列,179个氨基酸的胞外区序列,23个氨基酸组成的疏水跨膜区序列, 69个氨基酸的胞质尾区序列;其胞外区有5个N-糖基化位点和4个半胱氨酸残基形成Ig样结构的2个二硫键;依据Ig保守结构,Cys71和Cys113形成第一个Ig样EC1结构域,Cys152和Cys196形成第二个Ig样EC2结构域。克隆序列与发表的swFcγRⅡB(DQ026064)的核苷酸与氨基酸序列同源性分别为99.3%和98.3%,其胞质尾区多19个氨基酸的插入,与人类FcγRⅡb1剪接异构体相似。根据克隆的猪FcγRⅡB剪接异构体cDNA基因序列,利用Primer软件设计合成四对特异性引物,扩增信号肽加胞外区、胞外区、EC1结构域和EC2结构域的DNA片段,经kpn I和EcoR I双酶切后亚克隆到原核表达载体pET-32a中,分别构建了pET-ZY、pET-EY、pET-AY和pET-BY四种重组表达质粒,在IPTG诱导下成功表达swFcγRⅡb1-ZY、swFcγRⅡb1-EY、swFcγRⅡb1-AY、swFcγRⅡb1-BY四种融合蛋白,均以不溶性包涵体的形式存在。swFcγRⅡb1-ZY、swFcγRⅡb1-EY、swFcγRⅡb1-AY、swFcγRⅡb1-BY四种融合蛋白经纯化后分别免疫小鼠,制备抗融合蛋白抗体,经ELISA检测,效价分别为1:5120、1:5120、1:5120、1:10240。Western blotting检测表明,制备的多抗可以与各自的融合蛋白特异性结合,表明制备的多克隆抗体具有高度特异性。本试验将克隆的swFcγRⅡB剪接异构体cDNA基因亚克隆至pcDNA3.0真核表达载体中,构建了质粒pcDNA3-swFcγRⅡb1,利用脂质体瞬时转染COS-7细胞,经流式细胞术检测,发现swFcγRⅡB剪接异构体在COS-7细胞表面表达。同时转染细胞经G418筛选,4周后用间接免疫荧光检测,发现swFcγRⅡB剪接异构体能在COS-7细胞中稳定表达,表明建立了稳定表达swFcγRⅡB剪接异构体的COS-7细胞系。通过稳定表达COS-7细胞系的红细胞吸附试验,证明了swFcγRⅡB剪接异构体结合IgG免疫复合物的生物学活性。本试验研究结果将为进一步研究swFcγRⅡB剪接异构体功能作用及其介导的PRRSV侵入细胞的相互作用机制奠定基础。更多还原

【Abstract】 In PRRS studied to date,the process of infection for PRRSV existed the antibody -dependent enhancement phenomenon, the PRRSV infectted target cell which was enhanced by the link of deuto-neutralization dose antibodies and Fc receptors.Four different classes of Fc receptors have been defined:FcγRI(CD64), FcγRII (CD32), FcγRIII (CD16), and FcγRIV. FcgRII (CD32) is an FγR previously shown to exist three isoforms: FcγRIIA,FcγRIIB and FcγRIIC. FcγRIIB is an inhibitory FγR, Two transcript variants of FcγRIIB have been demonstrated in humans and mice, In this study,we cloned and characterizated swFcγRⅡB transcript variant,whitch established foundation to study and research the process of infection for PRRSV.According to the cDNA sequence of the porcine Fc gamma receptor IIB (swFcγRIIB ) in the GeneBank,a pair of primer was designed using primer.The gene of swFcγRⅡB transcript variant was amplificated from porcine peripheral white blood cells using RT-PCR. Amplificated gene fragment was inseted into pTG19-T vector to sequencing.The putative epitope of the swFcγRⅡB transcript variant’s amino acid sequence was predicted by DNAStar Protean.According to the seqense analysis,the swFcγRⅡB transcript variant’s cloning gene fragment is 951bp,and encodes 316 amino acids, The first 45 amino acids were predicted to be an N-teminal secretory signal peptide.The mature protein is composed of a 179 amino acid extracellular region followed by a hydrophobic region of 23 amino acids representing a putative trans-membranedomain and a 69 amino acid cytoplasmic tail. The extracellular region was found to include five potential N-glycosylation sites (Asn 34, Asn 44, Asn 135 Asn 142 and Asn 166) and four conserved cysteines that form the characteristic disulphide bonds of the immunoglobulin domains. From the conserved immunoglobulin domain structure it is suggested that Cys 71 is linked to Cys 113 to formdomain 1 (EC1), and that Cys 152 is paired with Cys 192 to form domain 2 (EC2).The homology of nucleotide and amino acid sequence between FJ608551 and DQ026064 is 99.3% and 98.3%.Its intracellular region is augmented 19 amino acids compared with the publicated sequence.Its amino acid sequence exists more than 3 putative dominant antigen epitopes. The porcine Fc gamma receptor IIB exists subgroup,the gene we got is one kind of RNA transcript variant of the porcine Fc gamma receptor IIB.It can be predicted that the RNA transcript variant of the porcine Fc gamma receptor IIB exists at least two kinds. The swFcγRⅡB transcript variant cDNA codes for a cytoplasmic amino acid sequence of 69 amino acids and so appears most similar to the FcγRIIb1 isoform in human.According to the cDNA sequence(FJ608551)of the porcine Fc gamma receptor IIB’s (swFcγRIIB’s)RNA transcript variant in the GeneBank, four pair of primers were designed using primer. The gene was amplificated from swFcγRⅡB transcript variant abscised intracellular region and transmembrane domain,extracellular region,EC1 region of swFcγRⅡB transcript variant, EC2 region of swFcγRⅡB transcript variant.The PCR products were cutted by kpnI and EcoRI, The swFcγRⅡB transcript variant truncated gene were cloned into pET-32a vector to generate the recombinant expressing plasmid pET-ZY,pET-EY,pET-AY,pET-BY.The swFcγRIIB -His fusion protein was successfully obtained with IPTG, The protein predominantly existed as inclusion bodies in the cytoplasm.After expression in E.coil and purification ,the purifide protein swFcγRIIb1-ZY, swFcγRIIb1-EY, swFcγRIIb1-AY, swFcγRIIb1-BY were used to immunizi mouse to obtain the antiserum. The results of ELISA and Western blotting indicated that the prepared ployconal antibody had high titer and specificity. The purifide protein of swFcγRIIB-His and its polyclonal antibody lays the foundation for fourther reserch on swFcγRⅡB transcript variant.According to the cDNA sequence(FJ608551)of the porcine Fc gamma receptor IIB’s (swFcγRIIB’s)RNA transcript variant in the GeneBank, a pair of primer was designed using primer. The gene was amplificated from the complete swFcγRⅡB transcript variant. The PCR product was cutted by kpnI and EcoRI, The swFcγRⅡB transcript variant gene was cloned into pcDNA3.0 vector to generate the recombinant expressing plasmid pcDNA-swFcγRIIb1.Thongh double digestion by kpnI/EcoR I and PCR identification,951 bp of DNA fragment was obtained,indicating that the recombinant plasmid pcDNA-swFcγRIIb1 was successfully constucted .The 951 bp fragment was then transfected into COS-7 cells by the liposome method, Flow-cytometric and Immunofluorescence detection analysis expression of the swFcγRⅡB transcript variant proteins on COS-7 cells,and the ligand affinity of was identified by Rosetting procedures.更多还原