【作者】 贾松涛；

【导师】 崔保安； 吴志明；

【作者基本信息】 河南农业大学 ， 预防兽医， 2008， 硕士

【摘要】 猪圆环病毒2型(Porcine circovirus type 2, PCV2)是引发猪断奶后多系统衰竭综合征的主要病原。PCV2主要由ORF1和ORF2构成,ORF2基因是PCV2的重要抗原基因,编码病毒的衣壳蛋白(Cap蛋白),在Cap蛋白上存在抗原表位,具有免疫原性,因此ORF2基因成为建立PCV2特异性血清学检测方法及核酸疫苗研究的目的基因。当前国内外尚没有PCV2疫苗可用,因此研制猪圆环病毒2型核酸疫苗对控制猪PCV2病的发生具有重要意义。设计含双酶切位点的引物采用PCR方法扩增PCV2的ORF2全长基因并克隆入pGEM-T easy克隆载体;采用重叠延伸PCR(splicing by overlap extension-PCR,SOE-PCR)技术将PCV2的ORF2全长基因和猪白介素2成熟肽基因( PoIL-2 )构建成重组嵌合基因(PCV2-linker-PoIL-2)并克隆入pGEM-T easy克隆载体;筛选含PCV2-linker-PoIL-2、PCV2 ORF2的阳性克隆质粒经限制性内切酶双切后连接入经相同酶切的pcDNA3.1(+)真核表达载体中以构建重组表达质粒(rpcDNA3.1/ PCV2-linker-PoIL-2、rpcDNA3.1/ORF2)。筛选阳性重组表达质粒rpcDNA3.1/PCV2-linker-PoIL-2、rpcDNA3.1/ORF2瞬时转染COS-7细胞,分别采用PCV2抗原间接免疫荧光试验和PoIL-2蛋白ELISA试验方法检测COS-7细胞中表达的重组融合蛋白(rCap-linker-PoIL-2、rCap )免疫反应活性;提取rpcDNA3.1/PCV2-linker-PoIL-2质粒和rpcDNA3.1-OFR2质粒分别对Balb/c小鼠和猪进行三次免疫,采用PCV2抗体ELISA检测方法以评价其免疫效果。结果表明:成功构建了单重组表达质粒pcDNA3.1/OFR2和PCV2-linker-PoIL-2嵌合基因及其共表达质粒rpcDNA3.1/PCV2-linker-PoIL-2 ; rpcDNA3.1/PCV2-linker-PoIL-2和rpcDNA3.1/OFR2质粒在COS-7细胞浆内进行了表达,表达的rCap-linker-PoIL-2蛋白可分别与抗PCV2和抗PoIL-2蛋白抗血清发生特异性免疫反应,表明rCap-linker-PoIL-2蛋白具有Cap蛋白和PoIL-2蛋白的双重免疫反应活性。rpcDNA3.1/PCV2-linker-PoIL-2质粒免疫小鼠后可诱导小鼠产生明显的抗PCV2抗体,第3次加强免疫后,免疫组抗体滴度最高是空白对照组的3.2倍,且显著高于rpcDNA3.1/OFR2质粒对照组; rpcDNA3.1/PCV2-linker-PoIL-2和rpcDNA3.1/ORF2质粒均可诱导猪体产生抗PCV2免疫应答,rpcDNA3.1/PCV2-linker-PoIL-2、rpcDNA3.1/ORF2质粒免疫组抗体滴度分别是空载体对照组的12.46倍、10.13倍,表明PCV2-linker-PoIL-2嵌合基因中的PoIL-2对rpcDNA3.1/PCV2-linker-PoIL-2质粒在小鼠体内的免疫起到了很好的免疫增强作用。本研究成功构建了PCV2-linker-PoIL-2嵌合基因及其重组表达质粒pcDNA3.1/PCV2-linker-PoIL-2;重组表达质粒在COS-7细胞中表达了具有Cap蛋白和PoIL-2蛋白双重免疫反应活性的融合蛋白,免疫小鼠和猪后可诱导小鼠和猪产生高的抗PCV2抗体,且其诱导的抗体水平明显高于pcDNA3.1/OFR2重组质粒。本研究为PCV2的DNA疫苗研究奠定了基础。更多还原

【Abstract】 Porcine circovirus type 2 (Porcine circovirus type 2, PCV2) are the main pathogens and trigger Postweaning multisystemic wasting syndrome.ORF1 and ORF2 are main open reading frames in PCV2,the ORF2 gene is an important antigen genes, the protein-coding (Cap protein), the existence of epitope in Cap epitope, with immunogenicity, so ORF2 gene is an objective gene which use establishing PCV2 specific serology and researching DNA vaccine of the PCV2. Current, no available PCV2 vaccine in domestic and foreign, the development of PCV2 DNA vaccine to control disease of PCV2 is of great significance.The primers with double restriction site were designed and amplify ORF2 gene of PCV2.The ORF2 gene was cloned into pGEM-T easy cloning vector. The recombinant chimeric gene of PCV2-linker-PoIL-2 was constructed by PCV2 ORF2 gene linked porcine interleukin-2 (PoIL-2) mature peptide gene via a 15-amino acid glycine-rich linker [linker(,G4S)3] by SOE-PCR (splicing by overlap extension-PCR). The recombinant chimeric gene was cloned into pGEM-T Easy vector and subsequently into eukaryotic expression pcDNA3.1(+) vector. The positive recombinant expression plasmid rpcDNA3.1/PCV2-linker-PoIL-2 and r pcDNA3.1/ORF2 were transfected into COS-7 cells by lipofectamine. Indirect immunofluorescent assay (IFA) and porcine IL-2 ELISA assay was used to detect the bioactivities for PCV2 Cap protein and PoIL-2 protein of the expressed recombinant fusion protein (rCap-linker-PoIL-2 protein), respectively. The positive recombinant expression plasmid were extracted and inoculated Balb/c mice and pigs. The immune effects induced by rpcDNA3.1/PCV2-linker-PoIL-2 and rpcDNA3.1/OFR2 was evaluated by ELISA assay for detection PCV2 antibody in Balb/c mice and pigs. The result showed the chimeric gene of PCV2-linker-PoIL-2 and its recombination expression plasmid were successfully constructed. The rCap-linker-PoIL-2 protein was expressed in the cytoplasm of COS-7 cells and displayed the specific immune responses for anti-PCV2 and anti-PoIL-2 antibodies, which indicated the rCap-linker-PoIL-2 protein possessing the duplex bio-activity of Cap protein and PoIL-2 protein. the mice have been immunited by rpcDNA3.1/PCV2-linker-PoIL-2 plasmid and have a clear anti-PCV2 antibodies,after the 3rd immunization,the immunity group antibody is the control group of 3.2 times, and significantly higher than the rpcDNA3.1/OFR2 plasmid control group; rpcDNA3.1/PCV2-linker-PoIL-2 and rpcDNA3.1/ORF2 plasmid can be induced in pigs production of anti-PCV2 immune response, rpcDNA3.1/PCV2-linker - PoIL-2, rpcDNA3.1/ORF2 plasmid immunohistochemistry antibody titers were empty vector control group of 12.46 times and 10.13 times that PoIL-2 in PCV2-linker-PoIL-2 chimeric gene played a very good immune-enhancing effect to rpcDNA3.1/ PCV2-linker-PoIL-2 plasmid immune in mice and pigs.The successful construction of the PCV2-linker-PoIL-2 chimeric gene and its pcDNA3.1 eukaryotic expression plasmid rpcDNA3.1/PCV2-linker-PoIL-2; the recombinant expression plasmid have expressed biological activity fusion protein of Cap-and PoIL-2 in COS-7 cells .The recombinant plasmid can induce mice and pigs have a high anti-PCV2 antibody in mice and pigs and the induced antibody levels were significantly higher than the pcDNA3.1/OFR2 recombinant plasmid.This study for the PCV2 DNA vaccine research laid the foundation.更多还原