【作者】 邢福珊；

【导师】 张彦明；

【作者基本信息】 西北农林科技大学 ， 临床兽医学， 2011， 博士

【摘要】 猪圆环病毒2型(Porcine circovirus type 2,PCV-2)是断奶仔猪多系统衰竭综合征(Post-weaning multisystemic wasting syndrome, PMWS)的主要病原,不但可以引起断奶仔猪发生衰竭、死亡,还与仔猪先天性震颤(Congenital tremors CT)、猪皮炎与肾病综合征(Porcine dermatitis and nephropathy syndrome,PDNS)和猪呼吸道疾病综合征(Porcine respiratory disease complex,PRDC)有关,更重要的是PCV-2的感染可以造成仔猪免疫抑制。PCV-2常和猪繁殖与呼吸综合征病毒、细小病毒、伪狂犬病毒混合感染,造成猪的免疫失败,继而引起其他病原的继发性感染,造成的间接损失非常大。目前,PCV-2已经成为严重阻碍养猪业发展的主要病原。PCV-2基因组为闭合环状单链DNA,全长1 767 bp或1 768 bp,含2个开放阅读框,即ORF1和ORF2。ORF1编码病毒复制相关蛋白。ORF2编码病毒衣壳蛋白,即病毒的结构蛋白,衣壳蛋白有很好的免疫原性,但在PCV-1和PCV-2之间不存在交叉保护。因此,研究PCV-2的ORF2基因对猪圆环病毒的检测和疫苗研究具有重要意义。杆状病毒是一种自然感染昆虫细胞的病毒,该病毒的吸附和侵入细胞的作用与囊膜糖蛋白gp64有关。gp64由N-端信号肽和具有跨膜区、胞质区的成熟蛋白组成。gp64已被开发成病毒表面展示系统以展示各种目的蛋白。将外源基因片段插入病毒衣壳蛋白的信号肽与成熟蛋白之间,经后加工作用信号肽被切除,形成的融合蛋白能稳定地表达在杆状病毒粒子表面,筛选后就可以得到能表达目的蛋白的重组杆状病毒,这种表达方式稳定、可靠,用来开发基因工程疫苗简捷、快速,现在已被广泛地应用到研发和实践。本研究对杆状病毒展示系统展示PCV-2衣壳蛋白进行了研究,并对展示的蛋白进行了动物免疫试验,获得以下结果:(1)设计一对特异性引物P1、P2,以提取的PCV-2 YL株DNA为模板,采用PCR方法成功获得了PCV-2 YL株的ORF2基因片段,将该片段胶回收后与pGEM-T载体连接,转化DH5α感受态细胞,经筛选获得阳性克隆,PCR和酶切鉴定正确的质粒测序。测序正确的质粒命名为pGEM-T-ORF2。结果显示目的片段大小为702bp,共编码234个氨基酸,表达的目的蛋白分子质量为28ku。DNAStar软件分析表明,PCV-2 YL株ORF2基因与GenBank中其他的PCV-2毒株的核苷酸同源性在89.9%～98%之间,编码的氨基酸同源性在88.5%～98.3%之间。(2)将pGEM-T-ORF2质粒和载体pET-32a用BamHI / XhoI双酶切后,连接构建pET-32a-ORF2重组表达质粒。重组质粒转染大肠埃希菌,用IPTG诱导表达。经SDS-PAGE检测表明ORF2在大肠埃希菌中得到表达,Western blot检测证实表达的蛋白能与猪圆环病毒2型阳性血清发生特异性结合。(3)参考Bac-to-Bac真核表达载体的酶切位点和ORF2基因序列设计一对引物,以pGEM-T-ORF2为模板进行PCR扩增,将回收的扩增产物与Bac-to-Bac真核表达载体分别进行XhoI、PstI双酶切,回收酶切片段并将其连接,构建了重组杆状病毒BscSC-ORF2。用重组病毒感染昆虫细胞后,经免疫荧光-共聚焦显微镜观察,重组衣壳蛋白能有效地表达在昆虫细胞膜上。将培养的病毒高速离心浓缩,经免疫金电子显微镜观察,证明重组蛋白表达在了杆状病毒囊膜上。(4)用重组杆状病毒免疫小鼠后,中和试验表明,小鼠产生了PCV-2的特异性抗体,抗体的中和效价达到1:12.8;淋巴细胞转化增殖试验表明,重组杆状病毒能诱发机体产生较强的细胞免疫应答。本研究成功构建了表面展示PCV-2 Cap蛋白的重组杆状病毒,为进一步应用该重组杆状病毒作为亚单位疫苗预防PCV-2的感染奠定了基础。更多还原

【Abstract】 Porcine circovirus type 2(PCV-2)is the major pathogen for the post-weaning multisystemic wasting syndrome ( PMWS ) and cause exhaustion and death in post-weaning piglets. PCV-2 also is relevant to congenital tremor (CT),porcine dermatitis and nephropathy syndrome(PDNS) and porcine respiratory disease complex(PRDC). PCV-2 can casue immnosuppression in infected piglets. PCV-2 also co-infected with porcine reproductive and respiratory syndrome virus(PRRSV), Porcine parvovirus(PPV) and Pseudorabies virus(PRV) in pig population and cause immune failure of infected pigs which will be followed by the secondary infetions of other pathogens.Hence we can see that PCV-2 infection has already become one of the major pathogens for the pig industry and can cause immeasurable indirect losses.The genome of PCV-2 is a closed circular single-stranded DNA of about 1 767 bp or 1 768 bp,which consists of two open reading frames(ORF),ORF1 and ORF2.ORF1 encodes virus replication associated protein. ORF2 encodes virus capsid protein(virus structural protein).This structural protein has fairly good immnogenicity,but it has no cross protection between PCV-1 and PCV-2. So a better understanding of the ORF2 proteinof PCV-2 will be very important for the PCV detection and vaccine development.The baculoviruses are a family of large rod-shaped viruses that naturally only infect the insect cells.The attachment and invasion process of the virus are associated with the envelope glycoproteingp64 which consists of N-terminus signal peptide and maturation protein(including transmembrane domain and cytoplasmic domain).gp64 has already been developed as the viral surface display system to display various objective proteins. The exogenous gene segment inserted between the signal peptide and the maturation protein.After the post-transcriptional processing,the signal peptide will be cutted off.The new fusion protein will be express steadily on the surface of the virion. After several times of selection,the recombinant baculoviruses with specific protein being expressed can be obtained.This expression pathway is stable and reliable.It is brief and fast when applied to develop genetically engineering vaccines.This technology has been widely applied to design and develop new genetically engineering vaccines.This sdudy display capsid protein of PCV-2 using baculovirus display system and animal immune experiment about the displayed protein. A conclusions of this study are following:(1)A pair of specific primers was designed( P1/P2). ORF2 gene of PCV-2 was amplified by PCR from PCV-2 YL strains and ligated with pGEM-T vector. The recombinant plasmids were transformed into Escherichia coli (E.coli)DH5α. The positive colonies were screened through the white-blue plaque selection. Then the bacteria were cultured in LB medium.The recombiant plasmid was extracted and identified by PCR and restriction enzyme digestion . The ORF2 gene of PCV-2 YL strains was sequenced and then compared with other PCV-2 strains available on GenBank. The correct plasmid was named as pGEM-T-ORF2. The results showed that amplified fragment was 702 bp,encode 234 amino acids and expressed a 28ku protein. The nucleotide identity and amino acids homology of PCV-2 ORF2 were 89.9%-98% and 88.5%-98.3% with other PCV-2 strains available on GenBank.(2)The ORF2 fragment was obtained from pGEM-T-ORF2 digested with BamHI / XhoI and inserted into plasmid pET-32a digested with tha same enzyme to construct the recombinant expression plasmid pET-32a-ORF2. E.coli competent cell was transformed with pET-32a-ORF2 and induced with IPTG.. The fusion protein was expressed in E.coli and reacted with the positive serum of PCV-2 by Western blot, it showed the expressed protein had fairly good antigenicity.(3)A pair of primer was designed according to the sequences of the eukaryotic expression vector Bac-to-Bac and the ORF2 of Porcine circovirus type 2. The complete ORF2 fragment was amplified from recombinant plasmid pGEM-T-ORF2 by PCR. The purified ORF2 fragment was digested with XhoI and PstI and inserted into the eukaryotic expression vector Bac-to-Bac digested with the same enzymes to construct the recombinant baculovirus BscSC-ORF2. The recombiant capsid protein was effectively expressed on the transfectd insect cell membrane observed with immunofluorescence-confocal microscope. The recombiant virus was obtained by high-speed centrifuge and the recombinant protein was observed on the baculovirus sac membrane by immuno-gold electron microscopy.(4)Animal immune experiments indicated that the recombinant protein could trigger fairly strong immune response.Neutralization test indicated that sera from mice immuned with the recombinant baculoviruses could effectively neutralize PCV-2 protein.Lymphocyte transformation prove experiment indicated that the recombinant baculoriruses could stimulate stronger cell mediated immune respone.PCV-2 capsid protein was expressed using baculorirus display system in this study.The reconbinant baculoriruse displaying PCV-2 capsid protein was successfully constructed and established a fundament for preventing PCV-2 infection with the recombinant baculoriruses. The recombinant baculoriruse can be developed a candidate of subunit vaccine for preventing PCV-2 infection.更多还原