【作者】 张善瑞；

【导师】 童光志；

【作者基本信息】 中国农业科学院 ， 预防兽医学， 2010， 博士

【摘要】 猪繁殖与呼吸综合征(Porcine reproductive and respiratory syndrome,PRRS)是目前严重危害养猪业的传染病之一,该病是由猪繁殖与呼吸综合征病毒(PRRSV)感染引起的,其主要临床特征是怀孕母猪发生流产、早产和死胎等严重的繁殖障碍;仔猪与育肥猪有明显的呼吸道症状。至今为止,PRRS仍在全球大部分地区流行,是猪主要的传染病之一。2006年5月以来,我国暴发最初称为“高热综合症”的猪病,给我国的养猪业造成很大经济损失。多项研究已表明,变异的北美洲型高致病性猪繁殖与呼吸综合征病毒(HP-PRRSV)是该病的主要病原。但是到目前为止PRRSV毒力增强的机制还不清楚,因此,非常有必要建立高致病性猪繁殖与呼吸综合征病毒(HP-PRRSV)的反向遗传学技术操作平台。本研究分别构建成功了HP-PRRSV HuN4和疫苗株F112的感染性克隆,并且构建了PRRSV的标记疫苗。本实验根据高致病性猪繁殖与呼吸综合征病毒HuN4株基因组序列设计并合成PRRSV特异性引物,进而应用RT-PCR技术分6段扩增了PRRSV HuN4株全基因组cDNA。将扩增的各个cDNA重叠片段分别克隆到pG1SK载体上。在扩增5’末端时,引入sp6启动子序列用于后期的体外转录得到病毒的转录本,在基因组3’末段Poly(A)尾引入SwaI酶切位点用于线性化包含HuN4全长cDNA的质粒,将HuN4基因组第14680位的A沉默突变为G产生一个MluI酶切位点作为鉴定拯救病毒的genomic marker。将含HuN4株基因组全长cDNA的质粒线性化后体外转录合成病毒RNA,转染BHK-21C细胞,24h后将细胞液上清接种Macr-145细胞,拯救出病毒。通过间接免疫荧光和检测genomic marker证明病毒拯救成功。比较亲本病毒与拯救病毒的病毒滴度、多步生长曲线,发现病毒学及生物学特性没有显著差异。以上结果表明PRRSV强毒株HuN4株感染性克隆构建成功,为研究PRRSV的分子生物学以及致病机制提供了技术平台。根据GeneBank中登陆的高致病性猪繁殖与呼吸综合征病毒HuN4株基因组序列设计并合成特异性引物,在扩增5′末端时引入sp6启动子核心序列,在基因组3′末段Poly(A)尾后引入NotI酶切位点用于线性化包含F112全长cDNA的质粒。应用RT-PCR技术分6段扩增了PRRSV疫苗株F112全基因组cDNA,将扩增的各个cDNA重叠片段胶回收后先通过合适的酶切位点分别克隆到载体pG2SK,再逐一拼接成全长cDNA。将全长cDNA克隆线性化后体外转录合成病毒RNA,转染BHK-21C细胞,24h后将细胞液上清接种Macr-145细胞,拯救出病毒。通过比较亲本病毒与拯救病毒的病毒滴度、多步生长曲线,结果发现病毒学及生物学特性没有显著差异。结果表明F112感染性克隆构建成功,建立PRRSV疫苗株F112的反向遗传学技术平台,为探讨PRRSV的致病机制以及研制有效的标记疫苗奠定了基础。PRRSV感染时机体内可以产生滴度很高的针对于NSP2的抗体,许多NSP2的B细胞表位已经被鉴定出来。利用PRRSV疫苗株F112感染性克隆,将NSP2上的B细胞免疫优势表位(第430-454位氨基酸)缺失替换为鼠肝炎病毒S2糖蛋白的优势B细胞表位5B19,构建标记疫苗株。通过体外转录,转染BHK21细胞,在Marc-145细胞上的传代,拯救出了病毒,通过RT-PCR测序和间接免疫荧光试验证明含遗传标记的突变株病毒拯救成功。试验结果表明PRRSV NSP2的B细胞表位(第430-454位氨基酸)对病毒的繁殖来说是非必须的,并且可以在NSP2区域插入外源表位进行表达,对于PRRSV新型疫苗的研制提供了基础数据和思路。更多还原

【Abstract】 Porcine reproductive and respiratory syndrome (PRRS) has become one of the most common and economically significant infectious diseases in theswine industry worldwide. It is characterized by mild to severe reproductive failure in sows and gilts and respiratory problems in piglets. Since May 2006, atypical PRRS (so-called porcine high fever syndrome (PHFS) in china) was pandemic in china. Several studies have confirmed that the causative agent of this outbreak was highly pathogenic PRRSV (HP-PRRSV), several HP-PRRSV strains were isolated and their reverse genetic systems were developed by different lab. Many questions of PRRSV, such as, the mechanism of the PRRSV virlence determents are still unclear. So it is viry crucial to development reverse genetic systems of PRRSV. In this study, reverse genetic systems of HP-PRRSV HuN4 and vaccine strain HuN4-F112 were developted, and a marker vaccine was developed based on the HuN4-F112 reverse genetic systems.A total of six fragments, covering the complete PRRSV HuN4 strain genome, were PCR amplified. All PCR-amplified fragments were gel purified, digested with the proper enzymes, and cloned into the pG1SK vector. The bacteriophage SP6 RNA polymerase promoter was engineered into 5’end of HuN4 genome, SwaI enzyme was engineered downstream of the poly (A) tail for the further linearization of the full-length cDNA. The A at position 14680 was mutated to G by PCR to create a restriction enzyme site MluI as the genetic marker. The completely assembled full-length cDNA clone was confirmed by sequence and enzyme digestion. Capped RNA was transcribed in vitro from a full-length cDNA clone of the viral genome and transfected into BHK-21C cells. The supernatant from transfected monolayers were serially passaged on Marc-145 cells. The virus rescued from this newly assembled cDNA clone was identified by IFA and the detection of the genetic marker. In vitro studies demonstrated that the cloned virus maintained growth properties similar to those of the parental virus. The availability of a full-length cDNA clone of PRRSV HuN4 strain lays a new ground for further investigation of PRRSV virulence and development of new potent vaccine.In order to develop a reverse genetic system for the research of PRRSV, a full-length cDNA clone was constructed in this study. A total of six fragments, covering the complete PRRSV HuN4-F112 strain genome, were PCR amplified. All PCR-amplified fragments were gel purified, digested with the proper enzymes, and cloned into the pG2SK vector. The bacteriophage SP6 RNA polymerase promoter was engineered into 5′end of HuN4-F112 genome, NotIenzyme was engineered downstream of the poly (A) tail for the further linearization of the full-length cDNA. The A at position 14680 was mutated to G by PCR to create a restriction enzyme site MluI as the genetic marker. Capped RNA was transcribed in vitro from a full-length cDNA clone of the viral genome and transfected into BHK-21C cells. The supernatant from transfected monolayers were serially passaged on Marc-145 cells. The virus rescued from this newly assembled cDNA clone were indistinguishable from the parental virus. The availability of genome sequence information and infectious cDNA clone of HuN4-F112 lays a new ground for further investigation of PRRSV virulence and development of new potent vaccine.Nsp2 protein of PRRSV is an immunogenic protein capable of eliciting specific antibody production during viral infections.The Nsp2 protein was found to contain a large number of linear B-cell epitopes in both the European type and North American type PRRSV strains. Based on the reverse genetic system of PRRSV vaccine strain HuN4-F112, a B-cell epitope of the S2 glycoprotein of murine hepatitis virus (MHV) was inserted in-frame to replace a linear B-cell epitope of nsp2(430-454aa). Recombinant viruses properly expressing the introduced MHV epitope were successfully generated, demonstrating that the B-cell epitopes not only is dispensable for virus replication but also can be replaced by foreign sequences. Our results provide proof of the concept that DIVA PRRSV vaccines can potentially be developed by replace of individual“marker”immunodominant epitopes.更多还原