【作者】 任宪刚；

【导师】 薛飞；

【作者基本信息】 中国农业科学院 ， 预防兽医学， 2009， 博士

【摘要】 牛传染性鼻气管炎(Infectious bovine rhinotracheitis,IBR)是由牛疱疹病毒1型(Bovine hepervirus-1,BHV-1)引起的以牛呼吸道病为主要临床症状的疾病,是造成养牛业损失的一种重要传染病。BHV-1为α疱疹病毒,基因组为135kb左右的双链DNA,像其他疱疹病毒一样,BHV-1基因组中有许多病毒复制非必需基因,可允许插入外源DNA,且BHV-1具有感染宿主范围小,弱毒株致弱背景明确的优点,是最有望成为构建多价牛传染病疫苗的理想载体,现已有很多外源病毒DNA成功地插入到了BHV-1基因组中。口蹄疫(Foot and mouth disease,FMD)是由口蹄疫病毒(Foot and mouth disease virus,FMDV)引起偶蹄动物的一种高度接触性、传染性疾病,FMD能够形成全球大规模流行,造成巨大经济损失。FMDV属于小RNA病毒科,基因组为单股正链RNA。研究证实结构蛋白VP1具有与细胞受体结合的主要功能,是病毒感染细胞的关键,且VP1蛋白可在体外实验及自然宿主中诱导产生中和抗体,是研究各种FMD基因工程疫苗的首要候选蛋白。本研究旨在构建表达FMDV(O/China/99)VP1基因的重组BHV-1,并对其部分生物学特性以及在动物体内的免疫原性进行研究,为研制口蹄疫及其他重要牛传染病的BHV-1病毒载体疫苗奠定基础。为了构建表达O型FMDV(O/China /99)VP1基因的重组BHV-1,利用人工合成的基因质粒与构建BHV-1 gE基因缺失转移载体时所用的质粒,构建了含VP1表达盒的gE基因缺失转移载体,该载体含有巨细胞病毒(cytomegalovirus,CMV)早期启动子控制下的VP1基因、以及用于同源重组的BHV-1gE基因上游与下游同源重组序列。采用磷酸钙介导转染法将该转移载体与自行构建的呈gE基因缺失、并引入大肠杆菌β-半乳糖苷酶基因(β-Galactosidase gene,LacZ)的重组病毒BHV-1/gE-/LacZ+基因组DNA共转染牛鼻甲细胞,采用反向病毒蚀斑筛选法,获得了表达VP1基因的重组病毒BHV-1/gE-/VP1,PCR检测结果表明VP1基因已经插入到了重组病毒BHV-1/gE-的基因组中,间接免疫荧光试验和Western blot证实了BHV-1/gE-/VP1中的VP1基因在感染的细胞中获得了表达。为了鉴定FMDV(O/China/99)VP1基因在BHV-1/gE-/VP1感染细胞中的表达,以原核表达系统表达了FMDV(O/China/99)VP1基因并制备了其多克隆抗体作为基础材料。首先采用PCR方法从质粒pUC57-VP1中扩增FMDV VP1基因,并将其克隆至原核表达载体pET-30a(+)中,获得重组质粒pET30a-VP1。重组质粒鉴定正确后,转化至大肠杆菌BL21感受态细胞,经IPTG诱导后,SDS-PAGE结果显示重组蛋白的分子量约为38ku,以包涵体的形式存在。Western blot与间接ELISA结果显示重组蛋白能够与FMD阳性血清反应。将初步纯化的重组蛋白免疫BALB/c鼠制备多克隆抗体,作为鉴定VP1基因在重组病毒感染的细胞中获得表达的基础材料。为了比较BHV-1/gE-/VP1与亲本病毒(BHV-1/gE-/LacZ+)之间的增殖能力、BHV-1/gE-/VP1的遗传稳定性以及在动物体内的免疫原性,测定了BHV-1/gE-/VP1和BHV-1/gE-/LacZ+在MDBK细胞上的半数组织培养感染量(50% tissue culture infective dose,TCID50);并将BHV-1/gE-/VP1连续在MDBK细胞上传15代,取5、10、15代病毒,提取病毒基因组DNA后扩增VP1基因;以及将1ml 1×107.0 TCID50的BHV-1/gE-/VP1和1ml 1×107.0 TCID50的BHV-1/gE-/LacZ+分别皮下免疫4只新西兰白兔后,每周采血,首免两周以同样剂量加强免疫,分别采用病毒中和试验和间接ELISA法检测免疫动物血清中的IBRV和FMDV VP1的抗体。结果表明BHV-1/gE-/VP1和BHV-1/gE-/LacZ+的TCID50分别为107.9/ml和107.3/ml;以5、10、15代病毒基因组DNA为模板均能扩增出VP1基因;中和试验和ELISA能够分别检测到BHV-1/gE-/VP1免疫动物血清中的IBRV和FMDV VP1的抗体。本研究构建了表达FMDV VP1基因的重组病毒BHV-1/gE-/VP1,且VP1基因能够在感染BHV-1/gE-/VP1的细胞中获得表达;与BHV-1/gE-/LacZ+相比,BHV-1/gE-/VP1的增殖能力无差异,VP1基因也能在BHV-1/gE-/VP1的传代过程中稳定存在;动物试验表明BHV-1/gE-/VP1能够诱导兔体产生抗IBRV和FMDV VP1蛋白的抗体。该研究为研制口蹄疫及其他重要牛传染病的BHV-1病毒载体疫苗奠定了基础。更多还原

【Abstract】 Infectious bovine rhinotracheitis （IBR） is a widespread viral disease of cattle that causes serious economic losses in the cattle industry worldwide. The causative virus is bovine herpesvirus type 1（BHV-1）, commonly known as infectious bovine rhinotracheitis virus, a member of the Herpesviridae family, alpha herpesvirinae subfamily. BHV-1 consists of double-stranded linear DNA with an approximate size of 135 kb, and foreign genes can be stably inserted into the genome of BHV-1, which makes the BHV-1 a promising candidate for the development of a live vaccine vector for economically important bovine diseases. Several recombinants expressing immunogenic foreign proteins have been reported as vaccines for other infectious diseases.Foot-and-mouth disease virus （FMDV） is the causative agent of a highly contagious and economically important disease affecting cloven-hoofed animals. FMDV consists of a single-stranded, positive-sense RNA genome of approximately 8,500 bases surrounded by four structural proteins, VP1, VP2, VP3 and VP4. It has been demonstrated that VP1 could induce neutralizing antibodies in the experimental and natural hosts and also involved in the interaction between virus and cell surface receptor. So the VP1 is a target protein for develop new vaccine for FMD. This research was aimed at constructing a recombinant BHV-1 expressing VP1 gene of FMDV as a candidate vaccine strain to develop a novel bivalent vaccine against FMD and IBR.In order to construct the recombinant BHV-1 which expressing FMDV VP1 gene, we constructed a BHV-1 gE gene transfer vector by inserting the synthetic VP1 gene of FMDV （O/China/99） under the immediate-early promoter of cytomegalovirus. The mixtures of parental virus （BHV-1/gE-/LacZ+） DNA and transfer vector were cotransfected into bovine turbinate cells using calcium phosphate-mediated transfection. Then the propagated viruses were harvested. The recombinant BHV-1 （designated BHV-1/gE-/VP1） was obtained by selection for white virus plaques. PCR results showed that VP1 gene was successfully inserted into the genome of BHV-1/gE-. The expression of VP1 in infected cells was proved by indirect immunofluorescence assay and Western blotting.In order to express foot and mouth disease viru（sFMDV）（O /China /99）VP1 gene and prepare the VP1 protein polyclonal antibody for providing a basic materials for identification of VP1 expression in cells infected BHV-1/gE-/VP1, the VP1 gene was amplified by PCR from the plasmid pUC57-VP1 and cloned into the prokaryotic expression vector pET-30a（+）. the recombinant plasmid pET30a-VP1 was transformed into E.coli BL21 after confirmation and then induced with IPTG, SDS-PAGE result showed that a protein approximately 38ku was expressed in inclusion body. Western blot and indirect ELISA results showed that recombinant protein can react with FMD positive serum. The BALB/c mice were immunized with pirmarily purified recombinant protein to prepare polyclonal antibody againist the VP1.To compare the multiplication capacity between BHV-1/gE-/VP1 and BHV-1/gE-/LacZ+, the 50% tissue culture infective dose （TCID50） was calculated, they were 107.9/ml and 107.3/ml respectively. To analyze the genetic character of BHV-1/gE-/VP1, 5,10,15 passages of BHV-1/gE-/VP1 DNA were taken as templet for PCR, the result showed that VP1 gene can be amplificated. Eight New Zealand white rabbits were randomly divided into two groups, the rabbits were inoculated subcutaneously with 1 ml 1×10^7.0 TCID₅₀ BHV-1/gE-/VP1 and 1 ml 1×10^7.0 TCID₅₀ vector virus respectively. The second inoculation was given with the same dose at two weeks post-inoculation and the blood samples were obtained once a week, and sera were collected to determine the presence of antibodies against IBRV and VP1 protein. The immunogenicity was confirmed in a rabbit model by virus neutralization test and ELISA.We successfully constructed BHV-1/gE-/VP1 which expressing foot and mouth disease virus VP1 gene. BHV-1/gE-/VP1 had similar multiplication capacity compared with BHV-1/gE-/LacZ⁺ and had stable genetic character. And the immunogenicity was confirmed in a rabbit model. This research provided a technical platform and basis for applying BHV-1 as vector for development recombinant vaccine of expressing FMDV VP1 gene and other foreign genes.更多还原