【作者】 朱远茂；

【导师】 相文华；

【作者基本信息】 东北农业大学 ， 预防兽医学， 2010， 博士

【摘要】 牛疱疹病毒Ⅰ型(Bovine herpesvirus type 1,BHV-1)和口蹄疫病毒(Foot and mouth disease virus,FMDV)均为牛重要病原,呈世界性分布,给养牛业造成巨大的经济损失。BHV-1基因组约为138 kb的双链DNA,含有多个病毒复制非必需基因,可允许外源基因的插入,是构建多价牛传染病疫苗的理想载体。2005年在我国数省区爆发FMDV Asial,造成了巨大的经济损失。FMDV结构蛋白VP1能刺激机体产生细胞免疫和体液免疫,是研究FMD基因工程疫苗的首选蛋白。本研究人工合成了Asial IND 49197(GenBank AY687334.1)VP1全基因,对VP1基因进行了原核表达并制备特异性的多克隆抗体。首先利用PCR方法扩增获得VP1全基因,将其克隆至原核表达载体pET-30a(+)中,在大肠杆菌BL21中进行表达。SDS-PAGE结果显示表达产物分子量约为31.6 ku,以包涵体形式存在。通过Ni-NTA Purification System纯化后进行Western blotting与间接ELISA分析,结果显示重组蛋白能够被FMD阳性血清识别,具有良好的反应性。将纯化的重组蛋白免疫新西兰白兔制备多克隆抗体,该抗体ELISA效价为1：20480,琼脂免疫扩散试验效价为1：64,病毒中和试验效价为1：64。在构建表达FMDV Asial VP1重组BHV-1时,利用前期已经构建好的转移载体pGEM T-gE-/LacZ+,该载体含有在巨细胞病毒(cytomegalovirus,CMV)早期启动子和BGH PolyA调节信号控制下的LacZ报告基因,同时含有BHV-1同源重组上下游臂,并利用人工合成含有VP1全基因质粒pUC57-AsiaVP1。以限制性内切酶XabⅠ和KpnⅠ双酶切质粒pGEM T-gE-/LacZ+和pUC57-AsiaVP1,通过回收目的片段、连接和转化等技术手段用VP1基因替换掉LacZ基因,最终获得含VP1表达盒且gE基因缺失的转移载体。采用磷酸钙介导转染法将该转移载体与前期获得的gE基因缺失并表达LacZ基因的重组病毒rBHV-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,获得表达VP1基因的重组病毒rBHV-1/gE-/AsiaVP1。PCR鉴定结果表明VP1基因成功重组到rBHV-1/gE-/AsiaVP1基因组中,间接免疫荧光试验和Western blotting证实VP1基因在rBHV-1/gE-/AsiaVP1感染的细胞中获得了表达。在MDBK细胞中连续传25代,PCR鉴定结果表明VP1基因在rBHV-1/gE-/AsiaVP1中稳定存在。rBHV-1/gE-/AsiaVP1与亲本病毒rBHV-1/gE-/LacZ+的TCID50测定结果分别是108.43/mL108.33/mL,表明他们之间的增殖能力差别相似。将15只2kg～3kg的健康新西兰白兔随机分成三组分别于颈部皮下注射1ml 108.33TCID50 /mL rBHV-1/gE-/LacZ+,1ml 108.33 TCID50/mL rBHV-1/gE-/AsiaVP1和未免疫对照组,首免3周后以同样剂量加强免疫一次。6周后用IBRV强毒(IBRV/JL06)滴鼻攻毒,每只兔子剂量为0.5 ml 107.67 TCID50/mL。IBRV中和抗体检测结果表明首免后1周可产生1：4～1：16不等的抗体,加强免疫后抗体滴度均能达到1：128,并一直持续到本实验结束(8周),rBHV-1/gE-/AsiaVP1或rBHV-1/gE-/LacZ+所产生的IBRV中和抗体滴度基本没有差别。VP1抗体检测表明免疫rBHV-1/gE-/AsiaVP1 1周后可产生抗VP1蛋白的ELISA抗体,效价可达1：256以上,免疫2周后抗体达到高峰,并一直持续到本实验结束(8周)。攻毒后鼻拭子TCID50测定结果显示经rBHV-1/gE-/AsiaVP1或rBHV-1/gE-/LacZ+免疫的实验动物组排毒高峰出现在2～3天,排毒时间可持续5天,排毒最高时的平均TCID50/0.1mL分别为1.372和1.246；而未免疫实验组排毒高峰在攻毒后的第3-5天,排毒时间可持续8天,排毒最高时的平均TCID50/0.1mL为2.643。临床观察结果显示三组实验兔在攻毒后均出现有呼吸道临床症状如呼吸困难、精神沉郁等,但rBHV-1/gE-/LacZ+、rBHV-1/gE-/AsiaVP1免疫组比未免疫组要轻。攻毒后第6天每组安乐死2只实验兔,对采集的心、肝、脾、肺、肾、脑和淋巴结等组织进行病理学观察、间接免疫荧光(Immunofluorescence assay,IFA)检测和免疫组化(immunohistochemistry,IHC)检查,结果显示rBHV-1/gE-/AsiaVP1或rBHV-1/gE-/LacZ+免疫组比对照组组织病理变化轻,荧光强度弱,组化阳性细胞少。综上所述,本研究原核表达了FMDV Asial VP1基因并制备了兔多克隆血清,同时构建了VP1重组BHV-1。IBRV/JL06攻毒试验表明该重组病毒能够减少临床表现、缩短强毒排毒时间、降低强毒排毒强度并降低组织病理变化程度,表明具有保护作用。该重组病毒同时能刺激机体产生1：256以上的特异性VP1抗体。因此本研究构建的表达VP1重组病毒为研究BHV-1和FMDV二价活载体疫苗奠定了基础。更多还原

【Abstract】 Bovine herpesvirus type 1 (BHV-1) and Foot and mouth disease virus (FMDV) are important cattle pathogens that have a worldwide distribution and are the cause of significant economic losses to the cattle industries. BHV-1 consists of double-stranded linear DNA with an approximate size of 138 kb. Foreign genes can be stably inserted into the genome of BHV-1, which makes the BHV-1 to be a promising live vaccine vector controlling other economically important bovine diseases. Several recombinant BHV-1 expressing immunogenic foreign proteins have been reported as vaccines for other infectious diseases. In April of 2005, an outbreak of FMD caused by serotype Asia 1 began in China. It has been demonstrated that VP1 of FMDV could induce neutralizing antibodies in the experimental and natural hosts. The VP1 is a target protein developing new vaccine for FMD.To express foot and mouth disease virus (FMDV) full VP1 gene and prepare polyclonal antibody against VP1 protein, the VP1 gene was obtained from plasmid pUC57-AsiaVP1 containing VP1 gene of Asia 1 IND 49197 strain by PCR and cloned into the pET-30a(+) for expression in E. coli BL21. SDS-PAGE result showed that protein with a molecular weight of approximately 31.6ku was expressed in inclusion body. The purified recombinant protein showed reactivity to FMD positive serum samples and no reactivity to normal bovine sera in indirect enzyme linked immunosorbent assay (ELISA) and Western blot analysis. These assays demonstrated that the recombinant protein VP1 had very good antigenicity. Antiserum to the VP1 protein was prepared by inoculating rabbits for four times with purified recombinant VP1 protein. The titer of the rabbit anti-FMDV-VP1 serum was 1:20480 in ELISA,1:64 in agar gel immunodiffusion test, and 1:64 in virus neutralization test, respectively. Recombinant protein obtained in this study can be developed to establish a diagnostic test for detecting FMDV Asia 1 antibodies. Polyclonal antibodies can be used to further study the structure, function and epitope mapping of FMDV serotype Asia 1 VP1 gene.In this study, we chose recombinant rBHV-1/gE-/LacZ+acquired previously as parental virus, LacZ gene as inserted site for foreign gene, and constructed recombinant BHV-1 expressing FMDV VP1 gene without reporter gene by way of reversal plaque selection. Firstly, we synthesized plasmid pUC57-AsiaVP1 which contains full FMDV Asia 1 (IND 49197) VP1 gene. Secondly we constructed recombinant transfer vector pGEM T-gE-/AsiaVP1 by substituting LacZ gene of pGEM T-gE-/LacZ+acquired previously for VP1 gene. The vector consists of VP1 gene under the immediate-early promoter of cytomegalovirus(CMV) and BGH polyA, and recombination arms, and without report gene. Thirdly, the mixtures of parental virus genome DNA and transfer vector were cotransfected into bovine turbinate cells (BT) using calcium phosphate-mediated transfection. Finally, the recombinant BHV-1 (rBHV-1/gE-/AsiaVPl) with VP1 gene but without BHV-1 glycoprotein E (gE) gene and LacZ reporter gene was obtained by selection for white virus plaques. PCR results showed that VP1 gene was successfully inserted into the genome of rBHV-1/gE-/AsiaVP1. The expression of VP1 in infected cells was proved by indirect immunofluorescence assay and Western blotting, and the VP1 is stable in serial passage (from1 to 25) of rBHV-1/gE-/AsiaVP1. The 50% tissue culture infective dose (TCID50) of rBHV-1/gE-/AsiaVP1 and rBHV-1/gE-/LacZ+ were 108.43/mL and 108.33/mL respectively, which demonstrated that the distinction of multiplication capacity between the two recombinants was quiet.In this study, we constructed and characterized the immune responses and vaccine efficacy conferred by the rBHV-1/gE-/AsiaVP1. Three groups of adult New Zealand white rabbits each with 5 were inoculated subcutaneously twice 3-week intervals with 1mL 108.33 TCID50 rBHV-1/gE-/AsiaVP1, 1mL 108.33 TCID50 rBHV-1/gE-/LacZ+and control, respectively, and all rabbits were infected with 0.5mL 107.67TCID50 wild virus IBRV/JL06 by the intranasal route 6 weeks post vaccination. The neutralizing antibodies(1:4-1:16) against IBRV could be detected one week post vaccination, and the levels of neutralizing antibodies significantly rose to 1:128 post booster and sustained to the end of this study(eight weeks). There was no difference in the levels of neutralizing antibodies between the two groups vaccinated with rBHV-1/gE-/AsiaVP1 or rBHV-1/gE-/LacZ+. The antibodies(1:256) against FMDV could be detected one week post vaccination by rVP1-ELISA, and the levels of antibodies slightly rose two weeks post inoculation and sustained to the end of this study(eight weeks). The virus was excreted in their nasal secretions for 5 days pi with an average peak of 101.372TCID50/0.1mL or 101.246TCID50/0.1mL on the second day pi, while the non-vaccinated group for 8 days and 102.634TCID50/0.1 mL on the third day pi. The two vaccinated groups showed slightly respiratory clinical signs consisting of labored breathing, conjunctivitis and depression than non-vaccinated group post infection (pi). In conclusion, the rBHV-1/gE-/AsiaVP1and rBHV-1/gE-/LacZ+can protect against clinical disease, which was confirmed by the histological evidence, immunohistochemistry reaction and immunofluorescence assay.In summary, the full VP1 gene of FMDV IND 49197 strain was expressed with prokaryotic expression vector pET-30a (+) in E. coli BL21and preparation of its polyclonal antibodies with rabbits. Moreover, recombinant virus expressing above-mentioned VP1 gene was successfully constructed which could induce rabbits to produce antibodies against FMDV and BHV and protect against clinical disease of BHV. It demonstrated that the newly recombinant BHV-1 rBHV-1/gE-/AsiaVP1 might further be an attractive candidate vaccine for preventing FMDV and BHV-1 simultaneously.更多还原