【作者】 赵宇军；

【导师】 相文华；

【作者基本信息】 中国农业科学院 ， 预防兽医学， 2005， 博士

【摘要】 猪戊型肝炎(swine HE)是一种新发现的人畜共患病。该病于1997年首次在美国发现,由于该次发现的病毒与美国当地的人戊型肝炎病毒基因组同源率非常高,达到97%,因此对其的人畜共患性提出质疑。而后的动物种间的病毒传播、不同工作人群之间的抗体流行率的差别研究及食用生猪肝后直接引起的人戊型肝炎等研究结果证明猪戊型肝炎是一种新的人畜共患病。国外的研究报道表明猪戊型肝炎的流行非常广泛,不仅在发展中国家普遍存在,而且在发达国家中,猪群中的抗体流行率也很高。我国新疆地区在1988—1989年曾经发生过戊型肝炎的暴发和流行,发病人数达到20多万。目前我国的戊型肝炎流行主要呈散发状态,在华东、华北等地流行的戊型肝炎病毒主要是基因4型。而我国东北也有人戊型肝炎的流行,因此对该地区猪群中戊型肝炎的研究非常必要。 戊型肝炎病毒是一种无囊膜,单链,正义RNA病毒,其生物学特性比较脆弱,而且没有可靠的体外培养系统,目前尚无对该病的诊断方法。国内外通过重组表达的各种蛋白来建立诊断方法,但是均没有理想的结果。 本文利用分子生物学方法对黑龙江省猪群HE流行情况进行了较为详细的调查,并对猪HEVDQ01株排毒猪进行监测,收集其阳性血清;以SOE-PCR方法将DQ01ORF2拼接完整,对其进行遗传演化分析;通过原核表达获得重组蛋白,进行了免疫反应性的鉴定,初步建立了该毒株的ELISA检测方法;以pcDNA3.1为载体构建了DQ01结构蛋白核酸疫苗,并免疫Bal B/C小鼠,获取免疫血清。采用双基因噬菌体展示系统,构建了DQ01ORF2特异性肽库。试验结果表明DQ01ORF2系统发育关系与长春人戊型肝炎病毒最近达到87%,氨基酸残基同源率达到97%;将拼接完整的ORF2命名为DQ01ORF2,以DNAStar分析其抗原性及水溶性片段,以pET32a为载体,将DQ01结构蛋白部分片段进行原核表达,经SDS-PAGE电泳和Western blot分析,该蛋白得剑表达并具有一定的抗原性,将该蛋白命名为DQ01Ag01,以DQ01Ag01为诊断抗原,建立的ELISA方法对黑龙江省6个猪场的血清学检验表明该方法与RT-PCR检测结果一致。将整个DQ01ORF2构建的核酸疫苗进行Vero转染,以间接荧光免疫反应检测,证明外源片段在细胞内得到瞬时表达,免疫Bal B/C小鼠,共免疫四次,结果表明有20%的小鼠产生了相应的抗体,将构建的核酸疫苗命名为pDQ01ORF2。将DQ01ORF2以DnaseI进行酶切,消化至80bp人小片段,将小片段插入噬菌粒pC89,电击转化至大肠杆菌XL1-Blue,建立了噬菌体展示肽库,经随机性检测、库容量检验,肽库达到筛选抗原表位的要求。以DNA疫苗免疫小鼠产生的阳性血清进行生物淘洗后,将洗脱后的40个单克隆噬菌体与猪HEV阳性血清反应,有23个单克隆噬菌体可以与猪HEV阳性血清产生强烈反应,证明该肽库可以用作猪HEVDQ01农壳蛋白抗原表位的筛选和鉴定。更多还原

【Abstract】 Swine hepatitis E is an important public health problem, it was first described in 1997 in USA by Meng. The consequence study found that not only in developing country but also in developed country ,there was more popular HEV antibody exist in developed country than it was expected. Swine HEV is a non-envelope , single strain and positive RNA virus . Because the swine HEV found in USA is very closed to HEV of human being , US-1 and US-2. So the question if the virus can infect human being came to be in close attention . As there was not a reliable method for swine HEV propagation, the diagnose had to rely on the recombined protein .but almost all the method was not very successful. In 1988-1989, there were 20,000 people that infected hepatitis E in south of Xinjiang. Although the hepatitis E were found in north of China and east of China, the condition of swine hepatitis E was not reported in the northeast China.In this study, with SOE-PCR method, we put five fragement of swine HEV DQ01 together to make a complete virus coat protein gene DQ01ORF2. After we analyzed the antigenic index and hydrophilicity of the coat protein gene with DNAStar protein software, we selected a fragment to do prokaryocyte expression in E.coli. With the complete coat protein gene we developed DNA vaccine to make HEV coat protein antibody which could be used for phage-display peptide libraries biospanning. By inoculated Bal B/C mouse, the serum anti-HEVORF2 protein was made. With two gene phage display system, we digested DQ01ORF2 into 80bp sized fragment to established a DQ01ORF2 gene fragment phage display libraries.The results showed that DQ01 is closed to changchun human being HEV strain. The nucleotide sequence identity between them is 87%, the deduced amino acid sequence identity is 97%. By inserting a fragment into pET32a, we got a soluble protein about 33kD fused with the 109aa Trx·Tag^TM thioredoxin protein, the protein was named DQ01Ag01. Analyzed with SDS-PAGE and western blot, we found that DQ01Ag01 could react to HEV positive serum, and the result was corresponded to the RT-PCR method. On the basis of this, we established ELISA that could be used to diagnose if swine serum was HEV positive. With the whole DQ01ORF2 inserted to plasmid pcDNA3.1, a DNA vaccine was made, named pDQ01ORF2. By transfected pDQ01ORF2 into Vero cells, we demonstrate the DNA vaccine could be expressed in Vero cell. Then the vaccine was inoculated on Bal B/C, and the antibody was collected and titered by ELISA based on DQ01Ag01, the titer of the serum was 1:600. With the established double gene phage display system and the mouse serum, two rounds of biospanning was done and the serum-affinity phage was saved. By indirect ELISA, we demonstrated the phage display libraries could be used for swine HEV DQ01 strain epitope characterization.更多还原