【作者】 王宏俊；

【导师】 杨汉春； 张培君；

【作者基本信息】 中国农业大学 ， 预防兽医学， 2007， 博士

【摘要】 鸡传染性鼻炎是由副鸡禽杆菌(Avibacterium paragallinarum, Apg)所致鸡的一种重要的细菌性疾病。副鸡禽杆菌有A、B、C三种血清型,其有效保护性抗原成分至今尚不明了。本文利用原核表达系统成功表达了副鸡禽杆菌三个血清型菌株的血凝素蛋白,并分析了重组血凝素蛋白的免疫原性;应用噬菌体肽库技术筛选和鉴定出副鸡禽杆菌的2个免疫优势模拟抗原表位,分析了模拟抗原表位的免疫原性,研究结果为进一步研制与开发鸡传染性鼻炎新型疫苗奠定了基础。根据副鸡禽杆菌A、B、C三种血清型代表性菌株Hp8、Dalian和Modesto的血凝素基因序列,分别设计特异性引物,采用PCR技术扩增出副鸡禽杆菌三种血清型菌株的血凝素基因,序列测定结果表明,三种血清型的血凝素基因全长分别为1 035bp、1 038bp和1 026bp。分别将PCR扩增产物克隆到pET-32a (+)载体,构建出原核表达重组质粒pET- A、pET- B和pET- C。表达产物经Western-blotting及血凝和血凝抑制试验鉴定,结果表明,重组血凝素可以与相应的副鸡禽杆菌阳性血清特异性结合,并且都具有凝集鸡红细胞的活性。用纯化的重组血凝素作为免疫原分别免疫鸡,其血凝抑制抗体滴度均达到1:10以上;用相应菌株攻毒后,部分免疫鸡获得了保护,大部分试验鸡出现鼻炎症状,三种重组血凝素的保护率分别为3/10,2/10和2/10。由此表明,重组血凝素蛋白具有良好的免疫原性,但不能诱导足够的免疫保护。结果提示,血凝素可能不是副鸡禽杆菌的惟一保护性抗原。利用Biosun和Hopp-wood分子生物学软件分析了副鸡禽杆菌Hp8株血凝素蛋白的B细胞表位,结果发现在血凝素蛋白的第86位氨基酸和第186位氨基酸的附近区域可能是2个表位区,以其前后15个氨基酸作为线性表位序列( A1-YDDFGRAKLRQDGET ,A2-NKVGRWEKDGSRVDY)。采用基因工程技术将其分别展示到大肠杆菌GI826的鞭毛上,经抗原抗体结合试验表明,构建的重组菌不具有与副鸡禽杆菌抗体结合活性,提示选定的2个血凝素蛋白的线性抗原肽缺乏免疫活性。利用噬菌体随机12肽库,以A型副鸡禽杆菌多克隆抗体和C型副鸡禽杆菌单克隆抗体纯化IgG筛选。结果表明,用多抗筛选获得的噬菌体克隆中,50%具有核心氨基酸序列A-DP(M)L,单抗筛选获得的噬菌体克隆中,80%以上带有共有氨基酸序列Y-P-Q(A)WW。含有上述共有序列的噬菌体克隆不仅可以与抗体特异结合,并与副鸡禽杆菌抗原竞争结合抗体。将编码YGLLAVDPLFKP和YSPHQWWLSGAV的DNA片段分别插入质粒pFliTrx,转化大肠杆菌GI826并在鞭毛上成功展示,构建的重组菌能与相应抗体特异结合。用2个重组菌免疫鸡产生的相应抗体分别能与A型副鸡禽杆菌0083株和C型副鸡禽杆菌Modesto株结合,攻毒试验结果表明,免疫鸡分别获得了4/8和3/8的免疫保护。结果提示,鉴定出的副鸡禽杆菌的2个免疫优势模拟表位可作为候选表位,具有研制与开发鸡传染性鼻炎新型疫苗的潜力。更多还原

【Abstract】 Infectious coryza (IC), caused by Avibacterium paragallinarum (Apg), is one of the important bacterial disease for poultry. Avibacterium paragallinarum has been classified into A, B, and C serovars. The protective antigens of this pathogen have not yet been clearly identified. In this dissertation, the haemagglutinin genes of the three serovars were cloned and successfully expressed. The immunogenicity of the expressed products was identified, respectively. Two immunodominant mimotopes were screened by means of phage display technique and their immunogenicities were analyzed, respectively. Our studies provide a foundation for developing a new vaccine against infectious coryza.According to the published hemagglutinin protein (HagA) gene sequences of Apg Hp8, Dalian and Modesto strains, three pair of primers were designed and synthesized. The corresponding HagA gene of Apg serovar A, B and C was amplified by PCR. The amplified products were sequenced, showing that the complete HagA gene fragment of Apg Hp8, Dalian and Modesto was 1035bp, 1038bp and 1026bp in length, respectively. The HagA gene fragments were cloned into the expression vector pET-32a (+). The recombinant expression vectors pET-A, pET-B and pET-C were successfully constructed. The expressed products were confirmed by Western-blotting, Hemagglutination test and Hemagglutination Inhibition (HI) test. The three recombinant haemagglutinin proteins (rHagA) could react with postive sera to the corresponding Apg serotype, and agglutinate chicken red blood cells. The immunogenicity of purified recombinant haemagglutinin was analyzed in chickens as immunogen. All the vaccinated chickens generated haemagglutination inhibition (HI) titers of more than 1:10, however only fewer chickens survived and most of them showed clinical signs of coryza after challenged with virulent Apgs. The rHagAs could provide protection rate of 3/10, 2/10 and 2/10 of immunized chickens against virulent Hp8, Dalian and Modesto, respectively. Our data suggest that recombinant haemagglutinins have good immunogenicities but failed to elicit enough immune protection for chicken, implying that HagA might not be a unique protective antigen of the pathogen.B-cell epitopes of haemagglutinin protein of Apg strain Hp8 were analyzed with bio-softwares Biosun and Hopp-wood. Two putative epitopes, located around area of No.86 and No.186 amino acid, named A1 that was composed of 15 amino acids YDDFGRAKLRQDGET and A2 with 15 amino acids NKVGRWEKDGSRVDY were displayed on bacterial flagella of E.coli.GI826 by means of genetic engineering technique, respectively. It was shown that the recombinant bacteria could not bind to chicken antiserum against Hp8. The results indicate that the two predicted linear epitope peptides have no immunogenicity.The phage clones binding to purified polyclonal antibody against Apg serovar A and monoclonal antibody against Apg serovar C were selected from the 12-mer random peptide library. More than 50% of the phage clones selected with polyclonal antibody were carrying consensus peptide motif sequence A-DP(M)L, whereas more than 80% of the phage clones selected were carrying consensus peptide motif sequence Y-P-Q(A)WW when the monoclonal antibody was targeted. The phage clones containing the peptide motifs not only reacted with the corresponding target antibodies but also competed to bind with Apg antigens. The DNA fragments encoding the peptide sequences, YGLLAVDPLFKP or YSPHQWWLSGAV were cloned into the expression vector pFliTrx, respectively. The recombinant plasmids were transformed into E. coli GI826, resulting in two recombinant E. coli expressing the peptides on the bacterial flagellum successfully. The recombinant bacteria could react with corresponding antibodies of Apg. Chickens vaccinated with the epitope-expressing recombinant bacteria developed specific serological antibody that could react with 0083 strain (serovar A) or Modesto strain (serovar C) Apg. Vaccination and challenge trial demonstrated that the 4 of 8 chickens and 3 of 8 chickens injected with recombinant E. coli were protected against homologous challenge of Apg 0083 and Modesto, respectively. These results indicate a potential for the use of the two immunodominant mimotopes as candidate epitopes in the development of a novel infectious coryza vaccine.更多还原