【作者】 刘培欣；

【导师】 孔宪刚；

【作者基本信息】 东北农业大学 ， 预防兽医学， 2011， 博士

【摘要】 新城疫（ND）是危害世界养禽业的重要疾病之一,目前对其病原新城疫病毒（NDV）进行了大量的研究,可是对于NDV编码蛋白的结构与功能以及致病机理还未完全阐明。应用单克隆抗体仍是对NDV编码蛋白进行结构与功能分析、表位鉴定以及强弱毒鉴别诊断的基本手段之一。本研究分别构建了含有NDV基因Ⅶ型QY97株F基因全长及部分基因片段FA（1-357bp）、FB（336-1047bp）和FC（948-1662bp）的原核表达载体。经IPTG诱导后,在大肠杆菌中成功地表达了含F蛋白部分片段（FA、FB和FC）分子量分别为30.6ku、43.5ku和43.7ku的3个融合蛋白,其中FA片段对应F2蛋白,FB和FC分别对应部分重叠的F1蛋白的两个片段。Western-blot结果表明表达的融合蛋白FB和FC具有良好的反应原性。以原核表达的融合蛋白FB和FC免疫小鼠,获得了针对NDV F蛋白FB和FC片段的单克隆抗体各1株,分别命名为FB-B9和FC-B5。以NDV QY97株全病毒免疫6周龄BALB/c雌性小鼠,采用细胞融合技术制备单克隆抗体。经间接ELISA和间接免疫荧光试验筛选,共获得了15株NDV杂交瘤细胞。其中5A12属于IgG2a亚型,18C6属于IgG2b亚型,31B4和33F12属于IgM亚型,其余均属于IgG1亚型。这15株单克隆抗体均无中和活性和血凝抑制活性。采用夹心ELISA对不同单抗与10株属于不同基因型的NDV进行交叉反应,结果表现出不同的反应性。用纯化的NDV QY97株作为抗原,与所获得的单抗进行Western-blot反应,结果表明18C6、24F9和25C12与病毒55ku左右的片段反应,因此这3株单克隆抗体针对的是NDV结构蛋白的线性表位,可能是针对NDV NP蛋白或者F蛋白的单抗,其它几株单克隆抗体与病毒蛋白没有明显的反应,可能是针对NDV结构蛋白构象表位的单抗。选取10株分属于基因Ⅰ型、基因Ⅱ型、基因Ⅵ型、基因Ⅶ型和基因Ⅸ型的毒株作为代表株,进行了识别线性表位的3株单抗18C6、24F9和25C12与不同NDV毒株之间的间接ELISA和Western-blot交叉反应。试验结果与夹心ELISA结果相符,单抗18C6只与CK/CH/HLJ/1/06（ZY）、CK/CH/HN/1/07（LHN）、Mallard/CH/HLJ01/06（JL01）和QY97株有反应性,24F9与除基因Ⅵ型PG/CH/JS/1/06（Y4）毒株以外的所有毒株有反应性,而25C12与10株NDV均有反应性。进一步利用原核表达系统表达了系列截短的NP蛋白多肽片段,采用Western-blot对18C6和24F9所识别的线性表位进行定位。结果发现这两株单抗都是针对NP蛋白的C-末端,18C6识别的抗原表位位于NP蛋白的第473-481位氨基酸之间,其多肽序列为（473）^PPPTPGASQ⁴⁸¹;24F9识别的抗原表位位于NP蛋白的第470-478位氨基酸之间,其多肽序列为（470）^HPEPPPTPG⁴⁷⁸。两个抗原表位相互重叠,具有共同的核心序列（473）^PPPTPG⁴⁷⁸。本试验扩增了4个分离株的NP基因,进行了23株NDV NP基因的序列分析,结果表明,NP基因全长序列比较保守,但是在C-末端的401-489位氨基酸差异较大,在弱毒株之间比较序列同源率较高,均在83.5%以上;强毒株之间的序列同源率与基因型相关,同一基因型内同源率均高于84.4%,但是强弱毒之间的序列同源率有的仅为67%。NP蛋白401-489位氨基酸之间的差异说明NP基因在病毒进化过程中也呈现一定的变异规律。单抗24F9所识别的470-478位氨基酸序列在各基因型毒株中也有所不同,在NDV基因Ⅶ型毒株的序列中相对比较保守,为（470）^HPEPPPTPG⁴⁷⁸;而在基因Ⅵ型毒株中的序列为（470）^H（P/L）EP（L/P）（S/P）HE⁴⁷⁸,第477位的组氨酸未在其它基因型毒株中出现;在弱毒株中的序列为（470）^Q（S/L）GPPPTPG⁴⁷⁸,与强毒株比较至少有三个氨基酸的差异。本研究采用全病毒免疫小鼠制备了15株NDV单克隆抗体,采用原核表达的F蛋白FB和FC多肽免疫获得了针对F蛋白的单抗2株。所获单抗与NDV不同基因型毒株呈现不同的反应性。进行了NP基因的序列分析,发现NP蛋白的C-末端氨基酸序列在不同基因型强弱毒之间有所不同,利用获得的单抗在NP蛋白C-末端上鉴定了两个具有共同核心基序的重叠表位。本研究为NDV编码蛋白进行结构与功能分析、表位鉴定以及强弱毒鉴别诊断等方面的深入研究奠定基础。更多还原

【Abstract】 Newcastle Disease Virus （NDV） is an important pathogen which causes great losses to poultry industry in the world. Currently much research has been done on NDV; however the structure and function of the proteins encoded by NDV and the mechanism on pathogencity of the virus are not fully elucidated. Using monoclonal antibodies （MAbs） is still a basic tool to do research on the structure and function analyses of the proteins encoded by NDV, epitope identification and differentiate diagnosis of NDV.The prokaryotic expression vector containing the whole F gene and its partial segment of FA（1-357bp）, FB（336-1047bp）and FC（948-1662bp）was constructed, after induction by IPTG, the fragment of the fused protein was expressed with a molecular weight of 30.5ku, 43.6ku and 43.7ku, respectively. Among them FA corresponding to F2, FB and FC corresponding to partial overlapped two fragment of F1. The result of Western-blot showed that expressed protein FB and FC have good reactivity. Two MAbs were obtained by immunization with the prokaryotic expressed fragment FB and FC and named as FB-B9 and FC-B5, respectively.6-week-old female BALB/c mice were immunized with purified NDV QY97 strain for the preparation of MAbs by cell-fusion technology. After screening by indirect ELISA and IFA, 15 MAbs were obtained. Among them, MAb 5A12 belongs to IgG2a subgroup; MAb 18C6 belongs to IgG2b subgroup; MAbs 31B4 and 33F12 belong to IgM subgroup; and the others belong to IgG1 subgroup. All these MAbs have no neutralization and HI activities. Cross-reactivity of MAbs with 10 NDV strains of different genotype were detected by sandwich ELISA and different reactivity was found.The purified NDV QY97 strain was used as antigen to do Western-blot with MAbs obtained in the study. The results of western-blot showed that 18C6, 24F9 and 25C12 reacted with 55ku protein fragment of NDV, so these three MAbs may react with linear antigenic epitope on structural proteins of NDV; the protein recognized by MAbs is NP or F protein. While other MAbs showed no obvious reactions with NDV proteins, these MAbs may react with conformational antigenic epitope on structural proteins of NDV.Ten NDV strains from genotype I, II, VI, IX and VII were used as typical strain and cross-reactivity of three MAbs which recognize linear epitope with different NDV strains were detected by indirect ELISA and Western-blot. The results are the same with the results of sandwich ELISA; MAb 18C6 could react only with NDV CK/CH/HLJ/1/06（ZY）, CK/CH/HN/1/07（LHN）, QY97 and Mallard/CH/HLJ01/06（JL01）. MAb 24F9 did not react with NDV strain in genotypeⅥ, but could react with strains in other genotype; while MAb 25C12 could react with all the strains used.Serials of truncated NP protein were expressed by prokaryotic expression system and used as antigen to map the linear antigenic epitope recognized by MAbs 18C6 and 24F9 by Western-blot, respectively. The results showed that these two MAbs could react with C-terminal of NP protein. Antigenic epitope recognized by 18C6 lines in amino acid sequence of 473-481 and its sequence is （473）^PPPTPGASQ⁴⁸¹; while the antigenic epitope recognized by 24F9 lines in amino acid sequence of 470-478 and its sequence is （470）^HPEPPPTPG⁴⁷⁸. Two antigenic epitopes are overlapped and they have the same core sequence （473）^PPPTPG⁴⁷⁸.The results of NP gene analysis showed that NP gene is relatively conservative, but the differences are obvious in the C-terminal 400-489aa of NP protein. The homology of amino acid sequence in C-terminal 400-489aa of avirulent strain is high and is 83.5% to the lowest level. The homology of amino acid sequence in this segment in the virulent strains is related to genotype, it is above 84.4% in the same genotype; while the homology is 67% between some virulent and avirulent strain. The difference in 400-489aa in the C-terminal of NP protein showed the variation of NP has its pattern in the virus evolution process.There are some differences in amino acid sequence of the antigenic epitope corresponding to the position of 470-478 recognized by MAb 24F9 in NDV strains from different genotypes. It is conservative in the NDV strains from genotype VII and the amino acid sequence is （470）^HPEPPPTPG⁴⁷⁸; while the amino acid sequence is （470）^H（P/L）EP（L/P）（S/P）HE⁴⁷⁸in NDV strains belong to genotype VI, His in position 477 was not seen in the virus from other genotypes; the amino acid sequence of avirulent virus strains are（470）^Q（S/L）GPPPTPG⁴⁷⁸, there are at least three amino acid difference with virulent strains.15 MAbs were obtained by NDV immunization and 2 MAbs were obtained by immunization with prokaryotic expressed F protein peptide segment in this study, respectively. Different reactivity was found among MAbs with NDV of different genotype. Sequence analysis of NP gene showed that there are some differences in amino acid sequence in the C-terminal of NP protein in the virulent and avirulent NDV strains from different genotypes. Two overlapped epitopes were identified by two MAbs and they have same core sequence. This study laid foundation for the further study on the structure and function analyses of the proteins encoded by NDV, epitope identification and differentiate diagnosis of NDV.更多还原