【作者】 卫玮；

【导师】 张元兴；

【作者基本信息】 华东理工大学 ， 生物化工， 2010， 博士

【摘要】 大黄鱼是我国重要的网箱养殖经济鱼类之一,目前各种病害频繁发生阻碍了大黄鱼养殖业的可持续发展,其中尤以鳗弧菌引起的败血症对规模化养殖构成较大威胁。本课题组前期采用无标记缺失突变技术,成功构建了新型鳗弧菌减毒活疫苗MVAV 6203,其特点是在消除鳗弧菌内源性毒力质粒的基础上敲除了染色体上aroC基因,使之在营养缺陷条件下难以存活。本工作将上述鳗弧菌减毒活疫苗免疫大黄鱼,构建了疫苗免疫后大黄鱼诱导前后差异表达基因的抑制性差减杂交文库,对该文库中随机挑取的300个克隆经PCR鉴定、斑点杂交筛选和DNA测序,共得到179个阳性克隆。序列分析表明,94个基因与GenBank己知的功能基因有较好的同源性,编码了39个基因,其中18个基因为免疫应答相关基因；37个基因与GenBank中未知功能的基因同源；而剩余的48个基因则未找到任何同源性片段。并且该差减文库筛选到的大多数基因在大黄鱼中都未有文献报道。进一步对大黄鱼补体系统的相关基因进行了克隆鉴定和功能相关研究。首先克隆了大黄鱼补体因子Bf／C2A和Bf／C2B的全长cDNA,其开放阅读框(Open reading frame, ORF)分别为2259 bp和2295 bp。同源比对和系统进化树分析显示前者与已报道的青鳉补体Bf／C2因子的亲缘关系最近,后者与已报道的鲤鱼补体Bf／C2B因子的亲缘关系最近,同时两者间的同源性也达28.8%。功能结构域预测发现,两者具有相似的结构域,均包括三个补体调节蛋白结构域(CCP),一个范德华A结构域(vWFA):和一个丝氨酸蛋白酶结构域。系统进化树分析和功能结构域中二硫键和糖基化位点的分布特点表明,补体因子Bf／C2A更接近于哺乳动物的补体C2,而Bf／C2B则更接近于补体B因子。荧光定量PCR的结果显示在鳗弧菌减毒活疫苗免疫后,大黄鱼的肝脏、脾脏和肾脏组织中补体因子Bf／C2A和Bf／C2B的表达量均有不同程度的上调,说明其在大黄鱼的免疫应答中起到重要作用。克隆得到了大黄鱼补体C1抑制因子(C1INH),其cDNA全长为2046 bp,编码一599个氨基酸的蛋白。进化树分析显示大黄鱼补体C1抑制因子与虹鳟的补体C1抑制因子形成一个簇。结构预测表明大黄鱼补体C1抑制因子具有两个低同源性的免疫球蛋白(Ig)结构域和一个相对保守的丝氨酸蛋白酶抑制剂结构域。原位杂交和半定量Western blotting的结果显示在鳗弧菌减毒活疫苗免疫后,大黄鱼的肝脏、脾脏和肾脏组织中补体C1抑制因子的表达均有不同程度的提高,说明补体C1抑制因子也可能在大黄鱼的免疫应答中起重要作用。而体外表达的大黄鱼补体C1抑制因子全长蛋白可以粘附鳗弧菌,间接说明其在同细菌的相互作用中具有一定的功能。最后克隆得到了大黄鱼凝血酶原类似基因的全长cDNA序列,并将其氨基酸序列与其它动物的凝血酶原类似基因进行了同源性及进化树分析,考察了其在鳗弧菌减毒活疫苗免疫前后各个组织中的表达差异,发现其在免疫后的脾脏和肾脏组织中有上调表达,说明凝血酶原类似基因在大黄鱼对细菌的免疫应答中也起到一定作用。综上所述,首次对鳗弧菌减毒活疫苗免疫大黄鱼前后的表达差异基因进行了筛选和鉴定,对多个免疫相关基因功能的初步研究及其在疫苗免疫前后的表达变化分析将有助于对大黄鱼免疫系统的理解及未来的健康养殖。更多还原

【Abstract】 Large yellow croaker (Pseudosciaena crocea) is a species of jewfish that has been widely maricultured in China. The sustainable development of large yellow croker culture has been affected by disease outbreaks. The lethal hemorrhagic septicemia caused by Vibrio anguillarum has threatened the whole culture industry. An unmarked deletion mutation technique is performed in our group and a new type of attenuated live Vibrio anguillarum vaccine MVAV 6203 is constructed successfully. The vaccine is virulent plasmid pEIB1 cured and aroC gene deleted to make it hard to survive in the condition of nutritional deficiency.In this work, a subtractive cDNA library was constructed by suppression subtractive hybridization (SSH) in large yellow croaker stimulated by attenuated live V. anguillarum vaccine. Approximately 300 clones were selected randomly from the library. A total of 179 different expressed sequence tags (ESTs) were screened by PCR identification, dot blotting screening and DNA sequencing. The sequence analysis showed that 94 genes have homology by comparing with the existing genes in GenBank and encoded 39 genes. Among them,18 genes were immune-relative genes. Meanwhile,37 were unknown genes and 48 had no similarity with other known sequences. Most of the sequences characterized here were reported for the first time in large yellow croaker.This is the first time that the identification and functional research of complement genes are studied in large yellow corker. The full-length of LycBf/C2A and LycBf/C2B cDNAs contained an ORF of 2259 bp and 2295 bp. LycBf/C2A clustered with medaka Bf/C2, while LycBf/C2B shared a relatively close ancestral linkage with carp Bf/C2. LycBf/C2A and LycBf/C2B shared 28.8%identity with each other. Both of the deduced proteins contained three complement control protein (CCP) modules, a von Willebrand factor A (vWFA) domain, and one serine protease (SP) domain. The 3D modelling of the two genes was performed and important functional sites were studied. Both the structure and phylogenetic analysis suggested that LycBf/C2A was close to human factor B more than to human C2 while LycBf/C2B was more human C2-like. More evidences are needed to distinguish one from another. Finally, in the large yellow croaker infected with attenuated live Vibrio anguillarum vaccine, the expression levels of LycBf/C2A and LycBf/C2B were remarkably up-regulated in liver, spleen and kidney, indicating that the two complement factors played a pivotal role in the immune response to bacterial challenge.The full-length cDNA of complement component 1 inhibitor (C1INH) was 2046 nucleotides encoding a protein of 599 amino acids. The phylogenetic analysis suggested that large yellow croker C1INH formed a cluster with rainbow trout C1INH. The deduced protein contains two N-terminus immunoglobulin domains without significant homology to other species and one C-terminal serpin (serine protease inhibitor) domain. Western blotting and in situ hybrization analysis showed that the expression of lycC 1INH was obviously up-regulated in liver, spleen and head kidney challenged with attenuated live Vibrio anguillarum vaccine. This indicated that lycC1INH might be involved in the immune response of large yellow croaker to bacterial challenge. The full-length protein of C1INH expressed by cell-free system could bind to Vibrio anguillarum, which indicated that C1INH might play roles in bacteria and host interaction.Finally, the full-length cDNA of thrombin gene was cloned from large yellow croaker. The homology of amino acid sequence was analyzed and phylogenetic tree was constructed. The expression of thrombin gene was obviously up-regulated in spleen and head kidney after immunization with attenuated live V. anguillarum strain, suggesting that thrombin might be involved in the immune response to bacterial challenge in large yellow croaker.On the whole, the differentially expressed genes were screened and identified in large yellow croaker immunologically responsed to attenuated live Vibrio anguillarum. The study of immune related genes and their expression analysis before and after vaccine immunization will benefit for the understanding of large yellow croker immune system and health culture.更多还原