Screening And Identification Of Differentially Expressed Genes In Large Yellow Croaker Immunologically Responsed To Attenuated Live Vibrio Anguillarum

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.