Hannai Martin Lysozyme And Two Protease Inhibitor Of Cloning And Expression

Haliotis discus Hannai lno is an economically important mollusk species cultivated in the northern provinces of China. Since the 1980s, cultured and wild abalone have suffered from abnormal deaths, resulting in the considerable reduction of abalone output throughout the world. Understanding the immune defense mechanism of mollusk is crucial for managing diseases and developing sustainable abalone culture.It is believed that imune responses is the key reaction in aminal immune process, and the inhibitors of cysteine proteases and serine protease play a central role in the invertebrate especially in arthropod immune response. They are mainly involved in signaling and amplification cascades that lead to the activation of specific defense mechanisms. In invertebrate,the lysozyme can act as an important role in the innate immune system as immune effectors which activited by the pathogen.Firstly, the sequence of chicken-type lysozyme gene was indentified and from Haliotis discus Hannai using Race method. The full-length cDNA of HdLysC consisted of 586 bp, containing an open reading frame of 441bp which encoded 147 amino acids. Sequence comparison showed that the HdLysC shares 21.4 ~ 30.8% similarity with the c-type lysozyme of invertebrate and 20.1~30.9% similarity with that of vertebrate.The genomic structure of HdLysC was more similar with vertebrate than invertebrate. Quantitative real-time RT-PCR detected HdLysC expression in all organs examined and the expression profiled in gills of abalone challenged with bacteria Vibrio anguillarum. The number of HdLysC mRNA transcript was found to be most abundantly expressed in mantles and weakly expressed in haemocytes, an increased expression of HdLysC was also observed after bacterial stimulation. The HdLysC was succeed expressed in E.coli, the recombinant protein showed bacteriolytic activity on both Gram-positive and Gram-negative bacteria , and the more effective activity was against V.anguillarum among the Vibrio spp. we used.Secondly,two kinds of protease inhibitors of abalone were obtained and characterized in abalone.The full-length cDNA of cysteine protease inhibitors(CPIs) was 1049bp,and it contained an open reading frame of 813 bp, encoding a 271-amino acid protein which possessed structural features of the Family cystatins, including three evolutionally conserved motifs known to interact with the active sites of cysteine peptidases: Gly at the N-terminus (Gly64), Gln-X-Val-X-Gly motif (Q202VVAG206) and Try at the C-terminus (W254). The full-length cDNA of serine protease was 1446 bp with a 1230bp ORF encoding 410 amino acids, and the structure of deduced amino acid sequence was similar with the Serpin family.A quantitative reverse transcriptase real-time PCR assay was developed to detect the mRNA expression of the two kinds of protease inhibitors in different tissues and the expression pattern in abalone challenged with bacteria Vibrio angullarum. The expression of these protease inhibitors was detected in mantles, gills, digestive tract, haemocytes and muscles, and a higher-level mRNA expression of HdSpi was detected in the tissues of digestive tract, and increased expression of these protease inhibitors also observed after bacterial stimulation.In addition, Genome walking strategy were used to obtain the promoter region of the three genes.And a number of putative transcription factor binding sites that related with immunity and cancer was identified, including Oct-1, Oct-2.1, Sp1, WT1, AP-1and AP-2alph.Moreover, Flow cytometry was used to assess the cellular immunity parameters of the abalone which maintained in different temperature seawater(8℃,14℃,20℃and 26℃). There was no significant difference on total hemocyte count ,hemocyte mortality and the proportion of differen type hemocyte cell, and the hemocyte of the abalone maintained 20℃and 8℃showed a higher phagocytic activity and respiratory burst activity respectively. And the expression of immunity related genes of abalone including CAT, HdLyC, HdCpi and HdSpi were also analysis after bacterial challenge, the expression features of these gene indicated the close relationship between the abalone immunity ability and temperature changing.