| As a kind of worldwide shellfish, Oyster (C. gigas) is also the largest breeding shellfish in our country. It is widely distributed in many provinces of China such as Guangdong, Guangxi, Hainan, Liaoning, Hebei, Shandong, Jiangsu, Zhejiang and so on. Oyster meat is a kind of healtHy food that contains the rich and high quality protein, but low fat. And it also has functions as preventing cardiovascular disease, anticoagulant, hypolipidemic, anti-thrombosis and improving the immune. Oyster breeding has vital significance in agricultural economy. But in the process of oyster breeding, it easily suffers diseases, and severely influence the development of aquaculture oyster.Lysozyme is an important effective factor in C. gigas innate immune system which involved in a variety of immune response. And it forms a hydrolytic system in the process of bacteriolysis which destroys and eliminates the invasive pathogens in vivo. Furthermore, it achieves the immune defense to play the following functions, such as antibacterial, antiviral, anti-tumor effect, as well as stopping bleeding, reducing swelling, corrosion prevention and accelerating to repair tissus and so on. Lysozyme is widely existed in animals, plants and microorganisms, and it is a natural antimicrobial agent that can specifically act on the β-1,4 glycosidic bond between N-acetylglucosamine (NAG) and N-acetylmuramic acid (NAM). In this way, it can damage peptidoglycan bracket in order to make microbial cells split up under the effect of osmotic pressure and cytoplasm leak, finally lead cells to die. Lysozyme has a good inhibitory effect on Gram-positive bacteria and some Gram-negative bacteria, but has no effect on the cells of human and animal, so it is identified as an non-toxic, harmless and safe additives by WHO and many countries. Therefore, it is more and more widely used in medicine, food, biotechnology and the process of feeding animals. Lysozymes in aquatic animals are divided into three categories according to their sources:i-type lysozyme, c-type lysozyme and g-type lysozyme. Lysozyme from C. gigas belongs to i-type lysozyme. If with lysozyme is extracted with raw oyster tissues, it will be short of oyster supply and the lysozyme yield is low, so it is difficult to achieve industrial production. Therefore, using DNA recombinant technology to gain lysozyme from prokaryotic expression system is an effective way to solve the contradiction of supply and demand.Based on the full-length cDNA of C. gigas lysozyme in GenBank (Accession no.: AB179775), the lysozyme gene with open reading frame (ORF) from C. gigas was cloned by the RT-PCR method and named Cg-lysozyme(CgLys in short). Bioinformatic software analysis showed that the ORF with the length of 414bp encoded 137 amino acids (aa), including a signal peptide of 20 aa at N-terminus and a mature peptide of 117 aa, the molecular weight of the mature protein was of 13.2 kDa. Phylogenetic analysis showed that the lysozyme from C. gigas clustered with the i-type lysozyme of other bivalve species. The mature peptide was the cloned into pMD18-T vector and was sequenced. The recombinant plasmid was sequenced and digested by BamH I and Sal I. The target gene wsa subsequently connected to the pET-32a(+) vector, which was digested with the corresponding restriction endonuclease. The recombinant plasmid pET32a(+)-CgLys was tranformed into E.coli BL21 (DE3) pLysS and then induced by isopropylthio-β-D-galactoside (IPTG) and purified through chelating affinity, obtained the purified recombinant protein CgLys.The results showed that a new anticipated fusion protein of CgLys (ca.18 kDa) was strongly expressed on tHe gel of SDS-PAGE. Further analysis showed that the expressed fusion protein existed as a soluble form. These results will simplify the next purification steps and facilitate the production of oyster lysozyme in a large scale. |
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