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Analysis Of Structure And Expression Of The Lysozyme Gene From The Sea Cucumber Stichopus Japonicus

Posted on:2013-06-30Degree:MasterType:Thesis
Country:ChinaCandidate:Y H ChangFull Text:PDF
GTID:2180330467484002Subject:Fermentation engineering
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Compared the i type lysozyme of sea cucumbers to the hen egg-white lysozyme(c type), we found that both of them can hydrolyse the β-1,4glycosidic bond between N-acetylmuramic acid and N-acetylglucosamine of the peptidoglycan which is the main component in the gram-positive bacteria cell wall. As a result, the enzyme will cleavage the cell and lead to the cell death. Since the gram-negative bacteria contain little peptidoglycan, the c type lysozyme of white eggs has little effect on the gram-negative bacteria. However, it is reported that the i type lysozyme of sea cucumbers has obvious antimicrobial activities against the gram-negative bacteria. As a serious of analysis of the gene structure and amino acid (aa) sequence, we found that the mature peptide in the i type lysozyme of sea cucumbers has glycosidase activity between amino acids (aa)26and65aa, and isopeptidase activity between80aa and125aa. However, it is seldom reported on the polypeptides of the recombinant lysozyme of sea cucumbers, which will hinder the further research of the i type lysozyme multi-function of sea cucumbers. In conclusion, the analysis of structure and expression of the lysozyme gene from the sea cucumber Stichopus japonicus will provide a basis to develop the new type lysozyme with wide spectrum antibacterial activity.This project was first to clone three target genes from the lysozyme cDNA of Stichopus japonicus (Genbank accession no:EF036468). They are the SjLys gene representing the gene of the sea cucumber lysozyme, the SjLys-N gene representing the gene of the sea cucumber N-terminal lysozyme and the SjLys-C gene representing the gene of the sea cucumber C-terminal lysozyme. The genes of SjLys、SjLys-N and SjLys-C represent a range of307bp to681bp,307bp to465bp and466bp to681bp, respectively. According to the sequence analysis, the lysozyme gene of sea cucumber encodes the total145aa, including125aa of mature peptide and21aa of signal peptide.Therefore, the target genes of SjLys、SjLys-N and SjLys-C encodes125aa,50aa and78aa, respectively, for the recombinant protein expression in the study. The total RNA was isolated and extracted from the intestinal tissue of the sea cucumber Stichopus japonicus. Using the gene sequence of the sea cucumber lysozyme as template, three pairs of specific primers, HS-Q-1and HS-Q-2, HS-N-1and HS-N-2, HS-C-1and HS-C-2, were designed for amplifying the target genes of SjLys, SjLys-N and SjLys-C by RT-PCR, respectively. Next, the three genes were connected to the expression vector of pET-32a(+) so as to construct the recombinant plasmids of pET-32a(+)-SjLys, pET-32a(+)-SjLys-N and pET-32a(+)-SjLys-C. Then they were transformed into E. coli Rosetta(DE3) pLysS. The following step was to induce and express the recombinant proteins of SjLys, SjLys-N and SjLys-C. The optimal conditions of induction are as follows:the genetic engineering strains of pET-32a(+)-SjLys/E. coli Rosetta(DE3)pLysS, pET-32a(+)-SjLys-N/E. coli Rosetta(DE3)pLysS and pET-32a(+)-SjLys-C/E. coli Rosetta(DE3)pLysS were grown in the liquid LB medium containing100μg/mL ampicillin (Amp) and34μg/mL chloramphenicol (Cam) and10g/L glucose. After overnight (14-16h) growth at37℃with vigorous shaking, the culture was diluted1:100in50mL of LB/Amp/Cam medium supplemented with5g/L glucose and grown at37℃with an orbital shaker at160rpm. The expression of the recombinant proteins of SjLys, SjLys-N and SjLys-C were then induced at OD600nm of0.6by adding IPTG to a final concentration of0.5mmol/L. The culture cells were continuously shaken at120rpm for15h,14h and10h at28℃to express the three recombinant proteins, respectively. After inducing cultivation, the pellets of fermentation cells were harvested by centrifugation at10000rpm for10min at4℃and then were resuspened for analysis of the expression patterns of the three recombinant proteins by SDS-PAGE. The results were shown that the recombinant proteins of SjLys, SjLys-N and SjLys-C could be highly expressed, accounting for48%,62%and80%of the total protein, respectively. Another amazing results were found that the three expressed proteins are in partial solube form, which were taken up10%,46%and70%of the total protein, respectively. After Western blotting analysis, it is confirmed that the recombinant proteins of SjLys-C had a specific immune response with Penta-His antibody at the position of about31kD,23kD and26kD.The antibacterial activity of the purified recombinant proteins of SjLys, SjLys-N and SjLys-C was detected according to agarose radial diffusion assay. The results showed that the recombinant protein SjLys has an inhibitive activity on Micrococcus lysodeikticus. and the recombinant proteins of SjLys-N and SjLys-C have an inhibitive activity on both Micrococcus lysodeikticus and Vibrio parahaemolyticus. In addition, the recombinant protein SjLys-C after heating treatment could improve5~21%of activities against M. lysodeikticus and V. parahaemolyticus. These results have demonstrated that the genetic engineering strain pET-32a(+)-SjLys/E coliRosetta(DE3)pLysS, pET-32a(+)-SjLys-N/E. coli Rosetta(DE3) pLysS and pET-32a(+)-SjLys-C/E. coli Rosetta(DE3)pLysS could highly produce the recombinant proteins of SjLys, SjLys-N and SjLys-C with both soluble products and potent antibacterial activity. So they will be widely used in aquaculture, livestock and poultry breeding, feed additives and food processing industry. Therefore, the products in this study have great market potential and development value.
Keywords/Search Tags:lysozyme of sea cucumber, recombinant protein SjLys, recombinantprotein SjLys-N, recombinant protein SjLys-C, soluble protein, separationand purification, antibacterial activity
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