Expression Of Genetic Recombination For Sea Cucumber Lysozyme In Bacillus Subtilis

Lysozyme is present in various tissues of sea cucumber,because the sea sal,pressure,temperature and geographical environment makes the enzyme has other unique other characteristics that other sources of terrestrial organisms do not have the characteristics of lysozyme,the lysozyme research is currently on sea cucumber that construction of sea cucumbers are mostly genetically engineered E.coli gene bacteria,but as an expression of the E.coli strains produce inclusion bodies,purification more difficult,higher production costs and other issues,which is currently in the industry is difficult to promote the cause of the enzyme.Bacillus subtilis is a probiotic,non-pathogenic to humans,there is a strong extracellular secretion,and not through the exogenous proteins refolding of E.coli,thus simplifying the separation and purification of the foreign protein.In this study,missing six kinds of extracellular proteases from Bacillus subtilis WB600 as the expression host,the laboratory saved pMD18T-SjLys plasmid DNA as a template,the gene was amplified sea cucumber lysozyme SjLys,constructed recombinant cloning plasmid pMD18T-Simple-SjLys,the recombinant cloning plasmid pMD18T-Simple-SjLys and expression vectors pHT43 were used restriction endonuclease BamH I and Sma I double digestion,connecting recovered and purified recombinant plasmid pHT43-SjLys,will build a good pHT43-SjLys recombinant plasmid into competent cells of Bacillus subtilis WB600,the expression of lysozyme from sea cucumber Stichopus japonicus in Bacillus subtilis were constructed by the method of recombinant DNA technique,and the expression plasmid pHT43-SjLys of growth curve and stability were analyzed.The expression of lysozyme from sea cucumber Stichopus japonicus in Bacillus subtilis were constructed by the method of recombinant DNA technique,and the expression plasmid pHT43-SjLys of growth curve and stability were analyzed.Results showed that a specific strip about 400bp was visible in the result of agarose gel electrophoresis,which was consistent with expected size of sea cucumber lysozyme gene.Construction of the lysozyme was performed in a vector pHT43 and validated by double digestion with BamH I and Sma I,witch indicated the fragment size was consistent with the expected result.The growth trend of engineering bacteria were consistent compared with the wild type strain WB600,and insertion of foreign genes did not affect the cell 's metabolism.In the absence of selection pressure,the recombinant plasmid was stability,and there was no gene rearrangement and lost.The results show that the recombinant sea cucumber lysozyme genetic engineering bacteria pHT43-SjLys/WB600 is constructed successfully.