ABSTRACT
The cDNA of an i type lysozyme was cloned from Stichopus japonicus (named as SjLys). The DNA fragment of the mature SjLys was subcloned into expression vector of pET-32a (+) to construct the recombinant plasmid of pET32a (+)-SjLys. The recombinant plasmid was then transformed into Escherichia coli BL21 (DE3) pLysS and induced by isopropylthio-beta-D-galactoside (IPTG). The recombinant protein expressed as inclusion bodies was denatured, partially purified and refolded to be an active form. The bacteriolytic activity of recombinant protein purified by the metal-chelating was 19.2 U/mg. The antibacterial activity of the purified recombinant SjLys (rSjLys) was analyzed. The rSjLys protein displayed inhibitive effect on the growth of the tested Gram-positive and Gram-negative bacteria. In particular, rSjLys had a strong inhibitive activity on Vibrio parahaemolyticus and Pseudomonas aeruginosa, both the most common pathogenic bacteria in the marine animals. The heat-treated rSjLys exhibited more potent activities against all tested bacteria. These results indicated that the S. japonicus lysozyme was the enzyme with combined enzymatic (glycosidase) and non-enzymatic antibacterial action, and it had a wide antibacterial spectrum. Therefore, it is suggested that the S. japonicus lysozyme should be one of the important molecules against pathogens in the innate immunity of sea cucumbers.
Subject(s)
Animals , Anti-Bacterial Agents , Metabolism , Pharmacology , Escherichia coli , Genetics , Metabolism , Genetic Vectors , Genetics , Muramidase , Genetics , Pharmacology , Recombinant Proteins , Genetics , Pharmacology , StichopusABSTRACT
Objective To study the anti-tumor effect of the proteoglycan extracted from abalone viscera (AVPF-I). Methods Hepatic carcinoma cell H22-bearing mice were randomized to negative control (physiological saline), positive control (cyclophosphamide) and three doses of AVPF-I groups. The tumor growth, cell mediated immune function including lymphocyte proliferation, phagocytosis of peritoneal macrophage, NK cell activity, and the level of serum TNF-?,IL-1and IFN-?were detected. Results A significant inhibition of the tumor growth was observed in the AVPF-I group(P
ABSTRACT
Objective To determine the purity,composition and antitumor activities of polysaccharide AHP-12 from abalone harslet.Methods Its purity was checked by HPLC with TSK-GEL G4000PWXL column and agarose gel electrophoresis;Molecular weight was determined by gel filtration chromatography;Sulfate content was identified by gelatine nephelometry and aminohexose content by chromatometry;Gas chromatograph was applied to determine monosaccharide composition;Antitumor activities were investigated by MTT method.Results AHP-12 from abalone harslet was a homogeneous polysaccharide both measured by molecular weight and electric property;The molecular weight was about 3?105;It was contained sulfate 13.07% and aminohexose 4.98%;AHP-12 was composed of rhamnose,fucose and galactose(the ratio in mole is 1∶2.2∶1.7);the activity determined by MTT method showed it could hamper the growth of HeLa cell.Conclusion AHP-12 was extracted and purified from abalone harslet.It was a homogeneous polysaccharide,which contained sulfate and aminopolysaccharide,and with weak antitumor activities in vitro.