| Background Vibrio parahaemolyticus(Vp) is a slightly halopHilic bacterium known to be the leading cause of gastroenteritis, arthritis and heart disease worldwide. The ratio of gastroenteritis caused by Vp is higher in all gastroenteritis. The gastroenteritis caused by Vp is reported each year in the littoral area in China. Several kinds of hemolysin produced by Vp are believed the major virulence factors. Among these hemolysins the thermolabile hemolysin is produced by Vp. Studies found not only environment isolated strains but also clinical isolated strains have the tlh gene that has specialty. The foreign researchers use the tlh gene to detect and monitor Vp, but the function of TLH studies was less, moreover, there have not the reports about TLH studies in China. So the function and the pathogenesis of TLH are not completely understood.Objectives To clone and construct expression vector according to the tlh gene exists universality and specialty, and then purify and obtain the biotic-activity TLH protein to study the functions, pathogenesis and immunity of TLH in Vp.Methods 1. PCR amplify the tlh gene using the China standard strain Vp 14-90 DNA chromosome as template and construct clone vector pGEM-tlh, the pGEM-t1h was identified by restriction analysis and PCR.2.The amplified tlh14-90 gene was sequenced and analyzed its biologic information by using biologic information Websites in Internet.3.The fusion expression vector pET32a+-tlh was constructed and expressed the fusion protein TLH14-90 induced by IPTG in DE3. ThepET32a-tlh and TLH14-90 were identified and analyzed by restriction analysis, PCR, SDS-PAGE and Western-blot.4.The induce conditions was optimized. The fusion protein TLH 14-90 that exists as inclusion body was dissolved into strong denaturant urea and purified by using an affinity chromatograpHy method.5.We obtained the biotic-activity TLH14-90 protein using gradient dialyzed refolding method and study the functions, pathogenesis and immunity of TLH14-90 through hemolysis, Rabbit ileal loop test (RILT), pathological changes of rabbit ileal, hemoliysis inhibit test, thermotolerance test, ELISA, Western-blot.Results l.The tlh!4-90 gene is 1257 bp and amplified by PCR. The tlh!4-90 gene has start and end codon and an ORF which be composed of 418 code .The tlh!4-90 has 99% homology with the tlh of WP1.2.The biologic information analysis results indicated that the tlh!4-90 encodes 418 amino acids that have 99% homology with the TLH of WP1. The TLH14-90 molecular formula is C2131H3184N548O649S16 and the molecular weight is 47.3929 KDa, and the Theoretical pi is 4.92. The predicted Secondary structure of TLH 14-90 is a a/D protein.3. The fusion protein TLH 14-90 was expressed stably and efficiently in DE3 representing 10% of the total bacterial protein, and the fusion protein molecular weigh is about 63KD analyzed by SDS-PAGE. The fusion protein existed as inclusion body in cytoplasm and the fusion protein has a 6 X his tag which facilitate purification.4.The inclusion body was dissolved into strong denaturant urea and purified by using an 6 X his Ni-NTA affinity chromatography method.. We obtained high purify protein(99%) by through using lower concentration imidazole, 0 -ME, Trition-x100 and optimized binding, wash and elution velocity of flow.5.Through reduced concentration of protein, gradient dialyzed renature, added oxidant-reducing agent which can form correct disulfide bonds between molecules and exterior active agents SDS which can stabilize thesemi-renatured product; furthermore, to prevent protein degradation and maintain biotic-activity, the whole process performed in lower temperature (4 C), We obtained the biotic-activity TLH14-90 protein6. The studies of renatured protein indicated the TLH14-90 is lecithin-dependent haemolysin which has haemolysis activity only when added lecithin and a thermolabile hemolysin which lost hemolysis function when hated 60 C, lOmin; and the TLH 14-90 has enterotoxicity, immunity and some protect antigen funct...
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