Studies On Cloning And Expression Of Metalloprotease And Syncytotoxin And The Pathogenic Mechanism Of Metalloprotease And Cytolys In Vibrio Vulnificus Infections

Vibrio vulnificus(V.vulnificus,Vv) is a common inhabitant of estuarine and marine waters throughout the world.It can be isolated under a wide range of salinity and temperature conditions from oysters,clams,mussels,and fish,as well as from sediment and plankton.Based on phenotypic characteristics and host criteria, V.vulnificus isolates have been grouped into three different biotypes.We studied strains from biotype 1 which have been associated with pathogenicity in humans, having a positive indole reaction,and are serologically heterogeneous.This gram-negative bacterium is an opportunistic human pathogen,mostly causing disease in persons with underlying chronic illnesses.It is often found in patients with a high plasma iron level.In immunocompromised individuals,V.vulnificus is often fatal. Ingestion of V.vulnificus infected shellfish can lead to gastroenteritis and primary septicemia,while contact with existing skin lesions can result in edema,erythema, and necrotizing wound infections.These diseases share the characteristics that the bacteria multiply extremely rapidly in host tissues resulting in extensive tissue damage and high fatality rate.Thus,the fatality rate in early stage of V.vulnificus infection.disease is very important.Metalloprotease(VVP) and cytolys(VVC) are two necessary factors closely related to the pathogenicity of V.vulnificus.But their pathogenic mechanism are still not clear.Moreover,the genes that express the two protein respectively,vvp gene and vvhA gene,both have high conservatism among different V.vulnificus.strains.Thus,The two factors can be used as the targets in V.vulnificus experimental immunological detection.Getting sufficient and highly purified protein of VVP and VVC is prerequisite on both studies of pathogenic mechanism and immunological detection.While the natural expression of VVP and VVC is very low in V.vulnificus.So getting sufficient and highly purified protein by some routine biochemical methods.While exogenous gene can be highly espressed in a suitable system through engineered methods.Then the sufficient and highly purified proteins can be obtained.OBJECT:The study was undertaken to obtain the sufficient and highly purified protein of VVP and VVC,prepare and identify the polyclonal antibodies against VVP and VVC and explain the pathogenic mechanismof VVP and VVC in V.vulnificus infection and to provide an foundation for experimental detection clinical therapy after V.vulnificus infection.METHOD:First of all,the vvp gene and vvhA gene was amplified from the template of V.vulnificus genome with PCR and subcloned into a pGEM-T vector to form pGEM-T-vvp and pGEM-T-vvhA construct.On the basic of pGEM-T-vvp and pGEM-T-vvhA construct,sequencing was carried out and sequences of vvp gene and vvhA gene were used to search in the gene bank and protein databases at BCBI by using BLAST.Secondly,the fragment ofvvp gene and the fragment of vvhA gene were excised from pGEM-T-vvp and pGEM-T-vvhA construct respectively with incision enzyme BamH I and Xho I and ligated into the equivalent sites of pET-32a(+) vector to form the new plasmid pET-32a-vvhA and pET-32a-vvp which was transformed into E.coli BL21(DE3).The resultant construct was confirmed by enzyme digestion and PCR analysis.Expressions from pET-32a-vvhA and pET-32a-vvp were induced by IPTG and protein was separated and analized by SDS-PAGE.The expressed proteins were further confirmed by Western-blot with the antibody against His-tagged.Thirdly,orthogonal design was used in the experiment of optimizing the inducing conditions of expression of rVVP and rVVP in E.coli BL21(DE3).The inducing conditions contained time,temperature,concentration of IPTG and agitation speed.Then the recombination proteins were expressed under the optimized inducing condition.The induced E.coli BL21(DE3) were mechanical lysised by sonication.Then the recombination proteins rVVP and rVVP with His-tag in the cell lysis were purified by binding to Ni-IMAC resin.The antigenicity of recombination proteins were detected by ELISA and Western-blot using antibody against V.vulnificus.At last,the polyclonal antibody against VVP and VVC were prepared and the pathogenic mechanisms of VVP and VVC in V.vulnificus infection were studied by established animal models.The tissues were made to paraffin blocks then were sliced and HE stained.The paraffin blocks of tissue were also detected by histological chemistry through polyclonal antibody against VVP and VVC.RESULTS:The fragment of vvp gene is 1830bp.Its contents of A,T and(G+C) were respectively 25.96%,24.54%and 49.5%.It has a very high homology with other V.vulnificus strains by sequence analysis.The fragment of vvhA gene is 1356bp. Its contents of A,T and(G+C) were respectively 27.65%,23.89%and 48.45%.It has a very high homology with other V.vulnificus strains by sequence analysis.The total proteins of E.coli BL21(DE3) induced were separated by SDS-PAGE. The results showed that the recombination proteins rVVP and rVVP could be expressed.A 70-kD band and a 86-kD band was observed in gels,matching the predicted molecular weight.The recombination proteins rVVP and rVVP were both expressed with His-tag confirmed by Western-blot.The optimized inducing condition of expression of rVVP was 4h-inducing time, 37℃-inducing temperature,1mmol/L concentration of IPTG and 250rpm-agitation. The optimized inducing condition of expression of rVVP was 4h-inducing time, 37℃-inducing temperature,1mmol/L concentration of IPTG and 250rpm-agitation. The two proteins could be highly expressed under the optimized inducing conditions. The concentration of two purified proteins with His-tag was respectively 1.90mg/ml (rVVC) and 1.69mg/ml(rVVP) and they both had good antigenicities.The purified polyclonal antibodies against VVP and VVC were specific and their potency respectively reached 1:12800.The injury of lung,kidney and liver were most severe among the main viscera of mice when V.vulnificus were peritoneal injected.The pathological changes of lung injury were characterized by a lot of abnormal red blood cells existing in the bronchus cavity and stroma,the cells of bronchiolar epithelium falling off into the cavity and extravasate appearing in the alveolus.The pathological changes of kiney were characterized by degeneration and necrosis of the epidermic cells of renal tubule and hemorrhage.The pathological changes of liver were characterized by extensive degeneration of the liver cells. Only injury of lung occurred when VVC was alonely injected and The pathological changes of lung shared the same characteristics with the V.vulnificus injected.While when VVC was alonely injected,no injury was occurred.VVC was detected in tracheal epithelium cells and alveolar epithelium cells in the lung of mice by immunohistochemical method.But VVP was not detected in any tissue.CONCLUSIONS:1.Both vvp gene and vvhA gene have very high homology with other V.vulnificus strains.2.The active and highly purified VVC and VVP were successfully obtained by the methods of gene engineering.3.The specific polyclonal antibodies against VVP and VVC were were successfully prepared.4.VVC is necessary to the pathogenicity of V.vulnificus.The lung is the target organ of VVC when V.vulrtificus infection occurs.5.VVP is not necessary to the pathogenicity of V.vulnificus.6.There is no interaction between VVC and VVP during Vv infection.