Preliminary Study On The Function Of Internalin G In Listeria Monocytogenes

Listeria monocytogenes(LM)is a common food-borne pathogen,which can cause human listeriosis and pose a great threat to human and many kinds of animal health.The process of invasion of the host cell is extremely complex,involving a variety of protein molecules.Internalin(Inl)are a family of proteins encoded by genes that are closely related to LM virulence.There are 25 kinds of internalins known,and they are named as Inl A,Inl B,Inl C,Inl J,etc.Up to now,there have been few studies on InlG gene,and the biological function of InlG gene and the role of InlG gene in LM remain unclear.Therefore,the InlG gene of LM is selected as the research object in this study,the prokaryotic expression and reactogenicity of its expression products were analyzed.The immune protection of InlG gene in mice was studied by mice infection test.The homologous recombination technique was used to construct InlG gene deletion strains,and its biological characteristics are preliminarily studied.The main research contents are as follows:1.The prokaryotic expression and reactogenicity of the InlG gene of LM:the standard strain ATCC19111 was used as a template,specific primers were designed for PCR amplification according to the InlG gene sequence published on Gen Bank,then the recombinant plasmid pET-28a-InlG was constructed and was transformed into the expression strain E.coli BL21(DE3).Then the recombinant protein InlG was expressed by IPTG induction.The expressed InlG protein was analyzed by SDS-PAGE and Western-blot.The results showed that LM InlG gene was 1 473 bp,encoded a protein of 490 amino acids.InlG was expressed in E.coli BL21(DE3)and the relative molecular weight was about 70ku.The expression level was high in the supernatant,Western-blot results indicated that the target protein had good reactogenicity.2.The immune protection effect of the protein encoded by the InlG gene of LM:The purified recombinant protein mixed with adjuvant was injected into mice subcutaneously,and the titer was determined after immunization.The mice were infected by LM strain.Then the mice were dissected,liver and brain lesions were taken to make pathological sections,and the effects of LM on the pathological changes of mice were compared.The results showed that after four times immunizations,the serum antibody titer could reach1:52 000.It had good immunogenicity.When the mice were intraperitoneally infected with more than half of the lethal dose of LM,the protection rate in the group of mice immunized with the protein could reach 50%.The tissue sections showed that the damage of brain and liver tissue in the immunoprotein group was less than that in the injected normal saline group.3.The construction of InlG gene deletion strain and biological characteristics of LM:the upstream and downstream homologous arms primers of the InlG gene were designed from LM.The homologous arms of InlG gene were amplified by SOE-PCR method.After the sequencing was correct,the?InlG was linked to the shuttle plasmid p KSV7 to form the recombinant shuttle plasmid p KSV7-?InlG.p KSV7-?InlG was transformed into LM competent cells,and the screened positive bacteria were subcultured at 41?and chloramphenicol resistant medium for homologous recombination.After complete recombination,the p KSV7 plasmid was lost in subculture at 30?and without chloramphenicol resistance.The gene deletion strain without chloramphenicol resistance was screened out by PCR and its stability was tested.Wild strains were used as the control group,the function of InlG gene in LM was studied by the growth characteristics under different temperature and p H conditions,biochemical test,hemolysis test and LD₅₀ in mice.Results:The deletion strain LM-?InlG was successfully constructed with good genetic stability.It was found that InlG gene deletion had little effect on the LM growth characteristics and environmental tolerance,and there was no difference in biochemical characteristics between the two strains,indicating that InlG gene deletion did not affect LM metabolic function.Hemolysis test results showed?hemolysis,InlG gene deletion had no effect on the hemolysis of bacteria.The LD₅₀ of LM-?InlG was 1.0×10^6.9 CFU,and the LD₅₀ of LM was 1.0×10^6.6 CFU,which was 0.3 orders of magnitude higher than that of the wild strain.The mouse infection test showed that the virulence of LM was decreased after the deletion of InlG gene.The function of InlG gene was preliminarily investigated in this experiment.This study laid a foundation for further study of the function of InlG gene and the pathogenesis of LM.