Construction And Characteristic Analysis Of Lpp Gene Mutant In Aeromonas Veronii

Aeromonas veronii(A.veronii)is a zoonotic bacterium that can exist in a wide range of temperature and salinity.It can infect aquatic,terrestrial animals,and mammals including humans,which pose a huge threat to public.Lipoprotein(LPP)is a structural component of the membrane of bacteria and plays a role in maintaining the cell membrane's integrity;Studies have found that LPP is also involved in virulence related processes such as bacterial adhesion,which plays a key role in the initial stage of bacterial invasion of the host.However,there are few studies on LPP in A.veronii,and the function of LPP in A.veronii is unclear.The purpose of this subject is to study the functional role of LPP in A.veronii in the bacteria,to provide new ideas with pathogenic mechanism of A.veronii and develop protective vaccines to prevent the bacteria.Firstly,on the basis of whole gene sequencing,the lipoprotein gene(Lpp)was expressed in the prokaryotic system,then identified the expressed product and immunized New Zealand white rabbits to prepare anti-LPP polyclonal antibody to study its antibacterial effect.PCR amplification of Lpp gene and ligated to the p ET-32a(+)vector for p ET-32a(+)-Lpp plasmid.And the plasmid was transformed into BL21(DE3),and expression was induced with IPTG at a final concentration of1 m M.The expressed product was purified by His-tag nickel ion protein purification column,and identified by SDS-PAGE and Western blot.Rabbits were immunized with purified recombinant lipoprotein LPP to prepare antiserum.And the antiserum was subcutaneously inoculated to mice.The results revealed that p ET-32a(+)-Lpp was successfully constructed after restriction enzyme digestion and sequence determination;SDS-PAGE detection showed that the molecular weight of recombinant protein LPP was about 69 Ku;Western blot revealed that the purified recombinant protein LPP was a single band;The ELISA revealed that the antiserum has a titer of 1:102400.The results show that we have successfully prepared an anti-LPP antiserum,which can improve the survival rate of bacteria-bearing mice,reduce the amount of bacteria in the spleen,and have a certain protective effect on the mice.Secondly,in this study,the Lpp gene was deleted through homologous recombination,the Lpp mutant ?Lpp was constructed,and the complementary strain C-Lpp was constructed through functional complementation.PCR amplified the LPP gene's homology arms of up and down and then connected by overlapping PCR.The gel recovered product was connected to vector p MD18-T and transformed into DH5?.The results revealed that the vector p MD18-T-L-12 was successfully constructed.After connecting to the Pre112 suicide plasmid,the competent cells of DH5?-?pir and WM3064 were successively transformed.The results revealed that the recombinant suicide plasmid Pre112-L-12 was successfully constructed.The bacterial solution is used as the donor bacteria to combine and transfer with the wild strain WT of the recipient bacteria,and was screened and identified.The q RT-PCR results showed that the Lpp gene was not expressed;the genetic stability results showed that ?Lpp can be inherited stably.Then,the Lpp gene containing the promoter sequence was amplified,the recovered product was connected to vector p MD18-T,and DH5? were transformed.It revealed that the vector p MD18-T-L-3was successfully constructed.Then connected with PBBR expression plasmid,and transformed into DH5? competent cells.The results revealed that the recombinant expression vector PBBR-L-3 was successfully constructed.The bacterial solution is combined and transferred as the donor bacteria and the recipient bacteria mutant?Lpp,and then screened and identified.The q RT-PCR results showed that the Lpp gene expression was about 11 times that of WT;the genetic stability results showed that C-Lpp can be inherited stably.In summary,this study successfully constructs A.veronii lipoprotein mutant ?Lpp and complementary strain C-Lpp,which can be used in the next experiments.Finally,we analyzed the biological characteristics of ?Lpp and C-Lpp.It revealed that the growth rate of ?Lpp and WT was similar,and the hemolytic activity did not change,but the ?Lpp reduced the biofilm formation ability,missed the flagella,weakened the swimming ability,the number of colonies in the spleen of infected mice was significantly reduced,and increased the half lethal dose of bacteria to mice;The biological characteristics of C-Lpp have been restored to a certain extent,but the flagella is partially missing and the swimming ability is weakened.The results show that the deletion of the Lpp gene reduced the virulence of A.veronii in mice and affect the pathogenicity of the bacteria.In summary,the LPP antiserum prepared in this experiment can effectively protect mice against A.veronii infection.In addition,LPP participates in the pathogenicity of A.veronii and plays a key role.This research has laid a good foundation for revealing the pathogenic mechanism of A.veronii and developing a protective vaccine against the bacteria.