| Objective: Legionella pneumophila is a gram-negative brevis bacterium that is ubiquitous in nature and artificial water supply systems.In 1976,the U.S.Veterans Conference caused an outbreak.221 people were infected and 34 people died of Legionella pneumonia.The bacteria were isolated from the autopsy tissue and named after the infection.Legionella can be spread by atomized water particles,and it is one of the pathogens of community-acquired and hospital-acquired pneumonia.Legionella is composed of more than 56 species.LP is the most common species.It causes 90% of legionellosis cases.Serogroups 1,4 and 6 are considered the main pathogens of human diseases.80% of the reported cases are caused by LP serotype 1.Whether it is sporadic or outbreak,community-acquired or hospital-acquired,Legionella can cause fatal infections,especially in patients with weakened immunity.If you don't get timely and correct treatment,the mortality rate caused by LP infection can be as high as 50%.In addition to monitoring environmental Legionella,there are no ideal effective measures to prevent infection for susceptible people.Therefore,it is of great significance to develop safe and effective vaccine against LP infection without toxic side effects.The development of vaccines has always been a major advancement in modern medicine,while improving the level of public health and extending human life.Studies have proved that DNA vaccines are proven to provide effective protective immunity by directly injecting genetic material into a living host.Compared with conventional vaccines,the preparation process is simpler,the stability and safety are better,and the ability to induce immune responses Stronger.Different from traditional vaccines,DNA vaccines are bacterial plasmids carrying specific coding genes.These bacterial plasmids are responsible for the expression of antigens in the host and inducing immune responses.DNA vaccines can trigger humoral and cellular immune responses,which is an important advantage over commonly used vaccines.Legionella vaccines have now been studied more DNA vaccines,providing new ideas for the preparation of Legionella vaccines.The most commonly used DNA vaccine vector is the pc DNA plasmid series,and pc DNA3.1(+)is the more common eukaryotic expression vector.Combining pc DNA with a suitable vector can improve delivery efficiency,not only improve plasmid stability,but also enhance gene expression and immune response.In this study,PAL protein is one of the important outer membrane proteins of gram-negative bacteria.Almost all PAL proteins studied are highly immunogenic,making them a good candidate antigen for vaccine production.Looking at the literature,sequence analysis PAL gene is highly conserved in various LPs.PAL,like lipopeptide analogs,can activate cellular immunity and humoral immunity.Currently known LP virulence factors include FlaA,IP,LPS,Mip,Momp S and PilE,etc.These virulence factors promote the adhesion,invasion and colonization of LP and host cells,and at the same time promote the growth and reproduction of LP in the host.FlaA encodes the flagellin of Legionella pneumophila,which not only functions as a structural protein,but also functions as an effector.FlaA has strong immunogenicity,can stimulate the cellular immune response mediated by T cells,and play a protective role after being attacked by a lethal dose of Legionella.PilE gene encodes type IV fimbriae protein of LP,and PilE gene expression is enhanced,and the growth of Legionella in host cells and water environment will be enhanced.The expression of type IV fimbriae is also related to the ability of DNA transformation in LP.PilE and FlaA are important attachment factors and protective immunogens of Legionella pneumophila,which can avoid the attack of toxin-producing strains.We cultivated Legionella pneumophila strains,tested the virulence of the strains,and reorganized PAL,PilE and FlaA gene fragments,and constructed them with specific Linker(FlaA-Linker:(G4S)3-PilE-Linker: EASPPGE-PAL).On the nuclear expression vector pc DNA3.1,PAL,PilE,FlaA and PAL/PilE/FlaA recombinant plasmids were obtained respectively.At the same time,we also used Western Blotting method to detect the expression of plasmid amplification of transfected 293 cells,which is used to further evaluate the protective effect of recombinant PAL/PilE/FlaA DNA on the vaccine protection against Legionella pneumophila infection and provide a working basis and reference.Studies have shown that the immune protection of DNA vaccines is also affected by the route and method of vaccination.A number of studies have shown that intramuscular injection has the best immune protection effect.The purpose of injection is to deliver DNA plasmids to antigen-presenting cells in secondary lymphoid organs.Therefore,in this study,recombinant plasmids pc DNA3.1-PAL,pc DNA3.1-Pi IE,pc DNA3.1-FlaA,pc DNA3.1-PAL/Pi IE/FlaA were injected intramuscularly into mice to produce immune protection.Through intramuscular injection of DNA vaccine,there will be persistent antigen expression in muscle cells,which can induce T cell proliferation,cytokine release and antibody production,especially inducing the killing effect of cytotoxic T lymphocytes.The results of this study showed that in the animal experiment of BALB/c mice,the response of mice immunized with recombinant PAL/PilE/FlaA DNA vaccine after intramuscular injection showed that the immune memory of Ig G antibody induced by the vaccine was rapidly induced.Legionella,Brucella,Mycobacterium tuberculosis,Typhoid bacillus and viruses are all intracellular parasitic bacteria.In the immune response of humans and mice,cytotoxic T lymphocytes have very important biological significance for eliminating intracellular bacteria.In this study,the three DNA monovalent vaccines and recombinant DNA vaccines can induce CTL killing activity,which is significantly higher than the pc DNA3.1 empty plasmid group.Previous documents have proposed that after pathogenic microorganisms invade the body,the dominant cells in Th1 cells and Th2 cells in the immune response determine whether the body's immune response against antigens is based on cellular immunity or humoral immunity.It has been reported abroad that cellular immunity and cytokine production play an important role in the protection of Legionnaires' disease.In this study,we found that the levels of TNF-?,IFN? and IL-10 in serum increased.After immunization with recombinant PAL/PilE/FlaA DNA vaccine in the supernatant of spleen cells,TNF-?,IFN?,The levels of IL-12,IL-4 and IL-10 are significantly increased.It can be seen that the recombinant PAL/PilE/FlaA DNA vaccine can induce Th1 and Th2 immune responses in mice.In addition,the survival time and histopathological changes of mice vaccinated with recombinant PAL/PilE/FlaA DNA vaccine were significantly improved.Therefore,the recombinant PAL / PilE / FlaA DNA vaccine can induce mice to produce protective immunity against LP infection.Methods:1.Western-blotting method detects the expression products of pc DNA3.1-PAL,pc DNA3.1-Pi IE,pc DNA3.1-FlaA and pc DNA3.1-PAL/Pi IE/FlaA,and confirms the eukaryotic expression of recombinant plasmid pc DNA3.1-PAL,pc DNA3.1-Pi IE,pc DNA3.1-FlaA and pc DNA3.1-PAL/Pi IE/FlaA were transfected into 293 cells in vitro to obtain effective expression.2.Female BALB/c mice were randomly divided into 5 groups and were given recombinant plasmids pc DNA3.1-PAL,pc DNA3.1-PilE,pc DNA3.1-FlaA,pc DNA3.1-PAL/PilE/FlaA and empty plasmid pc DNA3.1(+)immunization 3 times.1,3,5,and 7 weeks after the last booster injection,the Ig G antibody titers in the serum samples were detected by ELISA.MTT method was used to detect the CTL killing activity of mouse splenocytes.3.Female BALB/c mice were randomly divided into 3 groups and were immunized with recombinant plasmid pc DNA3.1-PAL/PilE/FlaA,empty plasmid pc DNA3.1(+)and PBS for 3 times.Two weeks after the last booster immunization,Mice were intraperitoneally injected with a lethal dose of LP1(2×107CFU in PBS).16 h after LP injection,10 mice from each group were taken to observe the survival rate of mice for 10 consecutive days.On the 11 th day,the surviving mice were sacrificed,and the lung tissues were taken for HE staining to observe the pathological changes of the lung tissues of the mice;ELISA method was used to detect the levels of TNF-?,IFN? and IL-10 in serum samples,and the supernatant was taken to detect the levels of TNF-?,IFN?,IL-12,IL-4 and IL-10 after spleen cell separation(at 12 h,24h,48 h,72h respectively).Results: 1.Due to the slow growth of Legionella,on the 4th day of culture,those with smooth surface,grayish white,grayish blue,and purple are suspicious colonies.Pick the suspicious colonies from the plates and inoculate them on the BCYE and blood plates respectively,the colonies that do not grow on the blood plate are Legionella;transfect 293 cells with pc DNA3.1-FlaA,pc DNA3.1-PilE,pc DNA3.1-PAL,pc DNA3.1-FlaA/PilE/PAL and pc DNA 3.1(+)for 72 h,and then use rabbit Legionella pneumophila polyclonal The antibody performs Western-blotting analysis.p-PilE recombinant plasmid expresses a protein band with a relative molecular weight of about 15 k Da;p-PAL recombinant plasmid expresses a protein band with a relative molecular weight of about 19 k Da;p-FlaA recombinant plasmid expresses a protein band with a relative molecular weight of about 34 k Da;The p-PAL/PilE/FlaA recombinant plasmid expressed a protein band with a relative molecular weight of about 70 k Da;no positive hybridization signal was detected in the 293 cell protein sample transfected with the empty plasmid pc DNA3.1(+).2.At 1,3,5,and 7 weeks after the last booster injection,blood was collected from 3 mice each group,and Ig G antibodies in the serum samples were detected by ELISA.The results showed that the pc DNA3.1 empty plasmid group did not produce antibodies.The Ig G antibody titer increased significantly from the first week to the fifth week after the last booster immunization,and the Ig G antibody titer decreased significantly from the fifth week to the seventh week.Ig G antibodies produced by pc DNA3.1-PAL,pc DNA3.1-PilE,pc DNA3.1-FlaA,pc DNA3.1-PAL/PilE/FlaA recombinant plasmid group at 1,3,5,and 7 weeks after the last boosted immunization Both were significantly higher than the pc DNA3.1(+)empty plasmid group,and the difference was significant(P<0.01).The pc DNA3.1-PAL/PilE/FlaA recombinant plasmid group induces the body to produce Ig G antibody titer significantly higher than the pc DNA3.1-PAL,pc DNA3.1-PilE,pc DNA3.1-FlaA recombinant plasmid group,the difference is significant(P <0.05);MTT proliferation test was used to detect CTL killing activity,and the recombinant plasmids pc DNA3.1-PAL,pc DNA3.1-PilE,pc DNA3.1-FlaA,pc DNA3.1-PAL/PilE/FlaAL were transfected,and the selected positive cell clones were used as target cells,and the OD value at 570 nm wavelength was measured with a microplate reader,and the killing activity of CTL was calculated according to the formula.The killing activity of CTL produced by the recombinant plasmid group of pc DNA3.1-PAL,pc DNA3.1-PilE,pc DNA3.1-FlaA,and pc DNA3.1-PAL/PilE/FlaA was significantly higher than that of the pc DNA3.1(+)empty plasmid group.The difference is significant(P<0.01).The killing activity of CTL produced by the pc DNA3.1-PAL/PilE/FlaA recombinant plasmid group was significantly higher than that of the pc DNA3.1-PAL,pc DNA3.1-PilE,pc DNA3.1-FlaA recombinant plasmid group,and the difference was significant(P<0.05).3.After the last boosted immunization,the experimental mice were divided into three groups: PBS control group,pc DNA3.1(+)empty plasmid group,and pc DNA3.1-PAL/PilE/FlaA recombinant plasmid group.Mice were injected intravenously with a lethal dose of LP(2×107CFU).10 mice in each group were monitored for 10 days to observe the survival rate and do survival analysis.The results showed that the survival rate of the mice in the pc DNA3.1-PAL/PilE/FlaA recombinant plasmid group was 100%,while the survival rate of the mice in the PBS control group and pc DNA3.1(+)empty plasmid group was 0%;After 16 hours of intravenous injection of lethal dose of LP(2×107CFU),serum was collected from 5 mice in each group.Compared with the pc DNA3.1(+)group,the levels of serum TNF-?,IFN? and IL-10 in the PAL/PilE/FlaA recombinant plasmid group were significantly increased,and the difference was significant(P<0.01);In the lethal dose of LP-infected mouse spleen cell culture supernatant,after 12,24,48 and 72 hours of culture,the levels of TNF-?,IFN?,IL-12,IL-4 and IL-10 in the PAL/PilE/FlaA recombinant plasmid group were significantly increased,compared with the pc DNA3.1(+)group,the difference was significant(P<0.01);The mouse lung tissue was separated,and then after the steps of fixation,dehydration,embedding,and sectioning,HE staining was performed,and the morphological changes of the mouse lung tissue were observed under a 200× microscope field of view.Under the microscope: 1)PBS control group: a large amount of fibrinous exudation,obvious proliferation of epithelial cells,widened lung interstitium,and obvious edema.2)pc DNA3.1(+)empty plasmid group: similar to the PBS control group,a large amount of fibrinous exudation,obvious proliferation of epithelial cells,widened lung interstitium,and obvious edema.3)pc DNA3.1-PAL/PilE/FlaA recombinant plasmid group: a small amount of fibrinous exudation,insignificant epithelial cell proliferation,mild edema of lung tissue and other pathological changes.Lung injury score: the score for alveolar edema,hemorrhage and inflammatory infiltration is 1-3(0: none,1: mild,2: intermediate: 3: severe),the highest score is 9 points.The results showed that the lung injury scores of the PBS control group and the pc DNA3.1(+)empty plasmid group were significantly higher than those of the pc DNA3.1-PAL/PilE/FlaA recombinant plasmid group.Conclusion:1.Using Legionella pneumophila LP1 type genomic DNA as template,PAL,PilE,FlaA,PAL/PilE/FlaA genes were obtained after PCR amplification;The recombinant plasmids pc DNA3.1-PAL,pc DNA3.1-Pi IE,and pc DNA3.1-FlaA for the eukaryotic expression of Legionella pneumophila PAL,Pi IE and FlaA genes were successfully constructed,and PAL/Pi IE/FlaA fusion gene eukaryotic expression recombinant plasmid pc DNA3.1-PAL/Pi IE/FlaA;the eukaryotic expression recombinant plasmid pc DNA3.1-PAL,pc DNA3.1-Pi IE,pc DNA3.1-FlaA and pc DNA3.1-PAL/Pi IE/FlaA were transfected into 293 cells in vitro to obtain effective expression.2.Mice immunized with pc DNA3.1-PAL plasmid group,pc DNA3.1-PilE plasmid group,pc DNA3.1-FlaA plasmid group,pc DNA3.1-PAL/PilE/FlaA recombinant plasmid group can induce the production of Ig G antibodies,the pc DNA3.1-PAL/PilE/FlaA recombinant plasmid group induces the body to produce Ig G antibodies with the highest titer;Mice immunized with pc DNA3.1-PAL plasmid group,pc DNA3.1-PilE plasmid group,pc DNA3.1-FlaA plasmid group,and pc DNA3.1-PAL/PilE/FlaA recombinant plasmid group have higher specific CTL killing activity pc DNA3.1(+)control group;The specific CTL killing activity of pc DNA3.1-PAL/PilE/FlaA recombinant plasmid group was higher than that of pc DNA3.1-PAL plasmid group,pc DNA3.1-PilE plasmid group and pc DNA3.1-FlaA plasmid group.3.After infection of mice with Legionella pneumophila,the survival rate of mice in the pc DNA3.1-PAL/PilE/FlaA recombinant plasmid group was significantly higher than that in the pc DNA3.1(+)vector plasmid group and the control group;mice infected with Legionella pneumophila;after infection of mice with Legionella pneumophila,,blood samples and spleen cells of surviving mice were taken for immunoassay.The levels of TNF-?,IL-12,IL-4,IL-10,and IFN? in the pc DNA-PAL/PilE/FlaA recombinant plasmid group were higher than those in pc DNA3.1.(+)empty plasmid group and the control group;the pathological changes of lung tissue after infection with Legionella pneumophila showed that the lung injury in the pc DNA-PAL/PilE/FlaA recombinant plasmid group was lighter than that in the pc DNA3.1(+)empty plasmid group,showing a small amount of fibrinous exudation,the proliferation of epithelial cells is not obvious,and the lung tissue is mildly edema. |
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