Protective Efficacy Against Ureaplasma Urealyticum Immunized With Recombinant GrpE And DnaJ

Objective:In this study,after the recombinant proteins of U.urealyticum(Uu)GrpE and DnaJ were obtained by genetic engineering,the anti-infective protection mechanism of GrpE and DnaJ recombinant proteins was preliminarily explored in the model of reproductive tract infection in mice.It lays a foundation for the research of subunit vaccine against Uu infection with preventive and therapeutic effects.Methods:The GrpE and DnaJ genes of ureaplasma urealyticum were sub-cloned into prokaryotic expression vector(pET28a)to construct pET28a/GrpE and pET28a/DnaJ.The expression of pET28a/GrpE and pET28a/DnaJ in E.coli BL21 was induced by different time,temperature and IPTG concentration.The expression and products of recombinant proteins GrpE and DnaJ were purified by Ni2+-NT-A column,and identified by SDS-PAGE and Immunbloting.The recombinant GrpE and DnaJ proteins were injected into female BALB/c mice by quadriceps femoris at 6-8 weeks(both mice were immunized at a dose of 50 μg/mouse every 2 weeks for 3 times in total).Serum samples were collected from venous blood of mouse tail after each immunization,and serum IgG and its subtype antibody levels in BALB/c mice were detected by ELISA.The proliferation level of spleen lymphocytes in BALB/c mice was detected by CCK-8 method.IL-4,TNF-α,IFN-γ and IL-10 in the supernatant of BALB/c mouse spleen cells were detected by ELISA.Flow cytometry was used to analyze the splenic T-lymphocyte phenotype of BALB/c mice immunized with recombinant GrpE and DnaJ proteins.After the 14 days of immunization,the mice were given estradiol subcutaneous injection into the neck of 500 μg/each,and the vagina was inoculated with 1 ×107 CFU/mL Uu serotype 8 bacterial solution,and the weight,appetite,hair removal,redness,edema and increased secretion of each mouse were recorded.After 21 days of infection,BALB/c mice were sacrificed,the reproductive tract,liver,spleen,kidney and lung tissues of the mice were isolated,and DNA was extracted.The Uu load in each tissue was detected by RT-PCR,and the cervical tissue inflammatory factors and pathological tissue sections of BALB/c mice in each experimental group were analyzed.Results:1.The prokaryotic plasmids pET28a/GrpE and pET28a/DnaJ were successfully constructed,and the target bands with the expected size were amplified by PCR,which were 657 bp and 1128 bp,respectively.The optimal expression conditions of recombinant proteins GrpE and DnaJ were obtained by changing the IPTG concentration,induced temperature and time,and they were all expressed in supernatant in soluble form.Immunbloting was used to verify that pET28a/GrpE group and pET28a/DnaJ group showed specific immunoreactive bands at 25.7 kDa and 41.79 kDa respectively,while the empty plasmid group(pET28a)and the control group showed no obvious immunoreactive bands.2.After immunized BALB/c mice with recombinant proteins GrpE and DnaJ,the corresponding specific antibody was produced on the 14 th day,and the antibody level gradually increased,reaching the peak on the 42 nd day,and the IgG2a level was significantly higher than that of IgG1.CCK-8 results showed that the spleen lymphocytes of the immunized mice were significantly increased.3.After immunized BALB/c mice with recombinant GrpE and DnaJ,the expression levels of TNF-α and IFN-γwere significantly increased(P<0.01),while there was no significant difference between IL-4 and IL-10.Streaming results show that the GrpE and DnaJ immune group in the spleen of IFN-γ increase in the number of CD4+and CD8+ T cells,and the proportion of CD4+ T cells was much higher than that of CD8+T cells(P<0.05),and compared with control group,the contents of IFN-y GrpE and DnaJ immune increased significantly,while no difference between the level of IL-4,further confirmed the GrpE DnaJ immune BALB/c mice and its Th1 cells can be induced by immune response;4.One week after Uu infection,all the mice showed hair loss around the vagina,increased secretions,vaginal opening redness and loss of appetite,but the symptoms of GrpE group and DnaJ group were relatively mild.After the Uu attack,the mice generally lost weight,and the consumption of food and water was reduced.The GrpE and DnaJ groups gradually regained their weight and appetite one week after infection,while the PBS and FA groups continued to lose weight.5.RT-PCR results showed that the Uu load levels in the infected sites of GrpE group and DnaJ group were significantly lower than those in the control group(P<0.05).Uu was not detected in isolated liver,kidney,spleen and lung tissues.The levels of IL-17a,IL-6,IL-10 and IL-1α in GrpE group and DnaJ group were not significantly different from those in the control group,while the expression levels of IFN-y(P<0.001),TNF-α(P<0.05),MCP-1(P<0.01)and IL-1β were significantly lower than those in the control group.6.Acute inflammation(a large number of polymorphonuclear leukocytes)was observed in the PBS and FA groups on the 7 th day after Uu infection,and it turned to chronic inflammation(a large number of lymphocytes or plasma cells infiltration)on the 21st day.GrpE and DnaJ groups,by contrast,inflammatory cells infiltration in the cervical tissue of mice significantly reduced,and compared with normal control group mice,can clearly identify the glands and cervical tissue structure,and maintain its integrity,and glands expansion,gland secretion to increase,pathological characteristics such as edema,hemorrhage and inflammatory cell infiltration reduction;Immuno-histochemical results showed that the Uu load of mice in GrpE group and DnaJ group was significantly lower than that in PBS group and FA group,and it was found that Uu could be found in cervical glands,stromal cells and cytoplasm,and inflammation was not observed in fallopian tubes or ovarian sacs,while mild to moderate inflammatory infiltration was observed in vagina and bladder.Conclusion:(1)The successful build BALB/c mice model of U.Urealyticum infection.(2)The GrpE and DnaJ can induce specific antibodies and Thl-biased type immune responses(3)The GrpE and DnaJ can reduce Uu engraftment of the uterine cervix,reduce the infection site of pathological damage against Uu infection,and provide protective effects.