| Bovine viral diarrhea(BVD)is one of the important bovine viral infectious diseases that can persist in cattle and causes bovine disease with complex and diverse clinical symptoms.Because of the unique pathogenic mechanism of BVDV,the host immune suppression can be caused,which leads to immune failure and other respiratory infectious diseases.At the same time,the reproduction,meat production and milk production performance of cattle infected with BVDV will be greatly affected,which will cause serious economic losses.At present,vaccination is mainly used to prevent BVD,including attenuated and inactivated vaccines.Due to the safety of attenuated vaccines,immunization is still dominated by inactivated vaccines.However,due to the susceptibility of BVDV mutations,inactivated vaccines have poor ability to respond to viral mutations,often leading to immune failure.Therefore,the development of a new vaccine which can effectively prevent BVDV infection is of great significance for the prevention and control of bovine viral diarrhea.This study is based on the characteristics of BVDV mainly through the infection of the digestive tract mucosa,inducing the body to produce an effective protective mucosal immune response,and constructing the body's first line of defense against BVDV transmucosal infection as the guiding research.Select BVDV protective antigen E2 protein as immunogen and cholera toxin B subunit(CTB)as immunological adjuvant,and use Lactobacillus casei as antigen delivery vector to construct genetically engineered lactic acid bacteria that express BVDV E2 protein and CTB.The recombinant Lactobacillus casei was immunized to animals by oral route,and the immunogenicity of the recombinant Lactobacillus system as oral mucosal immune agent was explored,in order to provide theoretical basis for the development of a new vaccine for bovine viral diarrhea.The specific research contents of this paper are as follows:Firstly,the constructive expression of BVDV E2 protein and histomorphic fusion expression of BVDV E2 protein and CTB gene engineering lactobacillus were constructed.The BVDV genomic RNA was extracted and reverse transcribed into cDNA.The E2 gene was amplified by cDNA as a template.The genomic DNA of Vibrio cholerae was extracted.The ctxB gene was amplified by PCR,and the PCR product was sequentially ligated into pMD-19T simple vector.Recombinant plasmids pMD-19Ts-E2'and pMD-19Ts-E2-ctxB were isolated and sequenced;The recombinant plasmids pMD-19Ts-E2'and pMD-19Ts-E2-ctxB were digested by SacI and Apal respectively.The target gene E2 and E2-ctxB fragments were ligated into the corresponding cleavage site of the Lactobacillus casei constitutive expression vector pPG-T7g10-PPT to construct recombinant expression plasmids pPG-E2 and pPG-E2-ctxB.The recombinant expression plasmid was transferred into Lactobacillus casei W56 by electroporation,and the recombinant Lactobacillus casei pPG-E2/Lc W56 and pPG-E2-ctxB/Lc W56 were successfully constructed by PCR and sequencing.The expression of the target protein was identified by Western blot and indirect immunofluorescence.The results showed that the target protein E2 and the fusion protein E2-CTB were constitutively expressed and displayed on the surface of the recombinant bacteria.Secondly,a model of oral infection of BVDV in BALB/c mice was established.BALB/c mice challenged 300?L of CP type BVDV with a titer of 105 3/0.1 mL(TCID50)via oral route.Mouse feces samples were collected every day,and the detoxification of mice was detected by RT-PCR.The monitoring was continued for 60 days.The results showed that BVDV could be detected in the excreted feces after 2 hours of challenge.During the 60 days monitoring period,mice continued to expel BVDV,indicating that BVDV can be effectively propagated in the intestine of mice.The temperature of mice was monitored continuously for 15 days with a mouse thermometer.The results showed that compared with the control mice,the body temperature of the mice in the challenge group decreased after the challenge.At the 3th day after the challenge,the body temperature gradually increased and "hot accumulation"appeared and finally returned to normal level.15 days after challenge,the lungs,liver,kidney,heart,spleen and intestine of the mice were collected for histopathological observation and viral load detection.The results showed that there were no obvious pathological changes in the organs of the mice before and after the challenge.The presence of BVDV was detected in spleen,lung and small intestine,and the viral load gradually increased,and the level tended to be stable after 7 days of exposure.Thirdly,BALB/c mice were used as animal models to systematically evaluate the immunogenicity of recombinant Lactobacillus casei and the adjuvant effect of CTB.BALB/c mice were randomly divided into four groups(35 in each group),including pPG-E2/Lc W56 group(test group),pPG-E2-ctxB/Lc W56 group(test group),and pPG/Lc W56 group.(control group)and PBS group(control group).The immunization procedure was as follows:Each group of mice in the experimental group was orally immunized with 200 ?L of recombinant lactic acid bacteria at a concentration of 1010 CFU/mL.The control mice were orally administered the same dose of pPG/Lc W56 and PBS.The immunization was performed 3 times,and the immunization time interval was 2 weeks,and each continuous immunization was 3 days.Fecal,intestinal mucus,vaginal douche and nasal fluid samples of each group of mice were collected before and after immunization.Detection of sIgA antibody levels by indirect ELISA.The results showed that the level of specific sIgA antibody increased significantly from the 14th day after primary immunization.The sIgA antibody level in the control group did not differ before and after immunization.The results showed that the two recombinant Lactobacillus strains could effectively induce the intestinal mucosal immune response based on antigen-specific sIgA and cause the,co-mucosal' immune effect.The induced sIgA antibody has the activity of neutralizing BVDV.The serum of each group of mice was collected,and the level of IgG antibody was detected by indirect ELISA.The results showed that compared with the control group,the mice in the experimental group produced significant levels of IgG antibodies after the second immunization.It is indicated that the constructed recombinant lactic acid bacteria can induce the body to produce a humoral immune response based on antigen-specific IgG.And the induced IgG antibody has neutralizing BVDV activity.Recombinant lactic acid bacteria pPG-E2-ctxB/Lc W56 induced antigen-specific sIgA and IgG antibody levels in the pPG-E2/Lc W56 test group.It indicated that CTB of cholera toxin B subunit can significantly enhance the oral immune effect of recombinant lactic acid bacteria,showing good adjuvant effect.The test results of spleen lymphocyte proliferation and CD4+?CD8+ T lymphocyte levels in the experimental group showed that the constructed recombinant L.casei could induce the cellular immune response.Through the detection of cytokine levels in serum,the results showed that recombinant Lactobacillus casei can stimulate the body to produce Th1,Th2 and Th17 type cellular immunity,mainly with Th2 type cell immunity.The recombinant lactic acid bacteria pPG_E2-ctxB/Lc W56 induced a significant cellular immune response to pPG-E2/Lc W56.Fourthly,the oral immune protection effect of recombinant Lactobacillus was preliminarily evaluated by using the established animal model of BVDV infection in BALB/c mice.On the 35th day after the first immunization,CP BVDV with a titer of 105.3/0.1 mL(TCID50)was injected orally to mice in each group.Fecal samples from each group of mice were collected every day to detect the clearance of the virus in the intestine of the mice.The results showed that the virus content in the feces discharged from the mice in the experimental group gradually decreased.Among the feces in the oral immunized pPG-E2/Lc W56 group,the presence of BVDV was not detected from the 6th day after challenge.The oral excretion of pPG-E2-ctxB/Lc in the excreted feces of the W56 group has not detected BVDV since the 4th day after challenge.The presence of the virus was continuously detected in the feces excreted by the control mice.The results showed that the BVDV in the intestine of the mice in the test group(pPG-E2/Lc W56 group and pPG-E2-ctxB/Lc W56 group)was quickly cleared.In the experimental group(pPG-E2/Lc W56 group and pPG-E2-ctxB/Lc W56 group),the viral load in the spleen,lung and blood of the mice was also gradually reduced.The viral load in the tissues of mice immunized with pPG-E2-ctxB/Lc W56 decreased significantly.In the control group,the viral load in each tissue remained high.In addition,no pathological changes in the tissues of the mice in each group were observed.The above experimental results show that oral recombinant lactic acid bacteria pPG-E2/Lc W56 and pPG-E2-ctxB/Lc W56 can provide effective antiviral protection to immunized animals.Oral immunization with pPG-E2-ctxB/Lc W56 has a better protective effect.Fifthly,the effects of oral recombinant Lactobacillus casei on the secretion of sIgA antibody in intestinal mucosa were investigated by flow cytometry and immunohistochemistry.BALB/c mice were used as animal models,divided into pPG-E2/Lc W56 immunization group(test group),pPG-E2-ctxB/Lc W56 immunization group(test group)?pPG/Lc W56 immunized group(control group)and PBS group(control group).Each group of mice in the experimental group was orally immunized with 200 ?L of recombinant Lactobacillus casei at a concentration of 1010 CFU/mL.The control group received the same dose of pPG/Lc W56 and PBS solution.After continuous immunization for 3 days,the Peyer's lymph nodes in the intestine of each group were isolated for 7 days after immunization for Bcl-6+ and IgA+ immunohistochemistry.The results showed that no marked Bcl-6+ and IgA+ cells were found in the PBS group.A small amount of Bcl-6+ and IgA+cells were seen in the pPG/Lc W56 immunized group.A large number of Bcl-6+ and IgA+ cells were seen in the pPG-E2/Lc W56 and pPG-E2-ctxB/Lc W56 immunized groups.The number of Bcl-6+ and IgA+ cells in the pPG-E2-ctxB/Lc W56 immunized group was higher than that in the pPG-E2/Lc W56 immunized group.At the same time,preparation of intestinal Peyer's lymphocyte suspension in each immunized group of mice.Percentage of Tfh cells?B220+IgM+ lymphocytes?B220+IgA+ lymphocytes and B220-IgA+plasma cells detected by flow cytometry.The results showed that the percentage of Tfh cells in the Peyer's nodules of the pPG/Lc W56 immunized group.?the pPG-E2/Lc W56 immunized group and the pPG-E2-ctxB/Lc W56 immunized group compared with the PBS control group,percentage of B220+IgM+ lymphocytes?B220+IgA+lymphocytes and percentage of B220-IgA+ plasmablasts were significantly increased.The levels from low to high were pPG/Lc W56?pPG-E2/Lc W56 and pPG-E2-ctxB/Lc W56,especially in the pPG-E2-ctxB/Lc W56 immunized group.These results suggest that the recombinant Lactobacillus casei pPG-E2/Lc W56 and pPG-E2-ctxB/Lc W56 constructed in this study can effectively promote the proliferation,maturation and differentiation of B lymphocytes(IgA + plasma cells)in intestinal Peyer's cells,and then induce the secretion of sIgA in mucosa.The induction effect is especially significant with pPG-E2-ctxB/Lc W56.It is confirmed that the BVDV E2 protein has good immunogenicity and the cholera toxin B subunit ctxB has good adjuvant activity.It was confirmed that Lactobacillus casei W56 used in this study had a good non-specific immune function.In summary,this study successfully constructed recombinant Lactobacillus pPG-E2-ctxB/Lc W56 fused to express BVDV protective antigen E2 protein and CTB as adjuvant.The results of animals immunized by oral route showed that the constructed genetically engineered Lactobacillus have good immunogenicity and immune protection.This study provides a theoretical and material basis for the further development of a new oral mucosal vaccine for bovine viral diarrhea. |
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