| The NP protein of avian influenza(AIV)is relatively conservative among different subtypes.The vaccine based on NP antigen protein can not only enhance humoral immunity,but also induce CTL(cytoxic T lymhocyte,CTL)reaction,enhance cell immunity,and thus help to clear the virus in host.Lactobacillus is a probiotic,which has the functions of regulating immunity,anti-tumor and anti infection.In addition,Lactobacillus can express exogenous genes and is widely used in biomedical field.The live vector vaccine based on Lactobacillus has the characteristics of economy,safety and simplicity,and it has great advantages to develop vaccine to prevent and control virus infection.Lactobacillus plantarum(Lb.plantarum)is a kind of foreign protein expression vector.The expression level of foreign antigen will directly affect the immune level.It plays a role by delivering foreign protein in the cytoplasm of the cell.However,the cell wall of the cell will limit the biological function of foreign protein outside the cell to a certain extent,affecting the immune effect of the body.Delivery of anchored proteins as surface display elements is conducive to the presentation of foreign antigen proteins by Lactobacillus plantarum.This construction strategy improves the expression efficiency of foreign proteins.Therefore,in order to compare the immunoprotective effect of intracellular expression and surface display expression of bacteria,we selected NC8-p SIP409-NP-DCpep new functional Lactobacillus expressing NP protein in cells and NC8-p SIP409-pgs A'-NP-DCpep new functional Lactobacillus expressing NP protein anchored in cells to evaluate the best immunoprotective effect induced by different protective antigen expression methods.In addition,new functional Lactobacillus can be used to immunize animals through oral and nasal immunization.According to literature reports,oral and nasal immunization of new functional Lactobacillus can improve the immune level of the body.Therefore,oral and nasal immunization were selected to explore the optimal immune mode of new functional Lactobacillus.This study is divided into three parts:(1)Construction of prokaryotic expression vector BL21-pET28a-NP and purification of NP proteinFirstly,using NC8-p SIP409-pgs A'-NP-DCpep plasmid as template,the NP gene fragment was obtained by PCR and gel recovery,and then connected with the cloning vector p EASY Blunt Zero and transformed into DH5?competent cells.Secondly,the NP gene fragment was obtained by restriction endonuclease digestion,connected with pET-28a vector and transformed into BL21(DE3)competent cells.The results of double restriction endonuclease digestion and sequencing showed that BL21-pET-28a-NP recombinant E.coli was successfully constructed.Finally,SDS-PAGE was used to detect the expression of the protein,and the concentration of the purified protein was measured.(2)Objective to compare the immunoprotective effects of different immune pathways of anchored and non anchored new functional LactobacillusMice were randomly divided into 9 groups,8 in each group,and divided into Saline-oral?Saline-nasal?NC8-p SIP409-pgs A'-oral?NC8-p SIP409-pgs A'-nasal?NC8-p SIP409-NP-DCpep-oral?NC8-p SIP409-NP-DCpep-nasal?NC8-p SIP409-pgs A'-NP-DCpep-oral?NC8-p SIP409-pgs A'-NP-DCpep-nasal?H9N2 inactived vaccine.The number of viable bacteria per mouse in immunized group was 1.0×10~9 CFU.The mice were immunized on the 1st,2nd,3rd,15th,16th and 17th day,and the immune indexes were detected on the 25th day.On the 26th day,the challenge experiment was carried out,and the immune indexes were detected on the 41st day.In order to detect the cellular immunity of mice after strengthening immunity,the activation of dendritic cells in the lymph nodes of Pai's lymph nodes was detected.The results showed that only the level of CD80 and CD86 in oral/nasal group was significantly improved compared with that of the normal saline group and the empty carrier group,and there was no significant difference between the mice in the expression mode group and the control group.The results showed that when MLN was detected,the difference of CD8~+IFN?~+was significant in the comparison of immune pathway between anchor oral group and anchor drop nose group,and the expression of CD4~+IFN?~+was significantly different between oral/intracellular expression oral/intranasal expression group and oral/intranasal group;In the comparison of immune pathway,the difference between the anchor/intracellular expression group and the anchor/intracellular expression nasal group was significant.The proliferation of T cells was detected after staining the MLN and spleen cells with CFSE for 72 hours with NP antigen protein.The results showed that the proliferation of CD8~+T cells in the oral group and the in cell expression group was significantly different in the comparison of expression patterns.When detecting spleen,the results showed that the expression of anchor expression in oral group and anchor group were significantly different from those of the oral group The proliferation of CD8~+T cells in the DNT group was significantly different.In addition,in order to evaluate the humoral immunity of mice after strengthening immunity,the B cells of the lymph node of Pai's lymph nodes were detected.The results showed that only the anchor expression group significantly increased the percentage of B220~+CD95~+PNA~+,the percentage of B220~+CD95~+PNA~+was not increased by the group of nasal drip and intracellular expression;the results of immunofluorescence showed that the percentage of B220~+CD95~+PNA~+was not increased by oral and intracellular expression groups The number of B220~+Ig A~+cells in ileum and lungs of mice in the group of new functional Lactobacillus was significantly increased by nasal anchor.Oral and nasal anchor expression of new functional Lactobacillus group could improve the secretion level of specific antibody SIg A and serum specific Ig G in mice feces.Two weeks after challenge,the specific cytokines of mice were detected.The results showed that compared with the anchored expression nasal drops,the anchored expression oral group had a higher expression of CD4~+IFN-?~+than the anchored expression nasal drops The difference of CD4~+IFN-?~+and CD8~+IFN-?~+between oral anchoring group and nasal anchoring group was very significant when MLN was detected,and the difference of CD4~+IFN-?~+and CD8~+IFN-?~+between oral anchoring group and oral intracellular expression group was significant.The results showed that the survival time and protection rate of mice were prolonged in the oral group.The pathological results of lung showed that there was no significant pathological change in the lung of the mice in the oral group,but the lymphocyte in the control group increased significantly.The results showed that oral immune pathway was better than that of nasal drip,and anchor expression was better than that of intracellular expression.That is,oral anchoring expression of NP protein new functional Lactobacillus could cause the level of cellular and humoral immunity to be increased and the immune effect was the best.(3)Evaluation of immune effect of oral anchored new functional Lactobacillus combined with inactivated avian influenza vaccineThe commercial inactivated vaccine of avian influenza can only induce the body to produce antibodies and then play a protective role,and the effect lasts for a short time.The new functional Lactobacillus can not only induce the body to produce antibodies,but also induce mucosal immunity.In the previous chapter,we found that the new functional Lactobacillus NC8-p SIP409-pgs A'-NP-DCpep had the best immune effect.Therefore,in this chapter,we evaluated the immune effect of NC8-p SIP409-pgs A'-NP-DCpep combined with intramuscular injection of inactivated vaccine,and enhanced the immune response of mice against H9N2subtype avian influenza virus infection.Mice were divided into four groups:NC8-p SIP409-pgs A'-oral?NC8-p SIP409-pgs A'-NP-DCpep-oral?NC8-p SIP409-pgs A'-NP-DCpep-oral+inactivated vaccine(oral immunization NC8-p SIP409-pgs A'-NP-DCpep combined with intramuscular injection of avian influenza inactivated vaccine)and H9N2 inactivated vaccine,with 8 mice in each group.The number of viable bacteria per mouse in immunized group was 1.0×10~9 CFU.The mice were immunized on the 1st,2nd,3rd,15th,16th and 17th day,and the immune indexes were detected on the 25th day.On the 26th day,the challenge experiment was carried out,and the immune indexes were detected on the 41st day.In order to detect the cellular immunity of mice after enhanced immunization,the specific cytokines in MLN and spleen of mice were detected.The results showed that the expression levels of CD4~+IFN-?~+in MLN,CD4~+IFN-?~+in spleen and CD8~+IFN-?~+in spleen of anchored expression group were significantly increased.The MLN and spleen cells of mice were stained with CFSE,and then co cultured with purified NP antigen protein for 72 hours.The results showed that the CD4~+T,CD8~+T in MLN and CD8~+T in spleen increased significantly in the anchored expression group.In addition,in order to evaluate the humoral immunity of mice after enhanced immunization,the activation level of B cells in mice's lymph nodes was detected,and the results showed that the number of B220~+Ig A~+cells in ileum of mice in the oral combined inactivated vaccine group was significantly increased;the level of HI antibody in serum of mice after immunization was detected The results showed that the level of HI antibody was significantly increased in the oral inactivated vaccine group one day before booster immunization.The results showed that the expression of CD4~+IFN-?~+and CD8~+IFN-?~+in MLN and lung were significantly increased in anchored expression oral combined with inactivated vaccine group;The results of weight loss rate and survival rate showed that the weight of mice in the anchored expression oral combined with inactivated vaccine group finally recovered to the initial weight,and the survival rate of mice reached 100%;the lung pathological results showed that there was no obvious pathological change in the anchored expression oral combined with inactivated vaccine group,while the lymphocytes in the control group increased significantly.Immunofluorescence assay showed that the number of virus antigen in lung of mice in the anchored expression oral inactivated vaccine group was significantly lower than that in the control group.These experiments proved that the immune response of SPF mice and the effect of anti-H9N2 subtype avian influenza virus infection were enhanced.The anchored expression was better than the intracellular expression.The oral immune pathway was better than the nasal immune pathway.The combined inactivated vaccine of new functional Lactobacillus NC8-p SIP409-pgs A'-NP-DCpep was better than the non combined immune pathway.This study laid a foundation for the prevention and treatment of avian infection with new Lactobacillus preparation. |
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