Detection Of Viral Gastroenteritis In Parts Of Jilin Province And Immunization Study Of Norovirus Genetic Engineering Candidate Vaccine

Background:Viral gastroenteritis,also known as viral diarrhea,is a common disease affecting people of all ages,which causes a serious global social and economic burden every year,and cause severe dehydration in infants and the elderly and causes complications and even death.It is mainly caused by viruses such as rotavirus and norovirus,which seriously endangers public health and daily life.Every year,viral gastroenteritis caused by Norovirus or rotavirus infection will cause a high economic burden,so researchers in various countries are working hard to develop vaccines.The rotavirus vaccine has been approved for use and has a good immune effect.Norovirus commercial vaccines have not been approved for use,and some studies are in clinical trials.Therefore,this study focuses on the Norovirus vaccine.Norovirus(NoV)is currently one of the major pathogens causing acute gastroenteritis worldwide,causing approximately 699 million infections each year.NoV is an RNA virus,which is easy to recombine,has strong environmental tolerance,has high infectivity,and is a potential pathogen that induces large-scale infection.In the winter of 2016,the GII.P16-GII.2 recombinant Norovirus began to spread in Asia.It has strong infectious and replicative characteristics,which illustrates the potential of the pandemic and has attracted attention.Innovation points:1.The epidemiological characteristics of viral gastroenteritis in Jilin Province were determined.2.Successfully obtained GII.P16-GII.2 norovirus virus-like particles(VLPs)and recombinant Lactobacillus plantarum Lac.VP1 expressing GII.P16-GII.2 norovirus disease VP1 protein,which can stimulate mice produce cellular and humoral immune responses.3.1t was confirmed that the recombinant Lactobacillus plantarum Lac.VP1 induces the maturation of dendritic cells(DCs)through the TRAF2/NF-?B signaling pathway for antigen presentation,and induces T cells to differentiate into Th1.Preliminary explanation of the immune mechanism of recombinant Lactobacillus plantarumObjectiveDetect the infection of viral gastroenteritis in parts of Jilin Province,and provide a reference for the prevention of viral gastroenteritis.Construct GII.P16-GII.2 recombinant Norovirus VLPs and recombinant Lactobacillus plantarum,and the immunogenicity of two genetically engineered vaccines was evaluated.Furthermore,the immune response mechanism of recombinant Lactobacillus plantarum Lac.VP 1 to DCs was further studied,which provided theoretical support for the development of a NoVel vaccine for norovirus and the study of immune mechanism.Research methods:1.Collect samples of patients with diarrhea in parts of Jilin Province and perform RT-PCR to analyze the infection of viral gastroenteritis in Jilin Province.2.VLPs of GII.P16-GII.2 recombinant Norovirus were prepared using baculovirus expression system,and the detection was proved by PCR,Western blot,indirect immunofluorescence(IFA)and transmission electron microscopy.3.PCR,Western blot,IFA,flow cytometry,and scanning electron microscopy and transmission electron microscopy were used to verify the successful construction of Norovirus recombinant Lactobacillus plantarum Lac.VP1.4.Analysis of in vivo colonization and proliferation of recombinant Lactobacillus plantarum Lac.VP1 by Western blot,CFDA-SE staining flow cytometry,and small animal imaging.Western blot,routine blood tests,and HE staining were used to analyze the in vivo pathological damage of recombinant Lactobacillus plantarum Lac.VPl.5.Through the detection of specific IgG,IgG typing detection,B lymphocyte activation,T lymphocyte activation and proliferation,T lymphocyte subtype detection,and CD8+memory T lymphocyte detection after immunizing mice,the analysis and evaluation of two Immunogenicity of a Genetically Engineered Candidate Vaccine.6.Analyzed the interaction between Lactobacillus plantarum Lac.VPl and DCs by flow cytometry,cytokine detection,qPCR,Western blot,IFA and immunohistochemistry,and analyzed Lac.VP1 activates the presentation mechanism pathway to DCs.Research results:1.The infection of viral gastroenteritis in parts of Jilin Province was detected by RT-PCR.The virus-induced diarrhea rate in Jilin Province was 27.62%,of which rotavirus was the main pathogen of infection,accounting for 58.14%.The viral gastroenteritis caused by Norovirus accounted for 9.30%.2.Through experimental methods such as PCR,Western blot,IFA,and electron microscope observation,it was proved that norovirus VLPs were successfully obtained using the baculovirus expression system.3.It has been proved by PCR,Western blot,IFA and other experimental methods that recombinant Lactobacillus plantarum can express Norovirus VP1 protein.The morphology of the recombinant Lactobacillus plantarum was observed by scanning electron microscope and transmission electron microscope.It was proved that the VP1 protein expressed by the recombinant Lactobacillus plantarum was successfully and anchored on the surface of the recombinant Lactobacillus plantarum.4.Western blot proved that continuous passage,induction time,artificial gastric juice and artificial intestinal juice had no effect on the expression of Norovirus VP1 protein by recombinant Lactobacillus plantarum,and proved the stability of Lac.VP1.The in vivo colonization of Lac.VP1 stained by CFDA-SE was detected by flow cytometry and small animal imaging system.Recombinant Lactobacillus plantarum Lac.VP1 can induce Caco-2 cells to overexpress tightly linked or closed proteins such as ZO-1,Claudin-1 and Occludin,which proves that Lac.VP1 can enhance the cell's resistance to pathogens.The blood safety,body weight/survival observation,and HE staining were used to determine the safety of recombinant plants such as Lac.VP1 in vivo.5.After immunizing mice,norovirus VLPs and recombinant Lactobacillus plantarum can produce specific IgG and induce activation of B lymphocytes.The T lymphocyte proliferation and activation test results of the immune group of VLPs and the recombinant Lactobacillus plantarum immunization group showed that the immune T lymphocytes can rapidly proliferate and activate when the pathogen is stimulated.At 28 days after immunization,Norovirus VLPs and the recombinant Lactobacillus plantarum immune group could produce CD8+memory T cells.Norovirus VLPs and recombinant Lactobacillus plantarum test results were significantly higher than the control group,indicating that Norovirus VLPs and recombinant Lactobacillus plantarum have good immunogenicity.6.Through incubation of recombinant Lactobacillus plantarum with DCs and the use of flow cytometry technology,it was proved that recombinant Lactobacillus plantarum Lac.VPl can induce the maturation of DCs,high expression of MHC-? as the first signal,and high expression of the common stimulating factors CD40,CD80 and CD86 serves as the second signal,and the secretion of cytokines such as IL-6 acts as the third signal to interact with T cells and induce Th0 to differentiate into Th1 lymphocytes.Detection of DCs in mesenteric lymph nodes and Peyer's lymph nodes after immunizing mice with recombinant Lactobacillus plantarum Lac.VP1 confirmed that DCs in the intestinal tissue can take up Lac.VP1 and migrate to mesenteric lymph nodes and Peyer's lymph nodes,acting on T cells to promote T cell differentiation.Western blot detection technology was used to detect changes in the expression of TRAF family proteins and NF-?B p65,NF-?B p50,phospho-IKK-?/?,IKB-? and phospho-NF-?B-p65 and other key proteins in NF-?B pathway after incubation of recombinant Lactobacillus plantarum Lac.VP1 with DC2.4 cells.It showed that the recombinant Lactobacillus plantarum Lac.VP 1 can activate the TRAF2/NF-?B signaling pathway and induce the maturation and activation of DC cells for antigen presentation.Fluorescence microscopy was used to observe that NF-?B p65 was highly expressed in DC2.4 cells of the Lactobacillus plantarum Lac.VP1 culture group and transferred to the nucleus.It was confirmed that recombinant Lactobacillus plantarum Lac.VP1 activated DCs NF-?B pathway for antigen presentation and immune induction.Conclusion:1.27.62%of gastroenteritis in parts of Jilin Province is caused by viruses,of which 58.14%and 9.30%are viral gastroenteritis caused by rotavirus and norovirus,respectively.2.Successful preparation of GII.P16-GII.2 VLPs by recombinant baculovirus expression system.3.Successful construction of recombinant Lactobacillus plantarum Lac.VP1 expressing GII.P16-GII.2 recombinant Norovirus VP1 protein.4.Recombinant Lactobacillus plantarum Lac.VP 1 can colonize and proliferate in the intestine of mice without causing intestinal tissue damage.5.Norovirus VLPs and recombinant Lactobacillus plantarum can produce spec-ific IgG and CD8+memory T cells and induce activated T lymphocytes and B cells at 28 day post immunization.It shows that Norovirus VLPs and recombinant Lactobacillus plantarum vaccine candidates have good immunogenicity and can induce cellular and humoral immune responses.6.Recombinant Lactobacillus plantarum Lac.VP1 can induce the differentiation and maturation of DCs.Mature DCs can take up Lac.VP1 and migrate to mesenteric lymph nodes and Peyer's patch lymph nodes to act on T lymphocytes and induce Th0 to Th1 lymphocyte differentiation.Recombinant Lactobacillus plantarum Lac.VP1 activates the TRAF2/NF-?B pathway of DCs for immune induction.