| Backgroud and ObjectiveNorovirus(NoVs)is a single-stranded positive-sense small RNA virus with a diameter of about 27 to 38 nm,which is the main pathogen of human acute gastroenteritis.Its entire genome consists of three open reading frames(ORFs),about 7.4-7.7 kb in length.The length of ORF2 is about 1.7 KD and encodes the major structural protein VP1.The molecular weight of ORF2 is about 56kDa,including the capsid region(S region)and the prominence region(P region).P region which can be divided into two sub-regions P1 and P2,P2 sub-region has a high degree of variability,but also NoVs and HBGA receptor binding sites.NoVs are contagious and can easily cause outbreaks all over the world and can occur throughout the year,especially in the cold season.Adult and school-age children are the main target of infection.Epidemiological surveys show that the global economic loss caused by Norovirus is 4.2 billion(95%of UI is 3.2 billion to 5.7 billion),and the direct economic loss of 60.3 billion(95%UI is 44.4 billion to 834 Billion)indirect social and economic losses of the United States dollar.At present,human histo-blood group antigens(HBGAs)on the intestinal mucosa are considered to be receptors or synergistic factors of NoV-infected organisms,and the affinity of different NoV genotypes to HBGA receptors is inconsistent.Raw foods such as bivalves and shellfish are considered as risk factors for acute gastroenteritis due to infection with No V,and there have been reports of large-scale acute gastroenteritis outbreaks caused by raw seafood such as oysters.The global outbreak of GII.4Sydney in 2012 led to the outbreak of epidemic.Shenzhen,Guangdong Province,China first reported in July 2012,and subsequently reported the new mutant strains in Jiangsu,Beijing and Sichuan in the following months.During the Novo-Prevalence of NoVs in the winter and spring 2014-2015,a new outbreak of human variant NoV GII.17 appeared outbreak that first appeared in Guangdong and Jiangsu Provinces,and the prevalence of outbreaks of human infection at that time had exceeded that of GII.4 Advantage of popular strains.NoV GII.3 strain was endemic in Guangdong in a small area in 2015,and GII.3 strains in this oyster culture area were the dominant strains of GII group with NoV pollution in oyster.So in this study,three strains of Norovirus were selected for P-particle expression,so as to conduct follow-up study.According to the previous survey results,our group found that oyster NoV pollution in Changsha oyster culture area in Guangdong Province was higher than that in other farming areas in Guangdong Province.Oyster oyster was the main occupation of the population in this area,and most of the residents in this area had raw shellfish Class of seafood such as eating habits,indicating that there may be repeatedly exposed to different types of NoV may be the population in the region.However,the incidence of acute gastroenteritis in this area is low.According to the previous study of the research group,the serum antibodies of this population in the population have a higher blocking ability against No38 of the VA387 strain than that of the general population.However,the resistance of this population to other strains of NoV Broken capacity is unknown.In this study,based on the characteristics of this special population and the previous research foundation of the research group,we selected the residents in this area as the appropriate target population and continuously monitored the serum antibodies in this population.At the same time,the age and gender conditions were matched at the same time,serum samples from the general population in four different inland areas of Guangdong Province were taken as samples.Continue to use the mature E.coli BL21(DE3)prokaryotic expression system expression NoV G?.4 Sydney strains,G?.17 strains,GII.3 P particles.To detect the level of serum antibodies against different types of NoV in this area and in the general population,to analyze the blocking effect of antibodies on different types of NoVs viruses in serum and to screen out the high blocking ability of serum antibodies in different types of NoVs Individuals to screen for a broad spectrum of studies on human monoclonal antibodies provide the basis for the study is expected to be safe and stable antiviral serum to protect people at high risk of emergency;continuous monitoring of the population in Guangdong G?.17 outbreak before and after the epidemic,the Antibody changes in the serum of the local population further demonstrated the duration of the protective antibody after NoV infection and provided the basis for the development of NoV vaccine.Methods1.Construction and identification of pGEX-4T-1 recombinant expression vectorGII.3 virus RNA was extracted from feces,and P region was amplified by PCR.The P region fragment and the expression vector pGEX-4T-1 double digestion after the connection,the connected plasmid go TOP 10 competent bacteria for amplification.Plasmids were extracted from the bacterial broth and the extracted plasmids were identified by double enzyme digestion with BamHI and NoTI and sequenced at the same time.2.Expression and purification of P particleThe plasmid witih the correct identification was transferred into BL21(DE3)competent cells and the NoV P particles were expressed by the prokaryotic protein expression principle.After the expression of the protein with a GST tag,it can be purified by GST chromatography on a gravity column.After purification,the GST-tagged protein was excised with human thrombin to obtain NoV P particle pure protein,which was analyzed and identified by 12%SDS-PAGE protein electrophoresis and Western blotting.3.The serum antibody of NoV in populationThree NoV P particles were used as coating antigen,and the antibodies against different strains in serum of experimental group and control group were detected by indirect enzyme-linked immunosorbent assay(ELISA).Using microplate reader 450nm wavelength for detection of absorbance,reading OD value,when OD450 value>0.25 bejudged as positive.4.Detected salivary HBGA receptor phenotype by ELISAThe pretreatment of saliva specimens coated with ELISA method,in each saliva were added different HBGA type monoclonal antibody.In the microplate reader 450nm wavelength reading OD value,OD450 value>0.08 that is judged as positive,according to the type of monoclonal antibody added to determine the type of HBGA receptor.5.Serum antibodies blocking G?.17 and HBGA receptor binding experimentThe HBGA type B type saliva has been coated,and an neutralization surrogate model was used to determine the ability of human serum antibodies to block the binding of NoV G?.17 to HBGA receptors.A blank control,positive control,and negative control were used to calculate serum Antibody blocking efficiency.According to the curve of the relationship between the individual serum dilution and the blocking rate,the serum dilution is calculated when the individual serum blocking rate is 50%(BT50).Results1.Construction and identification of pGEX-4T-1 recombinant expression vectorPlasmid PCR identification of about 6kb band,and the positive control known plasmid fragment size;enzyme digestion gel electrophoresis showed:The constructed recombinant plasmid was digested into two fragments were 4.9 kb of the Pegex-4T-1 vector fragment and the P region gene fragment of about 1000 bp in size,the result was in line with the expected,indicating that the construction of the recombinant plasmid Pgex-4T-1-P-G?.3 was successful.2.Identification of NoV P particleThis experiment co-expressed NoV G?.4 Sydney strain,G? 17,GII.3 a total of three virus particles P,10%SDS-PAGE in the electrophoresis results showed that:there are two obvious bands compared with Marker size 63 KD of GST-P particle and 36 KD of P particles,in line with the expected P particle size.Western Blotting results of the GIL 17 P particles and the GII.3 particles after the electrophoresis were respectively shown as follows:the bands were displayed at the sizes of 63 KD and 36 KD respectively,which proves that the P particle expressed by the prokaryotic expression system BL21(DE3)used in the study has the antigen Specificity.3.Detection of G?.4 Sydney strain serum antibody in populationThe positive rate of antibody in Shanwei population(N = 195)in 2015 was 100%(195/195),the mean value of antibody OD was 2.521(95%CI:2.419-2.642)(95%CI:2.493?2.672).The positive rate of antibody in the control group was 100%(210/210)and the OD mean was 2.249(95%CI:2.151?2.347)4.Detection of G?.17 strain serum antibody in populationThe positive rate of Shanwei population was 100%(195/195)in 2015 years,with an average of 1.578(95%Cl:1.529-1.627)and a positive rate of 100%(184/184)0.937?1.015).The positive rate of the control group was 55.2%(116/210)with a mean of 0.276(95%Cl:0.263-0.290)5.Detection of G?.3 strain serum antibody in populationThe positive rate of Shanwei population was 88.2%(15/19)with a mean of 0.492(95%Cl:0.463-0.520)and the positive rate of Shanwei population was 99.5%(183/184)in 2016 years with a mean of 0.885(95%Cl:00.836?0.935).The positive rate of the control group was 41.9%(88/210)with a mean of 0.249(95%Cl:0.236-0.261)6.Cross-blocking titer between GII?4 and G?.17 norovirusesAccording to the result of population test in oyster culture area in 2015,39 individuals with serum blocking ability of BT50 serum antibody titer?200 for GII.4 VA387 strain and G?.17 strain were screened out.7.GII.17 protective antibodies change in two years in Shanwei areaThe geometric mean titer of serum antibody was 68.45(95%Cl:54.4-86.13)at BT50 in Shanwei area in 2015.The geometric mean titer of serum antibody was 48.49(95%Cl:41.30-56.93).The titers of 2016 years decreased significantly compared with 15 years,and the difference was statistically significant.(t = 3.688;p<0.01)8.Duration of Immunity to G?.17 Norovirus among people with HBGA phenotypeThere were 53 surveillance samples for two consecutive years,accounting for 81.13%(43/53)for secretors individuals and 18.87%(10/53)for non-secretors individuals.In 2015,the results of serum antibody blocking test showed that BTsotiter?200 in non-secreting individuals was 0%(0/10),and the titer of BT50 titer?16.67 was 80%(8/10).BT50 titer?200 accounted for 48.84%(21/43)and BT50 titer?16.67 accounted for 34.88%(15/43)in secretors individuals,moreover in secretors there are 75%(21/28)persons' BT50 Titers ?200 in whose BT50 titer>16.67(N = 28),most of the population can be considered infected with norovirus,and has the protection.It is noteworthy that in 2016 85.71%(18/21)of the individual serum antibody titers dropped below 200 in individuals with BT50 titer ?200(N = 21)in 2015,five individual serum titers were as low as 16.67.The results show that after two years,individual serum antibody protection decreased significantly.Conclusions1.NoV GII.4 Sydney strain,GII.17 strain and GII.3strain P particle with good antigenicity were successfully identified;2.Serum antibody level of NoV response to different types of breeds was higher in aquaculture area than in control population,which indicated that there was more NoV exposure in aquaculture population than control population;3.The level of serum antibody blockage in aquaculture area was higher than that in control population,and some individual serum antibodies had the ability of blocking multiple types of NoV,indicating that the population had the conditions of screening all-human antibodies with cross-blocking ability;4.GII.17 one year after the epidemic,serum antibody titers dropped significantly,in the natural state of immunity protective antibodies short duration. |
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