| Human norovirus(HuNoV)is the main cause of food-borne diseases and severe diarrhea in children.After the rotavirus vaccine was developed,the virus was considered to be the main pathogen causing severe diarrhea in children.Although these viruses are important,little is known about them due to the difficulty of culture in vitro.It is estimated that they have caused more than one million visits and 200,000 deaths in poor areas of the world.The virus is mainly transmitted through fecal-oral or aerosol routes,including atomized virus particles in vomit,food,water sources,and environmental pollution,which can cause norovirus to spread in this way.Norovirus infection occurs in winter and spring,and people of all ages are generally susceptible,usually infants,children,the elderly,and people with weakened immune systems.The disease caused is self-limiting,with a duration of 24 to 48h.Due to the large number of genotypes of the virus,rapid mutation,and prone to genetic recombination,humans have limited understanding of the basic characteristics and pathogenic mechanisms of Norovirus,and this also greatly limits the research on norovirus antibodies.Therefore,understanding the immune response of human norovirus induced by natural infection and simulation is very important for the norovirus detection and the understanding of the immune response mechanism of norovirus infection in humans.The P domain of the Norovirus capsid protein is an important link that leads to the variability of the viral genome and antigen specificity.This part belongs to the hypervariable region in the capsid protein and carries the main antigenic site of the virus to the outside world.The viral protein expressed against the P domain can effectively cause the body's immune response,thereby making the preparation of antibodies more efficient and specific.As norovirus genotypes are opposed,we need to find the dominant subtype strains that have been circulating in recent years,and then we can get better applications.In the past ten years,the G?.4 subtype is the main cause of norovirus infectious gastroenteritis in most clinical cases,and has been one of the main prevalent strains in recent years,so in this research,this subtype is studied.The main contents of this research include:(1)Full-length genomes amplification and analysis of one murine norovirus and five microvirusCollect a diarrhea mouse(strain BALB/c)feces,and build a viral metagenomic library on it.After metagenomic sequencing analysis,it is found that there is a contig of Murine Norovirus(MuNoV)and bacteriophage(Microvirus)Analyzed the data using special software for metagenomics,completed the assembly of 5 circular phages in the library,and obtained their full-length sequences.And using Sanger sequencing,combined with the highthroughput sequencing results of metagenomics,the amplification of MuNoV was completed.As a result,the five Microviruses were named UJSM1,UJSM3,UJSM7,UJSM14,UJSM20.Their genome in lengths are 4737 bp,5121 bp,5211 bp,5 254 bp,and 4722 bp.Among them,UJSM7 and UJSM14 belong to Gokushovirinae,UJSM1 And UJSM3,UJSM20 belong to two potential new subfamilies,respectively.The MuNoV strain(named UJSMN01)has a genome in lengths of 7383 bp with three ORFs.Phylogenetic analysis shows that the gai strain belongs to the GV subtype and has the highest similarity(91%)with the Korean strain(FJ446719).The genome sequences of one MuNoV and five Microvirus share GenBank accession numbers as MW018367,MW073821,MW073822,MW073823,MW073824 and MW073825,respectively.(2)Prokaryotic expression,protein purification and renaturation of Norovirus G?.4 P domainBy consulting the literature,the nucleotide sequence encoding G?.4 P domain was designed and submitted to the company for synthesis,and named as Pujs.After being connected with the expression vector PET-28a,it was imported into E.coli prokaryotic expression system,and the protein was purified and renatured.Western Blot identification of G?.4 P domain protein showed an obvious immune band at the 35kDa origin.The protein concentration after renaturation was 0.22mg/mL.(3)Preparation of polyclonal antibodiesThe purified and renatured protein was mixed with adjuvant and immunized New Zealand white rabbits for 5 weeks.After the immunization,blood was taken from the heart and the serum was separated.The antibody titer by indirect ELISA was 1:819200.Using this antibody to detect Noro-positive samples,an effective degree of detection can be obtained.The research conclusion of this subject:We have discovered a murine norovirus strain using metagenomics,and amplified the full-length sequence of the strain and the 5 phage sequences in the sample.The molecular biological characteristics of norovirus Further research has been done and the virus database has been enriched.And for the predominant subtype strain G?.4 of human norovirus,the gene sequence was synthesized for the P domain containing the main antigenic site,and the expression vector of G?.4 P domain was constructed,and the E.coli prokaryotic expression system was used.The P protein was successfully expressed and named Pujs.Pro.Furthermore,New Zealand white rabbits were immunized,and polyclonal antibodies were produced after several weeks.After analysis and testing,the antibody had a high titer and could be used to detect virus particles in norovirus stool samples.This provides an important foundation for the study of the immune response mechanism between Norovirus and the receptor and the establishment of a platform for rapid detection of Norovirus. |
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