Expression Of Norovirus Multi-epitope Gene And Preparation Of Its Polyclonal Antibody

Norovirus is one of the most common causes of nonbacterial gastroenteritis in humans. Itcan lead to a major public health concern. Norovirus has high infectivity and variability, and itcan cause acute gastroenteritis in people of all ages, especially in vulnerable patient populations.Therefore, it is important to diagnose noroviruses rapidly and early to prevent and control theirspread. In recent years, most of studies on norovirus were expressing the capsid protein encodedby full-length ORF2as to preparing the monoclonal antibody, detected by means of ELISA. Theassay is accurate only for a single genotype norovirus, but it is not fit for determining a pluralityof genotypes sample at the same time, which limits its application. Multi-epitope gene is thatwhich uses recombinant DNA technology to concatenate multiple encoding epitope nucleotidefragment. It has made a breakthrough in HIV, CSFV and Mycobacterium tuberculosis, however,multi-epitope gene about norovirus has not been reported. This thesis use chemical synthesis,PCR amplification, enzyme digestion and connection method to combine four epitope sequencesof S region, and induce expression of recombinant fusion protein in a prokaryotic expressionsystem; to obtaine with the purified recombinant fusion protein and immune rabbit to getpolyclonal antibody; and use prepared rabbit anti-norovirus polyclonal antibody coated beads toget the immunomagnetic beads, then use immunomagnetic beads to enrich the norovirusparticles. The results are shown as follows:(1) According to the information of published literature, the three S region epitope close tothe N-terminal capsid protein were chosen to obtaine the preceding gene fragment A of norovirusmulti-epitope gene by chemical synthesis method; combined with the early research in ourlaboratory and NCBI sequence information, primers were designed according to the S region and1004norovirus cDNA as a template, then they were amplified to get the gene fragment B ofnorovirus multi-epitope gene; at last, A and B were connected to build norovirus multi-epitopegene. The nucleotide length was438bp, totally encoded145amino acids. It was connected withthe expression plasmid pET-32a (+) and transformed into E. coli BL21(DE3) prokaryoticexpression. After inducing for6h under30°C, we got a34kDa recombinant fusion proteins. BySDS-PAGE analysis, the proportion of target protein is about35%of all the bacterial protein,and most are soluble protein. Finally, the recombinant fusion protein was purified by affinitychromatography methods.(2)After concentrating the purified recombinant fusion protein, they were used to immunize rabbits with immunologic adjuvant. Taking the antiserum from the heart blood after immunizingfive times, then polyclonal antibody was purified using caprylic acid-ammonium sulfateprecipitation method. The antibody titer was determined to be1:51200by indirect ELISAmethod.(3) Prepare rabbit anti-norovirus polyclonal antibody coated beads, then optimize thecoupling conditions of beads and antibody. The optimal conditions are: the coupling media isPBS, pH of7.4, coupling time of3h, temperature of20°C, and the optimal antibody couplingconcentration is300μg/mL. The immunomagnetic beads were prepared to enrich the noroviruswithin anal swab. And the virus RNA were extracted before and after the enrichment, then theywere detected with real-time PCR method. The results show that the enrichment of norovirus testresults were positive, and the immunomagnetic beads had good adsorption of GI and GIIgenotypes virus, furthermore, the polyclonal antibody can specificly bind with norovirusparticles.