Expression Of Norovirus Capsid Protein In E.coli And Preparation Of Antiserum

Norovirus (NV) is a food-borne acute gastroenteritis causing virus. After being found in United States of America for the first time, its identification has been reported in many other countries all over the world. Recently, NV caused acute gastroenteritis is continuously breaking out. As a result, many countries carry out NV identification for their outputting food. However, the complex and fussy detecting method can not meet the demand of such identification. A quick, cheap and high throughput detecting methord is welcomed. Unfortunately, such a method is not available currently.The identification of NV is difficult; it is highly variable. Comparatively, capsid protein is conservative. If being expressed in prokaryotic system, a motif of 11 conservative amino acids can be laid out, serving as the epitope site. By immuning animals, antiserum with wide detecting ability can be obtained. This antiserum could overcome the disadvantage of variability of NV.In this experiment, the capsid protein was expressed in E. coli, and the antiserum was prepared. These results will certainly facilitate the development of NV detecting kits.The mainly result of this experiment were listed as the following:1. Total RNA was extracted with Trizol methord. The conservative fragment of RNA polymerase encoding gene was amplified with RT-PCR and sequenced. The fragment was 301 nucleotides in length. Analysis through DNA Star Megline, MEGA and online BLASTN, the strain represented by the sequence obtained was assigned to NV Gâ…¡.2. Two pairs of degenerate primers were designed based on the alignment of the capsid protein gene sequences of the representative strains of NV Gâ…¡-4 and NV Gâ…¡-3 respectively. The gene of capsid protein was amplified with this two pairs of primers respectively, only the degenerate primers of NV Gâ…¡-3 were effective. The PCR product was sequenced and the length of it was 1647 nucleotides, it had the same length to Gâ…¡-3 capsid protein gene. Analysis through DNA Star Megline, MEGA and online BLASTN, the similarity of the capsid protein gene to NV Gâ…¡-3 was higher than that to Gâ…¡-4.3. The capsid protein gene amplified can be translated completely. At its N-terminal, there was a conservative motif consisting of 11 amino acid residules, which was in the epitope.4. The capsid protein gene was inserted into pQE30, and transferred into E. coli M15. The recombinant protein was isolated and purified with Ni-NTA affinity chromatography with the molecular weight estimated with SDS-PAGE. The recombinant protein was 60kDa.5. The capsid protein was used to immune New Zealand rabbit with the antiserum prepared. The titer of the antiserum was 1:10000 as was determined with indirect ELISA (enzyme linked immunosorbent assay).