| Porcine Rotavirus(PoRV), which belongs to the family Reovirus, is a major cause of diarrhea inseveral kinds of young animals. RV, Porcine epidemic diarrhea virus(PEDV)and TransmissibleGastroenteritis Coronavirus(TGEV)always co-infect the piglets, which cause serious diarrhea, vomiting,fever, dehydration and RV exists in almost every farm. It can spread from sow to piglet via milk. Theprevalence of RV all over the world results severe economic losses to the swine industry.The genome of Rotavirus consists of11double-stranded RNA, encoding12proteins, includingstructural proteins VP1, VP2, VP3, VP4, VP6, VP7and nonstructural proteins NSP1, NSP2, NSP3,NSP4, NSP5. Nonstructural protein NSP4is encoded by gene10and is described as the first viralenterotoxin. It is a multifunctional protein. It can induce diarrhea in the newborn mice withoutpathological changes in order to promote the mature of RV in the ER. Fusion protein can increase theendogenous Ca2+concentration. The infected intestinal epithelial cells are damaged and result inmalabsorption. Its endotoxin activity may be another mechanism of diarrhea. In RV infected patient’sbody, NSP4IgA and IgG can transformed into each other. This indicate that NSP4can expose toimmune cells.In this study, NSP4protein gene of RV was amplified by reverse transcription-polymerase chainreaction(RT-PCR), then the gene was inserted into the prokaryotic expression vector pET-30a and therecombinant plasmid was named as pET-30a-NS. The generated recombinant plasmids were sequencedafter digestion. The correct plasmid was chosen to express protein. Recombinant protein was expressedby inducing pET-30a-NS transformed E.coli with IPTG. Analysis of SDS-PAGE showed the protein wassuccessfully expressed. The recombinant protein was purified by the method of rubber-cutting and theresult showed that the protein was purified as expected. The purified protein could be used in thesubsequent research. It was revealed by Western blot assay that the recombinant protein could react withanti-RV serum, demonstrating the recombinant protein has good reactivity.BALB/c mice were immunized with the purified NSP4protein to prepare MAb against NSP4protein of RV. After four times of immunity, spleen cells were prepared and merged with SP2/0cells.One strain of hybridoma secreting antibodies against NSP4protein was obtained, which was named as4F10. The ELISA result showed high antibody titer. MAb4F10could react with RV virion revealed byWestern blot assay. MA104cells infected with RV showed specific immunofluorescence with the MAb4F10as primary antibody by Indirect immunofluorescence assay(IFA).A series of peptide segments were synthesized according to the amino acids sequence of NSP4protein. Every segment consisted of16amino acids, and every two adjacent segments shared6sameamino acids. The antigenic epitopes were established by ELISA assay by MAb4F10, whichwere"61SKCSYK66". In order to analyze the homologous of NSP4protein among different strains,sequences of amino acids were aligned. The results indicated that the core epitopes of NSP4proteinwere completely conserved among different strains. So the epitopes could be supposed to be a possiblecommon epitope region. One strain of hybridoma secreting antibodies against NSP4protein was obtained, and antigenicepitopes were identified, which coincided with relative literature. This will contribute to further study ofthe function of protein, understanding the antigenic structure, establishment of diagnosemethods,designing of vaccine and so on. It will also offer information to the research of identificationof core epitopes, predominant epitopes, explanation of mechanism of RV. |
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