Expression And Immunological Analysis Of Group A Human Rotavirus Protein VP6

Rotavirus (RV) belongs to Rotavirus genus in family Reoviridae. RV is the major causative agent of acute gastroenteritis in human beings, mammals and birds. The complete virion consists of three concentric protein layers. VP6 is the structural protein located at the middle layers of virion. VP6 is encoded by RNA segment 6 (gene 6), and consists of 379 amino acids with a theoretical molecular mass of 45KDa. VP6 is the most abundant protein of the virion comprising about 51% of the total protein mass. the middle capsid layers of RV is assemblied by 260 VP6 trimers. The VP6 plays an important role as a physical adaptor and is nessesary in RV gene transcription.In this study, VP6 gene was derived from TB-Chen strain RV which was isolated from a two-year old child with acute gastroenteritis. Using complete gene of VP6 of TB-Chen as a template, a PCR was carried out. The amplified VP6 coding sequence was constracted on to pET plasmid and, a recombinant expression plasmid pET-VP6 was obtained and used to transform competent bacterial E.coli BL21(DE3) cells. The recombinant VP6 (rVP6) was highly expressed in E.coli BL21(DE3), accounted for 27.4% of the total bacterial cell's proteins. Western Blot results showed that rVP6 could be specifically recognized by rabbit anti-rVP6, anti-SA11 and anti-Wa antibodies. Immunofluorescence assay (IFA) results showed that after virus infection, VP6 protein of RV SA11 or Wa strain in infected MA104 cells could been recognized at 3h, and fluorescence was strongest at 12 h after infection. VP6 protein exsists mainly in the cytoplasm in particle forms. The results of neutralization test, showed that the anti-rVP6 antibodies elicited in rabbits had neutralizing activity against rotavirus SA11 and Wa infection, in vitro.The above results provide an usfull basis for futher studying the biology and immunity characteristics of VP6 and for an improbable rVP6 protein candidate vaccine.