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Rotavirus (RV), belonging to the family of Reoviridae, is the leading cause of severe viral gastroenteritis in infants and children worldwide. In developing countries RV infections cause approximately 600, 000 children deaths per year(15). In addition, rotaviruses also infect mammals and poultry. Therefore, RV not only threaten human health but also impact the development of the stockbreeding(14). RV are non-enveloped icosahedral viruses and their genome contains 11 segments of double-stranded RNA coding six structural proteins (Vp1, Vp2, Vp3, Vp4, Vp6, Vp7) and five nonstructural proteins (NSP1, NSP2, NSp3, NSp4, NSP5) (7). The Vp4 is the outermost spike protein of RV and accounts for 1.5% of the total viral proteins. The gene of Vp4 is made up of 2359 base pairs, coding a protein of 775 amino acids, with a molecular weight ~ 88 kDa. The Vp4 protrudes from the outer capsid glycosylated protein Vp7 and has many functions such as cell binding, entry, hemagglutination and neutralization. The Vp4 can be cleaved into Vp5* and Vp8* by the trypsin-like proteases, and virus infectivity is enhanced by this cleavage (6). From the above discussion, it is clear there are several theoretical and practical benefits to further study of this protein. This study reports the results of the gene cloning, recombi-nant expression and immunity characteristics of Vp4 of a human group A RV strain TB-Chen, which was isolated from a clinical patient in two years with acute gastroenteritis.
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Using the pBS-Vp4 as template DNA, PCR was performed. The PCR product with 2450 bp was detected at 1.2% agarose gel electrophoresis (Fig. 1).
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PCR product of Vp4 gene was digested with Nde I and Sac I and inserted into the corres-ponding site in the pETL DNA. The recombinant expression plasmid pET-Vp4 which contain RV Vp4 full coding gene was selected and confirmed by digestion and sequencing (Fig. 2).
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The 8%, 10%, 12% gradient SDS-PAGE was performed to detect the pET-Vp4 expression in the BL21(DE3) cells. The results of the SDS-PAGE showed that the pET-Vp4 was highly expressed in the BL21 (DE3) cells, and, as predicted, appeared as an ~87.6 kDa band on the gel (Fig. 3). The gel scanning analysis showed that the target protein accounts for about 17% of the total bacterial proteins.
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The results of the Western blot indicated that the rVp4 could be specifically recognized by the guinea pig antiserum against SA11 or against Wa strain (Fig. 4 and Fig. 5).
Figure 4. Western blot of recombinant rVp4 with antibodies against SA11 in guinea pig antiserum. 12% SDS-PAGE and Coomassie brilliant blue R250 stained proteins (A) and Western blot (B). 1, Protein marker; 2, rVp4; 3, BL21 (DE3) cells mock transformed with pETL; 4, Recombinant RV Vp6 (3).
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The results showed that some dimers of the rVp4 were formed during renaturation. The gel scanning analysis of figure indicated that the molecular weight of the dimer is 176.67kDa. Some trimers or polymer were also seen. When the samples were either treated by denaturation/heating at 100℃, all of the dimmers or polymers disappeared (Fig. 6).
Figure 6. SDS-PAGE analysis of the renatured rVp4 under different conditions. 1, Protein marker (kDa); 2, Denaturation rVp4, adding 5X loading buffer without heating; 3, Renatured rVp4, treated as in 2; 4, Denaturation rVp4, adding 5X loading buffer and heating 5 min; 5, Renatured rVp4, treated as in 4; 6, Denaturation rVp4, adding 5X loading buffer without mercaptoethanol and without heating; 7, Renatured rVp4, treated as in 6.
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It was estimated from above gel (Fig. 6) scanned by Imagemaster VDS system that dimers account for about 9.13% of the total renaturation rVp4. Some trimers and polymers could also be seen at the top line of the separation gel, accounting for about 30.17%. See Table 1.
Table 1. Results of gel scanning analysis of the renatured rVp4
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The neutralization test showed that the anti-rVp4 antibodies in guinea pig serum could protect MA104 cells from infection by RV strains Wa and SA11. The neutralization titer was 1:256 against Wa strain, and 1:160 against SA11. While the neutralization titers of antisera from Wa virus inoculated guinea pigs were 1:2 560 against Wa infection and 1:640 against SA11 infection. The neutralization titers of antisera from SA11 virus inoculated guinea pigs were 1:2560 against SA11 infection and 1:640 against Wa infection.