The human group C rotavirus VP6 gene

Cooke, Susan Jane (1994) The human group C rotavirus VP6 gene. University of Southampton, Doctoral Thesis.

Abstract

A rapid and simple method was developed, and applied to the purification of human group C/Bristol rotavirus dsRNA genome from faecal samples. Subsequently cDNA corresponding to the VP6 gene of this isolate was cloned and sequenced. A Northern blot hybridisation method was also developed which assigned this coding sequence to segment 5 of the human group C/Bristol and porcine group C/Cowden rotavirus dsRNA genomes. The complete human VP6 sequence contained an open reading frame of 1185 nucleotides (395 amino acids; deduced M_r 44.7kDa). The protein sequence demonstrated low identity with various group A VP6 sequences and a relatively high identity with the porcine group C/Cowden and bovine group C/Shintoku VP6 sequences. Amino acid sequence alignments between the human group C/Bristol and the published porcine group C/Cowden VP6 proteins revealed a region of extreme sequence divergence. Data obtained from the direct sequencing of Cowden VP6 cDNA was unable to confirm the reported nucleotide sequence. Sequence comparisons revealed a frame shift and three nucleotide changes within the Cowden VP6 gene.

Direct sequencing of PCR-amplified VP6 cDNA of isolates from Bel'em, Brazil and Preston, UK confirmed the presence of a group C rotavirus. The deduced amino acid sequence of the Brazilian strain was identical to that of the two UK human isolates.

The human group C/Bristol rotavirus VP6 gene was cloned into an E.coli expression vector, pMAL^TM-c2. One recombinant E.coli DH5α was chosen for the ability to express a VP6-fusion protein of expected molecular weight (approx. 95kDa) induced by the addition of IPTG. However, the levels of expression were low and intracellular degradation of the protein was observed.

This record has no associated files available for download.

Contributors

Download statistics