Studies on the VP7 and VP3 genes of human group C rotavirus

Samarbaf-Zadeh, Ali Reza (1996) Studies on the VP7 and VP3 genes of human group C rotavirus. University of Southampton, Doctoral Thesis.

Abstract

The sequence of cloned VP7 gene of human group C rotavirus was verified by automated DNA sequence analysis. The VP7 fragment was then cloned into pSP64 and the coupled in vitro transcription/translocation produced a 38kDa protein confirming the existence of an ORF in the segment. VP7 was expressed in E. coli using a vector (pMAL) which created a maltose binding protein fusion protein. The fusion protein is designed to be cleaved with the specific protease factor Xa. However, cleavage of the fusion protein with factor Xa was not successful. This material was not considered useful for developing a serological assay because immunisation of mice showed an antibody response only to MBP part of the fusion protein.

For the second part of the thesis, work was concentrated on characterisation of the rotavirus core protein genes VP1, VP2, and VP3. A group C rotavirus cDNA library was screened and clones representing the 5' termini of genome segment 1 and 2 were identified. Further analysis revealed a full-length recombinant of genome segment 4. The complete nucleotide sequence of genome segment 4 from human group C rotavirus (Bristol strain) was determined. Comparison of the nucleotide sequences of the genome termini with the consensus 5' and 3- terminal non-coding sequences of the human group C rotavirus genome revealed characteristic 5' and 3' sequence motifs. Human group C rotavirus genome segment 4 is 2, 166bp long and encodes a single open reading frame of 2,082 nucleotides (693 amino acids) starting at nucleotide 55 and terminating at nucleotide 2,136 giving a 3' untranslated region of 30 nucleotides. Analogy with the group A rotaviruses suggested that the genome segment 4 encodes the group C rotavirus guanyltransferase.

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