RESUMO
Strain TB-Chen is a group A rotavirus (RV) isolated from a Chinese infant suffering from gastroenteritis in hospital. The NSP5 and NSP6 of strain TB-Chen are encoded by the 10th gene segment (816bp in whole length) of the viral genome. The results obtained in this study showed that the NSP5 was encoded in the first open-reading-frame (ORF) of the gene segment (from 22bp to 624bp), and NSP6 was encoded in the second ORF (from 80bp to 355bp). The NSP5 protein consisted of 200 amino acid residues with a putative molecular mass of 21.9 kD, and a putative isoelectric point of 7.86. The NSP6 protein consisted of 92 amino acids with a putative molecular mass of 11 kD, and a putative isoelectric point of 9.65. This study further analyzed phylogenetic relationship of the NSP5/NSP6 ORF nucleotide sequence. The results showed that the NSP5s of group A rotavirus could be at least classified into 7 genotypes (H1-H7), the NSP6s could be at least classified into 8 genotypes (hl-h8); the genotypes of the NSP5 and NSP6 derived from strain TB-Chen was classified as H2 and h2. This was the first report on the genotype classification of the NSP6 of group A RVs, and it was proposed English letter "h" to represent genotype of the NSP6, e. g. strain 69M classified as H7h7, strains Wa and KU classified as H1h8.
Assuntos
Humanos , Evolução Molecular , Gastroenterite , Virologia , Genótipo , Fases de Leitura Aberta , Genética , Filogenia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Rotavirus , Classificação , Genética , Proteínas não Estruturais Virais , GenéticaRESUMO
Rotavirus capsid protein Vp4 plays an important role in the virus adhering and entering the cells. In this study, a Vp4 gene cloned from a rotavirus strain TB-Chen was highly expressed in E.coli BL21 (DE3). The results of the Western blot showed that the protein possesses specific immuno-reactivities and can be specifically recognized by guinea pig antibodies against rotavirus strain SA11 or Wa. Some Vp4 dimers were formed during renaturation. These data obtained from this study provide a strong basis for further study on the structure and function of the Vp4.
RESUMO
Little is known about the non-structural protein 6(NSP6)of rotavirus.This report describes expression of the NSP6 of a group A human rotavirus strain TB-Chen in bacteria,and its immunological properties and cellular distribution.The results showed that the recombinant NSP6(rNSP6)was expressed in high efficiency without any other proteins fused(possesses about 34.2% of total bacterial proteins).rNSP6 elicited mono-specific antibodies in immunized guinea pigs and the antibodies could react with the rNSP6 itself and the viral NSP6 proteins synthesized in SA11-or Wa-infected MA104 cells in Western blot and immunofluorescence assay.The NSP6 distributed evenly in the cytoplasm mainly around the nucleus of virus-infected cells,no viroplasm-like gathering observed;The top amount of NSP6 synthesized in SA11-infected cells or Wa-infected cells could be detected at 12h after infection.This is the first report about the high expression of entire NSP6(without any other proteins fused)in prokaryotic expression system and detection of NSP6 synthesis in virus infected cells by immunofluorescence assay.The results are important to understand the structure,biological properties and further application of the NSP6.
RESUMO
By means of genetic cloning and recombinant techniques, full genome cDNA sequences of rotavirus strain TB-Chen were isolated from an infantile hospitalized with acute gastroenteritis. Nucleotide sequences analyses showed that the full genome of strain TB-Chen contains 18613 nucleotides, encoding 5791 amino acids. Genotyping results showed that the strain TB-Chen belongs to genotype G2P[4]/NSp4[A]. This is the first report on a full genome of Group A rotavirus in China, and has important significance for deep understanding structure and functions of rotaviruses and developing rotavirus vaccines.
RESUMO
The structural protein VP6 of rotavirus form the middle layer of the triple-layered viral capsid, playing a key role in the organization of the virion. The gene of structural protein 6 of rotavirus strain TB-Chen isolated from a clinic sample was amplified using PCR from the reverse transcription product of RV genome RNA, using pET as expression vector, a recombinant plasmid pET-VP6 containing coding sequence of VP6 protein was constructed. The results showed that the VP6 was highly efficiently expressed in E. coli BL21(DE3) cells which were transformed with the recombinant plasmid pET-VP6.The expressed VP6 protein possessed 27.4% of total cells protein, with an approximately 45kDa of molecular weight, and could be recognized by guinea pig anti-SA11 antibody on Western blot. The results obtained provide important basis for further study on structure and function of the VP6 protein.
RESUMO
<p><b>OBJECTIVE</b>To evaluate in vivo immunological protective efficacy and safety of expressed recombinant rotavirus epitopes in mice.</p><p><b>METHODS</b>Using the Flock House virus capsid protein as a vector, three epitopes derived from rotavirus Vp4 amino acid 223-242 [rotavirus epitope A, (REA)], 243-262 [rotavirus epitope B, (REB)], and 234-251 [rotavirus epitope C, (REC)] were genetically engineered on the surface of the vector protein and expressed in pET-3 (E. coli BL21 [DE3]) system into multiple epitopes, REABC, which comprises REA, REB, and REC. Kunming strain mice were inoculated with the recombinant epitopes REABC, and then challenged perorally by cell culture-adapted rotavirus Wa (type G1P1A) and SA11 (type G3P2). Infection syndrome was observed, and virus antigen in stools of mice and serum neutralizing antibody activities were determined and analyzed.</p><p><b>RESULTS</b>The recombinant epitopes REABC significantly induced rotavirus specific neutralyzing antibodies against WA and SA11, reduced virus reproduction, elicitted immune memory in inoculated mice, and protected inoculated mice from challenge by WA or SA11 (P<0.001).</p><p><b>CONCLUSION</b>The recombinant epitopes have high immunological protective efficacy and mild side effects in mice. It may be used as an epitope-based vaccine candidate in human.</p>