RESUMO
Human adenovirus types 3 and 7 (HAdV-3 and HAdV-7) occur epidemically and contribute greatly to respiratory diseases, but there is no currently available licensed recombinant HAdV-3/HAdV-7 bivalent vaccine. Identification of serotype-specific neutralizing antibody (NAb) epitopes for HAdV-3 and HAdV-7 will be beneficial for development of recombinant HAdV-3/HAdV-7 bivalent vaccines. In this study, four NAb epitopes within hexon hypervariable regions (HVRs) were predicted for HAdV-3 and HAdV-7, respectively, by using bioinformatics. Eight hexon chimeric adenovirus vectors with the alternation of only one predicted neutralizing epitope were constructed. Further in vitro and in vivo neutralization assays indicated that E2 (residing in HVR2) and E3 (residing in HVR5) are NAb epitopes for HAdV-7, and E3 plays a more important role in generating NAb responses. Cross-neutralization assays indicated that all four predicted epitopes, R1 to R4, are NAb epitopes for HAdV-3, and R1 (residing in HVR1) plays the most important role in generating NAb responses. Humoral immune responses elicited by the recombinant rAdH7R1 (containing the R1 epitope) were significantly and durably suppressed by HAdV-3-specific NAbs. Surprisingly, the rAdΔE3GFP-specific neutralizing epitope responses induced by rAdMHE3 (R3 replaced by E3) and rAdMHE4 (R4 replaced by E4) were weaker than those of rAdMHE1 (R1 replaced by E1) or rAdMHE2 (R2 relaced by E2) in vitro and in vivo. Furthermore, rAdMHE4 replicated more slowly in HEp-2 cells, and the final yield was about 10-fold lower than that of rAdΔE3GFP. The current findings contribute not only to the development of new adenovirus vaccine candidates, but also to the construction of new gene delivery vectors.
Assuntos
Adenovírus Humanos/imunologia , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Proteínas do Capsídeo/imunologia , Epitopos/imunologia , Vacinas Virais/imunologia , Infecções por Adenovirus Humanos/genética , Infecções por Adenovirus Humanos/imunologia , Infecções por Adenovirus Humanos/prevenção & controle , Adenovírus Humanos/genética , Animais , Proteínas do Capsídeo/genética , Epitopos/genética , Feminino , Técnicas de Transferência de Genes , Camundongos , Camundongos Endogâmicos BALB C , Especificidade da Espécie , Vacinas Virais/genéticaRESUMO
A 3-kb HindIII fragment bearing the cry6Aa2 gene and the adjacent and intergenic regions was cloned from Bacillus thuringiensis strain YBT-1518. Two open reading frames (ORFs), namely, orf1 (termed cry6Aa2) and orf2 that were separated by an inverted-repeat sequence were identified. orf1 encoded a 54-kDa protein that exhibited high toxicity to the plant-parasitic nematode Meloidogyne hapla. The orf2 expression product was not detected by SDS-PAGE, but its mRNA was detected by RT-PCR. The orf2 coexpressed with orf1 at a high level in the absence of the inverted-repeat sequence, whereas, the expression level of orf1 was decreased. When orf2 was mutated, the level of orf1 expression was enhanced obviously. In conclusion, the inverted-repeat sequence disturbs orf2 expression, and the orf2 downregulates orf1 expression. This is an example of novel negative regulation in B. thuringiensis and a potential method for enhancing the expression level of cry genes.
Assuntos
Bacillus thuringiensis/genética , Proteínas de Bactérias/genética , Endotoxinas/genética , Regulação Bacteriana da Expressão Gênica , Proteínas Hemolisinas/genética , Sequência de Aminoácidos , Animais , Toxinas de Bacillus thuringiensis , Sequência de Bases , Clonagem Molecular , DNA Bacteriano/genética , Eletroforese em Gel de Poliacrilamida , Genes Bacterianos , Dados de Sequência Molecular , Fases de Leitura Aberta , Óperon , Plasmídeos , Sequências Repetitivas de Ácido Nucleico , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Tylenchoidea/microbiologiaRESUMO
On the basis of the sequencing of the N-terminal amino acid of the crystal protein, a nucleotide acid fragment harboring a novel nematicidal gene cry6Aa2 was obtained from Bacillus thuringiensis strain YBT-1518. This fragment contains two ORFs: orf1 and orf2, while a "stem-loop" exists between orf1 and orf2. BLAST showed the similarity of orf1 nucleotide acid sequence with cry6Aa1 is 98%, and has been deposited in the Genbank database (Acc. No. AF499736). The cloning fragment was transferred to crystal negative mutation strain BMB171 by E. coli-Bt shuttle vector pHT304. A 54kDa protein with similarity to strain YBT-1518 was detected in recombinant strain, and rice shaped crystal was detected with transmission electron microscope. Bioassay indicated the LC50 of recombinant strain against second lavae juvenile of Meloidgyne hapla is 9.47 microg/mL, nearly equal to the original strain YBT-1518 (10.74 microg/mL).
Assuntos
Anti-Helmínticos/farmacologia , Bacillus thuringiensis/genética , Proteínas de Bactérias/genética , Endotoxinas/genética , Proteínas Hemolisinas/genética , Proteínas Recombinantes/biossíntese , Animais , Toxinas de Bacillus thuringiensis , Proteínas de Bactérias/farmacologia , Sequência de Bases , Clonagem Molecular , Endotoxinas/farmacologia , Escherichia coli/genética , Proteínas Hemolisinas/farmacologia , Dados de Sequência Molecular , Proteínas Recombinantes/farmacologia , Tylenchoidea/efeitos dos fármacosRESUMO
OBJECTIVE: To explore a new method for determining hepatitis B virus (HBV) genotypes B-D with real-time fluorescence-based PCR. METHODS: The PCR primers and probes were designed according to the analysis of 143 complete HBV genomes from GenBank and on the basis of a search for genotype-specific nucleotide sequences which were conserved in the 3 HBV genotypes. Real-time fluorescence-based PCR was performed for HBV genotyping of 128 samples collected from Lanzhou. Three samples of each genotype of B, C and D were randomly selected and their S gene was sequenced for confirmation of the results of PCR-based method. RESULTS: Real-time PCR identified genotype B in 26 (20.3%), genotype C in 92 (71.9%), and genotype D in 10 (7.8%) cases. The sequencing results of the S gene of 18 PCR-produced clones were in complete consistence with those of fluorescence-based PCR. CONCLUSION: The real-time PCR method is convenient, highly sensitive, rapid and accurate, especially suitable in clinical and large-scale epidemiological studies.