RESUMO
AIM: To investigate the hotspots, direction, and the time course of evolution of hepatitis A virus in the process of consecutive cell culture passage in human KMB17 diploid cells. METHODS: Wild type hepatitis A virus H2w was serially propagated in KMB17 cells until passage 30, and the full-length genomes of H2w and its six chosen progenies were determined by directly sequencing RT-PCR products amplified from viral genomic RNA. Alignment comparison of sequences from H2w with its six progenies and phylogenetic analysis of the whole VP1 region from H2w, progenies of H2w, and other cell culture adapted hepatitis A virus were then carried out to obtain data on the molecular evolution of hepatitis A virus in the process of consecutive passage in KMB17 cells. RESULTS: Most of the mutations occurred by passage 5 and several hotspots related to adaptation of the virus during cell growth were observed. After that stage, few additional mutations occurred through the remaining duration of passage in KMB17 cells except for mutation in the virulence determinants, which occurred in the vicinity of passage 15. The phylogenetic analysis of the whole VP1 region suggested that the progenies of H2w evolved closely to other cell culture adapted hepatitis A virus, i.e. MBB, L-A-1, other than its progenitor H2w. CONCLUSION: Hepatitis A virus served as a useful model for studying molecular evolution of viruses in a given environment. The information obtained in this study may provide assistance in cultivating the next generation of a seed virus for live hepatitis A vaccine production.
Assuntos
Proliferação de Células , Diploide , Evolução Molecular , Vírus da Hepatite A/genética , Pulmão/virologia , Mutação , RNA Viral , Vacinas contra Hepatite Viral/genética , Linhagem Celular , Análise Mutacional de DNA , Bases de Dados Genéticas , Vírus da Hepatite A/crescimento & desenvolvimento , Vírus da Hepatite A/imunologia , Vírus da Hepatite A/patogenicidade , Humanos , Pulmão/citologia , Pulmão/embriologia , Filogenia , Fatores de Tempo , Vacinas contra Hepatite Viral/biossíntese , Fatores de Virulência/genética , Replicação ViralRESUMO
AIM: To study the renaturation, purification and binding activity of scFv of anti-nasopharyngeal carcinoma monoclonal antibody(mAb) BAC(5) expressed as inclusion body in E.coli. METHODS: The E.coli BL21(DE3) transformed with the pET 22b-scFv was cultured and pulvereged by ultrasonic cell disintegrator. The collected inclusion bodies were denatured with 8 mol/L urea and renatured by dilution refolding, step dialysis and gel filtration chromatography. Binding activity of renatured BAC(5)-scFv was determined by immunohistochemical staining and Western blot. RESULTS: BAC(5)-scFv purified though Ni-NTA His Bind chromatographic clomn showed high purity. The highest proteins recovery rate was obtained through gel filtration chromatography. It was proved by Western blot and immunocytochemical staining that the renatured BAC(5)-scFv protein could specifically bind to CNE2 cells. CONCLUSION: BAC(5)-scFv expressed as inclusion body retained good activity after being dissolved, purified and renatured, which paves the way for preparing large amount of BAC(5)-scFv to be used for the study of radioimmunoimaging and therapy of nasopharyngeal carcinoma.
Assuntos
Anticorpos Monoclonais/química , Anticorpos Monoclonais/metabolismo , Escherichia coli/metabolismo , Fragmentos de Imunoglobulinas/química , Fragmentos de Imunoglobulinas/metabolismo , Corpos de Inclusão/metabolismo , Anticorpos Monoclonais/genética , Anticorpos Monoclonais/isolamento & purificação , Western Blotting , Linhagem Celular Tumoral , Escherichia coli/genética , Humanos , Fragmentos de Imunoglobulinas/genética , Fragmentos de Imunoglobulinas/isolamento & purificação , Imuno-Histoquímica , Neoplasias Nasofaríngeas/imunologia , Dobramento de ProteínaRESUMO
OBJECTIVE: To evaluate the RT-PCR-ELISA method applied for testing live attenuated hepatitis A vaccine titer. METHODS: A solid phase hybridization-enzyme colorimetric detection method was used for detecting specific nucleic acid. Primer labeled with biotin was used to amplify viral gene fragment, then the product was quickly hybridized with the specific probe covalently coupled on DNA-binding microplate wells. Finally, peroxidase-labeled streptavidin was used in colorimetric detection. The results were judged by reading A value. Eleven batches of live attenuated hepatitis A vaccine titer were tested by this method. The results were compared with that of routine cell culture method (CCID50). RESULTS: The sensitivity was similar to routine cell culture method (P>0.05). This method was convenient, fast and specific. CONCLUSION: CCID50 method may be replaced by the RT-PCR-ELISA method in evaluating the titer of live attenuated hepatitis A vaccine.
