RESUMO
OBJECTIVE: To construct the HPV16 L1 prokaryotic expression plasmid and to optimize its expression. METHODS: A pair of primers was designed according to plasmid sequences of pGEX-KG and the HPV16L1 genes published by GeneBank. The DNA fragment of 1500 bp was amplified by PCR from the HPV recombinant plasmid with HPV16L1 gene, then cloned into pGEX-KG and transformed into the host E.coli strain JM109. The pGEX-KG-HPV16L1 plasmid was taken and transformed into BL21(DE3) for expression. Induced by IPTG at 37 degree, the expression product of HPV16L1 gene was identified by SDS-PAGE and Western blot. RESULTS: HPV16L1 fusion protein was expressed successfully in the form of inclusion bodies. The molecular weight was 83 kD. Meanwhile, the optimum condition of HPV16L1 fusion protein expression was induced with 1.0 mmol*L(-1) IPTG for 4 h. The fusion protein reacted specifically with antibodies against HPV16L1. CONCLUSION: The prokaryotic expression vector of HPV16L1 gene has been constructed and expressed in E.coli successfully.
Assuntos
Proteínas do Capsídeo/biossíntese , Papillomavirus Humano 16/genética , Proteínas Oncogênicas Virais/biossíntese , Proteínas Recombinantes de Fusão/biossíntese , Vacinas Anticâncer/biossíntese , Proteínas do Capsídeo/genética , Clonagem Molecular , Escherichia coli/genética , Escherichia coli/metabolismo , Vetores Genéticos , Proteínas Oncogênicas Virais/genética , Proteínas Recombinantes de Fusão/genéticaRESUMO
Although pneumococcus is one of the most frequently encountered opportunistic pathogen in the world, the mechanisms responsible for its infectiveness have not yet been fully understood. In this paper, we have attempted to characterize the effects of pneumococcal transformation on the pathogenesis of the organism. We constructed three transformation-deficient pneumococcal strains, which were designated as Nos. 1d, 2d, and 22d. The construction of these altered strains was achieved via the insertion of the inactivated gene, comE, to strains 1, 2 and 22. We then conducted a comparison between the virulence of the transformation-deficient strains and that of the wild-type strains, via an evaluation of the ability of each strain to adhere to endothelial cells, and also assessed psaA mRNA expression, and the survival of hosts after bacterial challenge. Compared to what was observed with the wild-type strains, our results indicated that the ability of all of the transformation-deficient strains to adhere to the ECV304 cells had been significantly reduced (p < 0.05), the expression of psaA mRNA was reduced significantly (p < 0.05) in strains 2d and 22d, and the median survival time of mice infected with strains 1d and 2d was increased significantly after intraperitoneal bacterial challenge (p < 0.05). The results of our study also clearly indicated that transformation exerts significant effects on the virulence characteristics of S. pneumoniae, although the degree to which this effect is noted appears to depend primarily on the genetic background of the bacteria.
Assuntos
Infecções Pneumocócicas/microbiologia , Streptococcus pneumoniae/genética , Streptococcus pneumoniae/patogenicidade , Transformação Bacteriana/fisiologia , Adesinas Bacterianas/biossíntese , Adesinas Bacterianas/genética , Animais , Aderência Bacteriana , DNA Bacteriano/química , DNA Bacteriano/genética , Células Endoteliais/microbiologia , Regulação Bacteriana da Expressão Gênica , Humanos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Mutagênese Insercional , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Streptococcus pneumoniae/metabolismo , Virulência/genéticaRESUMO
Spatial and temporal characteristics of airborne dust over Asia during springs of 2000, 2001 and 2002 were simulated with a mineral dust entrainment and deposition model (DEAD) embedded in a global model of atmospheric transport and chemistry (MATCH) using the real-time meteorological data as forcing fields. The results show a good agreement of the pattern of model-simulated atmospheric dust concentration with the distribution of surface-observed spring dust storm frequency and a significant correlation between the simulated dust aerosol optical depth (DAOD) and satellite-observed aerosol index (AI). These results validate applicability of the integrated model in simulating dust entrainment, transportation and deposition and describing spatial and temporal characteristics of dust loading over the Asian continent. In addition, an attempt was made to explore possible paths of dust transportation by use of correlation analyses between the simulated dust emission flux (DEF) and DAOD.