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Introducción: Infección persistente con el virus de papiloma humano de alto riesgo (VPH-AR) causa cáncer de cuello uterino (CCU). Existen ensayos moleculares para la detección y la genotipificación del gen L1 de VPH, sin embargo, L1 puede perderse durante la integración viral. La expresión e integración del oncogén E7 es fundamental para el desarrollo de CCU. Objetivo: Estandarizar una PCR multiplex (mPCR) del oncogén E7 (E7-mPCR) para genotipificación de los VPH-AR de mayor frecuencia en CCU (VPH-16, -18, -31, -33, -45 y -52). Métodos: Se obtuvieron cepillados cervicales de voluntarias y se analizaron amplificando por PCR el gen L1 con subsecuente hibridación reversa. Posteriormente, se escogieron 59 muestras positivas para VPH-AR y se analizaron por E7-mPCR. Resultados: Se evidenció una elevada concordancia entre los resultados del ensayo E7- mPCR y los de la PCR de L1 (concordancia observada de 95,1%, Kappa de Cohen = 0,88), encontrándose mayor número de infecciones por VPH- AR en el 15,8% con E7-mPCR. Conclusión: E7-mPCR es una herramienta diagnóstica con alta concordancia y económica que puede adaptarse a una plataforma de mayor complejidad para procesar y detectar mayor cantidad de muestras y genotipos de VPH-AR.
Introduction: The persistent infection of the high-risk Human Papiloma Virus (VPH-AR in Spanish) causes uterine cervix cancer (CCU in Spanish). There are molecular essays for detection and genotyping of gen L1 of VPH. However, L1 may get lost during the viral integration. The expression and integration of oncogene E7 is fundamental for the development of CCU. Objective: To standardize a multiplex PCR (mPCR) of oncogene E7 (E7-mPCR) for genotyping the VPH- AR of highest frequency in CCU (VPH-16, -18, -31, -33, -45 y -52). Method: We obtained cervix brushing simples from volunteers and we analyzed them by amplifying the L1 gene through PCR with a subsequent reverse hybridization. After that, we chose 59 positive VPH- AR samples and we analyzed them for E7-mPCR. Results: We found out a high concordance between the results of the essay E7-mPCR and those of L1 PC (Observed concordance was of 95.1%, Cohen's Kappa = 0.88), and we revealed a higher number of infections for VPH-AR in a 15.8% with E7-mPCR. Conclusion: E7-mPCR is an economic diagnostic tool with high concordance which can be adapted to a platform with more complexity to process and detect a higher number of samples and VPH-AR genotypes.
Introdução: a infecção persistente com o virus de papiloma humano de alto risco (HPV-AR) causa cáncer de colo do útero (CCU). Existem ensaios moleculares para detecção e para a genotipificação do gene L1 de HPV; contudo, L1 pode ser perdido durante a integrado viral. A expressão e integração do oncogênese E7 é fundamental para o desenvolvimento do CCU. Objetivo: padronizar uma PCR multiplex (mPCR) do oncogênese E7 (E7-mPCR) para genotipificação dos HPV-AR de maior frequência no CCU (HPV-16, -18, -31, -33, -45 e -52). Métodos: foram realizadas raspagens com escova cervical rodada em voluntárias e foram analisadas a partir da amplificação do gene L1 por PCR com subsequente hibridação inversa. Em seguida, foram escolhidas 59 amostras positivas para HPV-AR, as quais foram analisadas por E7-mPCR. Resultados: foi evidenciada elevada concordância entre os resultados do ensaio E7-mPCR e os da PCR de L1 (concordância observada de 95,1%, Kappa de Cohen = 0,88), encontrando-se maior número de infecções por HPV-AR em 15,8% com E7-mPCR. Conclusão: E7-mPCR é uma ferramenta diagnóstica com alta concordância e económica que pode ser adaptada a uma plataforma de maior complexidade para processar e detectar maior quantidade de amostras e genótipos de HPV-AR.
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Objective To understand the gene E sequences of prevalent strains of Dengue fever type Ⅰ virus in Zhaoqing City during 2014.Methods The medical record data and acute stage serum of the patients with Dengue fever in Zhaoqing City during 2014 were collected.Dengue virus was cultured and isolated by using the C6/36 cell culture.Gene E of positive strains was amplified with RT-PCR.The phylogenetic tree was drawn and the bioinformatics analysis was conducted.Results Of 36 samples,20 samples were positive in viral isolation and culture.The gene E sequences of 20 strains of type Ⅰ Dengue virus prevalence in Zhaoqing dur-ing 2014 were obtained;the homology of these sequences was close to that of the 2 prevalent strains found in Zhongshan City,but was distant from that found in Guangzhou City.Conclusion The epidemic situation of Dengue fever in Zhaoqing City is closely re-lated to the prevalence situation of Zhongshan and is characterized by imported prevalence.
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Bacterial ghosts are bacterial cell envelopes devoid of cytoplasmic contents while maintaining their cellular morphology, which can be used as a new vaccine and delivery vector. In this study, a clinical isolate of avian pathogenic Escherichia coli (APEC) strain DE17 was used to prepare bacterial ghost through three different ways. The results showed that the cleavage efficiency of DE17 bacterial ghost was 99.9% with the lysis plasmid containing the PhiX174 lysis gene E. Scanning electron microscopy showed that transmembrane tunnels were formed in the middle or both ends of the cell envelope of DE17. Furthermore, the DE17 bacterial ghost was prepared with one of cell penetrating peptides (CPPs) named MAP (KLALKLALKALKAALKLA), which will completely inactivate DE17 (OD₆₀₀=0.1) by 10 μmol/L MAP. The cell envelope showed a gully-like structure and obvious transmembrane tunnels were not found through the SEM. However, the DE17 could not be lysed by importing the lysis plasmid (pBV220-MAP), which was used to express MAP. The present study will benefit for research on bacterial ghost preparation methods and provide a reference for biosafety of bacterial ghost vaccines.
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Staphylococcal nuclease A (SNA) may be used to produce bacterial ghosts for further inactivation of host bacteria and elimination of residual genetic materials. It is still controversial if SNA without signal peptide can be secreted to extracellular matrix and if fusion with other peptide is required for its function in the cytoplasm of host bacteria. To clarify this dispute, a series of temperature-inducible plasmids carrying SNA alone or SNA fused with partial sequences of λ phage cro gene (cSNA) or Mycobacterium tuberculosis urease gene (uSNA) were constructed and evaluated in Escherichia coli. Results show that the percentages of inactivated E. coli by SNA, cSNA and uSNA after 4 h of induction were 99.9%, 99.8% and 74.2%, respectively. Moreover, SNA and cSNA in the cytoplasm of host bacteria were initially detectable after 30 min of induction, whereas uSNA was after 1 h. In comparison, SNA and cSNA in culture supernatant were initially detectable 1 h later, whereas uSNA was 2 h later. The nuclease activity in the cytoplasm or supernatant was ranked as follows: SNA > cSNA > uSNA, and the activity in the supernatant was significantly lower than that in the cytoplasm. Furthermore, host genomic DNA was degraded by SNA or cSNA after 2 h of induction but not by uSNA even throughout the whole experiment. In conclusion, this study indicates that SNA, cSNA and uSNA expressed in host bacteria all have nuclease activity, the enzymes can be released to culture media, and fusion with exogenous peptide negatively reduces the nuclease activity of SNA.
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Bacteriólisis , Bacteriófago lambda , ADN , Química , Escherichia coli , Vectores Genéticos , Nucleasa Microcócica , Química , Plásmidos , Señales de Clasificación de ProteínaRESUMEN
Objective: To investigate the methylation status of the promoter of E-cadherin gene and its protein expression in gastric cardiac adenocarcinoma(GCA). Methods: The methylation status of the 5′CpG island of E-cadherin was determined by a nested methylation specific PCR approach and E-cadherin protein expression was measured by immunohistochemical method in GCA tissues and adjacent tissues. Results: E-cadherin was methylated in 63 of 92 (68.5%) GCA specimens, which was significantly higher than that in paracancerous normal tissues (P < 0.001). Methylation occurred more frequently in patients at stage III and IV than those at stage I and II (P = 0.01). The frequency of methylation was significantly higher in poor differentiated group than moderate and well differentiated groups (P < 0.01). Immunohistochemical staining showed that E-cadherin expression was demonstrated heterogeneous, in 51 of 92 (44.6%) GCA specimens which was significantly different from matched paracancerous normal tissues. The positive rate of E-cadherin protein was significantly lower in patients at stage III and IV than those at stage I and II (P < 0.01). The positivity of E-cadherin was markedly lower in poor differentiated group compared with moderate and well differentiated groups, and the difference was significant (P < 0.05). Conclusion: Gene silencing caused by methylation of the 5′CpG island of E-cadherin gene may be one of the mechanisms for the tumorigenesis and development of GCA. The methylation status of E-cadherin could be used as a marker indicating the biological behaviors of GCA.
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Objective: We tried to construct and screen the most effective expression vector of siRNA targeting HPV-E6 oncogene and investigate its long-term influence on HPV-E6 gene expression in cervical cancer cells. Our aim is to discuss the molecular mechanism of E6 gene in the development of cervical cancer and explore the new approach to prevention of HPV infection and saving patients with cervical cancer. Methods: HPV16-E6 siRNA was transfected into CaSki cells mediated by FuGene 6. The expression of HPV16-E6 was detected by western blot; the influence of HPV16-E6 siRNA on cell proliferation was detected by MTT assay; the effect of HPV16-E6 siRNA on cell cycle and apoptosis was determined by flow cytometry. Results: HPV16-E6 siRNA significantly inhibited HPV16-E6 expression, suppressed the proliferation and cell cycle progression, and induced apoptosis of CaSki cervical cancer cells. Conclusion: HPV16-E6 siRNA effectively inhibited cell proliferation, arrested cells at G0/G1 phase, and induced apoptosis in CaSki cells.
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Chitinases genes from Metarhizium anisopliae which is an important entomopathogenic fungus were considered one of the key factors to invade their hosts. One Metarhizium anisopliae HN1 strain was isolated and screened. A chitinase gene was amplified by RT-PCR from Metarhizium anisopliae HN1, The whole length of this gene was 1275bp,and the nucleotide sequence of the gene was 96% similarity to that of the M. anisopliae E6 accessed in GenBank ( AF02749). The gene has been registered in GenBank and its accession number is DQ011865. The gene was subcloned into prokaryon expression vector pET-22b( + ), transformed this recombinant expression plasmid into E. coli strain BL 21 and effective expressed. The SDS-PAGE analysis indicated that the recombinant protein was 42kDa which is same to the reported article. The expression level of recombinant protein was about 63. 3% of whole expressed proteins , And when recombinant E. coli were crushed by freeze and supersonic wave , the activity assay indicates that the chitinase expressed in bacteria possesses biological activity.
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The HCV core gene and E1 gene derived from the plasmid pBRTM/HCV1 3011 by using polymerase chain reaction(PCR) was inserted into the backward position of cytomegalovirus(CMV) immediate early promotor element of framework plasmid (pAd.CMV Link 1) of adenovirus expression vector, then the recombinant plasmid pAd.HCV CE1 was obtained. The inserted DNA of pAd.HCV CE1 was confirmed to be HCV core and E1 gene by endonuclease, PCR and sequencing. HCV core gene was expressed transiently with lipofectamine 2000 coated in human hepatoblastoma 7721 cells which was confirmed by immunofluorescence. The results indicate that the recombinant framework plasmid of adenovirus expression vector pAd.HCV CE1 can express HCV core and E1 gene. This should be useful to pack adenovirus expression vector which can express HCV core and E1 gene.
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Objective:To investigate the clinical significance of E-Cadherin (E-CD) expression in breast carcinoma.Methods:The expression of E-CD gene in human breast carcinomas was studied by immunohistochemistry(SABC) in 92 patients and the relationship between the expression and the clinical pathologic parameters was observed.Results:The positive rate of E-CD expression was 48.9%(45/92).The group with negative E-CD expression has higher incidence of distant metastasis (34%,16/47)than the positive one (15.6%,7/45)(P
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0.05). On the day4, day5, the expression of E-selectin protein were greatly higher than that of normal endometrium (57.52 + 7.03, 62.91 + 7.31; P