Assuntos
Vacinas contra COVID-19/administração & dosagem , COVID-19/prevenção & controle , Imunidade Coletiva/efeitos dos fármacos , Pandemias/prevenção & controle , Vacinação/tendências , COVID-19/epidemiologia , COVID-19/imunologia , Vacinas contra COVID-19/imunologia , Humanos , Imunidade Coletiva/imunologiaRESUMO
Recombinant human interferon-α8 (rhIFN-α8) was obtained by synthesizing a codon-optimized gene in a two-step polymerase chain reaction (PCR) and expressing it in Escherichia coli. The gene encoding human IFN-α8 shows a high content of rare codons. These were replaced based on E. coli codon usage and balancing TA-GC ratio contents of the entire gene. The two-step PCR was performed using long (45-60 nucleotides) overlapped primers and two Taq polymerases (pfu clone and GC-rich system) and resulted in a DNA band of 504 base pairs (bp) corresponding to the calculated size of the IFN-α8 coding sequence; the pfu clone failed to amplify the gene in the correct size without unspecific bands. The full gene was cloned into the pBAD-TOPO expression vector. After cloning, the gene was reoriented by NcoI restriction digestion and religation. The ligated pBAD-TOPO-IFN-α8 (pBAD-IFNα8) plasmid carried the IFN-α8 gene under transcriptional control of the L-arabinose-inducible P(BAD) promoter. IFN-α8 expression was optimized with respect to L-arabinose concentration, temperature, and time of induction in shake flask cultures to maximize the yield of soluble IFN-α8. The produced IFN-α8 was characterized by polyacrylamide gel electrophoresis and immunoassays. After purification on DEAE-Sepharose, the yield was 100 mg/liter. The antiviral and anticancer activities of the IFN-α8 were evaluated in comparison with IFN-α2a, and the results are discussed.
Assuntos
Regulação da Expressão Gênica , Genes Sintéticos/genética , Interferon-alfa/genética , Interferon-alfa/metabolismo , Regiões Promotoras Genéticas/genética , Proteínas Recombinantes , Antineoplásicos/farmacologia , Antivirais/farmacologia , Linhagem Celular Tumoral , Clonagem Molecular , Regulação da Expressão Gênica/efeitos dos fármacos , Proteína Vmw65 do Vírus do Herpes Simples/efeitos dos fármacos , Humanos , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMO
Recombinant human consensus interferon-alpha (cIFN-α) was obtained by synthesizing a codon-optimized gene composed of the consensus nucleotides at each position in the human alpha interferon family and expressing it in Escherichia coli. The full cIFN-α gene was synthesized in two steps of assembly and amplification by polymerase chain reaction (PCR) using long (45-50 nucleotides) overlapped primers. The two-step PCR resulted in a DNA band of 504 base pairs (bp) corresponding to the calculated size of the cIFN-α gene. The synthetic gene was cloned into temperature-regulated Power3 expression vector. The ligated Power3-cIFN-α (Power3-cIFNα) plasmid carried the cIFN-α gene under transcriptional regulation of the heat-inducible λP(L) promoter. This expression system was optimized with respect to heat-shock temperature and time of induction in shake flask cultures. The produced cIFN-α protein was characterized by polyacrylamide gel electrophoresis and immunoassays. The majority of the expressed cIFN-α protein of about 19 kD in size accumulated in the form of inclusion bodies. After refolding and purification utilizing single-step ion-exchange chromatography on DEAE-Sepharose, the yield was 70 mg/L. cIFN-α anti-cancer activity was assayed and compared with the commercially available IFN-α 2a.
Assuntos
Escherichia coli/genética , Corpos de Inclusão/química , Interferon-alfa/isolamento & purificação , Engenharia de Proteínas/métodos , Sequência de Aminoácidos , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Códon , Sequência Consenso , Meios de Cultura , Primers do DNA , Escherichia coli/metabolismo , Vetores Genéticos , Temperatura Alta , Humanos , Interferon alfa-2 , Interferon-alfa/biossíntese , Interferon-alfa/genética , Interferon-alfa/farmacologia , Dados de Sequência Molecular , Plasmídeos , Reação em Cadeia da Polimerase , Regiões Promotoras Genéticas , Redobramento de Proteína , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/farmacologiaRESUMO
In an attempt to evaluate the levels of immunity against diphtheria in the Egyptian population, 709 healthy subjects aged 2 months-105 years and from six regions of Egypt were investigated. An ELISA was used to determine the serum concentration of anti-diphtheria IgG in each subject. Following widely-used criteria for defining levels of protection, 34.1% of the subjects were categorized as susceptible to diphtheria (with < 0.01 IU antitoxin/ml serum), 43.7% were considered to have basic protection (0.01- < 0.1 IU/ml) and only the other 22.1% appeared fully protected (> or = 0.1 IU/ml). The results revealed that most of the subjects aged 2 months-50 years had a basic or fully protective level of IgG against diphtheria, although males were slightly more likely to be unprotected than females (36.2% v. 31.6%) and certain age-groups appeared to be much more likely to be susceptible than others. If outbreaks of diphtheria like those seen in recent years in the former Soviet Union are to be avoided in Egypt, the most susceptible groups of the population need to be given booster immunizations.
