RESUMO
Recently identified interferon-epsilon (IFN-epsilon) belongs to type I interferons. IFN-epsilon is highly and constitutively expressed in the brain, but its biochemical and biological characteristics are poorly understood. In this study, full-length IFN-epsilon cDNA was cloned from human peripheral blood lymphocyte by RT-PCR, and was expressed in Escherichia coli (E. coli). Reverse phase high pressure liquid chromatography was used to purify recombinant human IFN-epsilon (rhIFN-epsilon) and to facilitate refolding of the protein. About 0.8mg of highly purified rhIFN-epsilon protein was obtained from 100ml of E. coli culture. Functional study of rhIFN-epsilon demonstrated that the antiviral activity of rhIFN-epsilon was 6+/-0.5x10(5)IU/mg, which was lower than that of rhIFN-alpha-2b in the WISH-VSV (WISH cells infected with vesicular stomatitis virus) assay system. As for the activity to promote NK cytotoxicity and antiproliferation activities, rhIFN-epsilon was about 60 times less potent than rhIFN-alpha-2b. However, oligonucleotide microarray analyses revealed dramatic differences in gene expression profiles of cultured human cells treated with IFN-epsilon and IFN-alpha-2b. Particularly, differential regulation of genes related to central nervous system by rhIFN-epsilon suggests a role for IFN-epsilon in maintenance of the structure and function of brain.
Assuntos
Interferon Tipo I/genética , Interferon Tipo I/isolamento & purificação , Antivirais/isolamento & purificação , Antivirais/farmacologia , Sequência de Bases , Encéfalo/metabolismo , Proliferação de Células/efeitos dos fármacos , Citotoxicidade Imunológica/efeitos dos fármacos , DNA Complementar/genética , DNA Complementar/isolamento & purificação , Escherichia coli/genética , Expressão Gênica/efeitos dos fármacos , Células HeLa , Humanos , Técnicas In Vitro , Interferon Tipo I/farmacologia , Interferon alfa-2 , Interferon-alfa/farmacocinética , Células Matadoras Naturais/efeitos dos fármacos , Células Matadoras Naturais/imunologia , Análise de Sequência com Séries de Oligonucleotídeos , Proteínas RecombinantesRESUMO
OBJECTIVE: To prepare human interferon-k (hIFN-kappa) and study its biological activities. METHODS: Whole length of hIFN-kappa's cDNA was cloned, and its sequence was chemically synthesized according to the optimized codons of E.coli, then was expressed in E.coli DH5alpha. After purified, the rhIFN-kappa protein was tested for its various kinds of biological activities. RESULTS: The purity of rhIFN-kappa was above 90%. In WHIS-VSV system, the antiviral activity of rhIFN-kappa was 2.0 x 10(6) IU/mg. Compared with rhIFN-alpha-2b, the biological activities of rhIFN-kappa were all feeble, including antiviral activity, promoting NK cell activity and anti-proliferation activity. CONCLUSION: Antiviral activities of rhIFN-kappa on cell lines of different species are different, different viruses show different sensitivity to rhIFN-kappa.
Assuntos
Antivirais/farmacologia , Proliferação de Células/efeitos dos fármacos , Interferon Tipo I/farmacologia , Proteínas Recombinantes/farmacologia , Animais , Linhagem Celular , Chlorocebus aethiops , Clonagem Molecular , DNA Complementar/genética , Eletroforese em Gel de Poliacrilamida , Escherichia coli/genética , Expressão Gênica , Humanos , Interferon Tipo I/genética , Interferon Tipo I/isolamento & purificação , Células K562 , Células Matadoras Naturais/citologia , Células Matadoras Naturais/efeitos dos fármacos , Células Matadoras Naturais/imunologia , Testes de Sensibilidade Microbiana , Plasmídeos/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Células VeroRESUMO
OBJECTIVE: To express recombinant human interferon lambda2 in E.coli and to study its antiviral activities. METHODS: According to preferred codons used in E.coli, the highly-expressed human interferon lambda2 gene was designed, synthesized and cloned into expression vector pBV220 and transfected into E.coli DH5alpha. The expressed product was purified by using CM FF and size exclusion chromatography. Its antiviral activities were tested on different cells. RESULTS: The expressed product was calculated about 15% of the total E.coli protein. The purified protein reached about 90% purity. Its specific antiviral activity was about 1.5 x 10(6) IU/mg on WISH/VSV test system. It was shown that the antiviral activity of the product on primates-origin cells seemed to be much higher than that on other non-primates-origin cells, indicating that interferon lambda2 possessed more stringent species specificity as compared with interferon-alpha2b. New interferon lambda2 showed similar anti-HBV activity as interferon-alpha2b. CONCLUSION: Recombinant human interferon lambda2 could be expressed on E.coli. The purified product showed more stringent species specificity and similar anti-HBV activity as compared with interferon-alpha2b.
Assuntos
Antivirais/farmacologia , Proliferação de Células/efeitos dos fármacos , Interleucinas/farmacologia , Proteínas Recombinantes/farmacologia , Animais , Linhagem Celular , Linhagem Celular Tumoral , Chlorocebus aethiops , Relação Dose-Resposta a Droga , Eletroforese em Gel de Poliacrilamida , Ensaio de Imunoadsorção Enzimática , Escherichia coli/genética , Escherichia coli/metabolismo , Vírus da Hepatite B/efeitos dos fármacos , Humanos , Interleucinas/biossíntese , Interleucinas/genética , Interleucinas/isolamento & purificação , Testes de Sensibilidade Microbiana , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/isolamento & purificação , Células VeroRESUMO
To clone KGF-2 gene, get hKGF-2 protein and detemine its activity. The cNDA of human KGF-2 was isolated from fetal lung by RT-PCR and cloned into pBV220 plasmid. The recombinant pBV220-hKGF-2 plasmid was transformed into E. coli (BL21), induced at 42 degrees C for the expression of hKGF-2. Recombinant human KGF-2 was purified from the ultrasonic-treated BL21 by heparin-Sepharose CL-6B treated column chromatography and cation exchange column chromatography. MTT method was used for the determination of its biological activity. SDS-PAGE showed that rhKGF-2 was expressed in E. coli BL21 as soluble protein of approximately 20kD. The rhKGF-2 protein can stimulate the proliferation of NIH3T3 cells significantly from 1 ng/mL to 10 ng/mL. HKGF-2 cDNA wasclned and highly expressed in E. coli BL21 and the purified rhKGF-2 showed the mitogenic activity on NIH3T3 cells.
Assuntos
Fator 10 de Crescimento de Fibroblastos/biossíntese , Fator 10 de Crescimento de Fibroblastos/isolamento & purificação , Clonagem Molecular , Escherichia coli/genética , Escherichia coli/metabolismo , Feto , Fator 10 de Crescimento de Fibroblastos/genética , Vetores Genéticos/genética , Humanos , Pulmão/química , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificaçãoRESUMO
BACKGROUND: To study the anti-SARS virus activities of different recombinant human interferons on the cell culture system. METHODS: Anti-SARS virus activities of interferons were determined by using CPE inhibition test in human skeletal muscle sarcoma (Rda) cell culture. RESULTS: The average minimum amount of interferon alpha 2b, alpha 1b, beta 1b or omega 1b to inhibit 50% CPE in Rda cell culture was (160.5+/-129.5) IU/ml, (149.0+/-71.7) IU/ml, (69.5+/-61.5) IU/ml, (87.3+/-47.1) IU/ml, respectively or (0.6+/-0.5) ng/ml, (10.6+/-5.1) ng/ml, (3.5+/-3.1) ng/ml, (0.9+/-0.5) ng/ml, respectively. CONCLUSION: All the tested recombinant interferons showed anti-SARS virus activities on the Rda cell culture with different sensitivities.
