Résumé
<p><b>OBJECTIVE</b>To express recombinant human interferon lambda2 in E.coli and to study its antiviral activities.</p><p><b>METHODS</b>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.</p><p><b>RESULTS</b>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.</p><p><b>CONCLUSION</b>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.</p>
Sujets)
Animaux , Humains , Antiviraux , Pharmacologie , Lignée cellulaire , Lignée cellulaire tumorale , Prolifération cellulaire , Chlorocebus aethiops , Relation dose-effet des médicaments , Électrophorèse sur gel de polyacrylamide , Test ELISA , Escherichia coli , Génétique , Métabolisme , Virus de l'hépatite B , Interleukines , Génétique , Pharmacologie , Tests de sensibilité microbienne , Protéines recombinantes , Pharmacologie , Cellules VeroRésumé
<p><b>OBJECTIVE</b>To prepare human interferon-k (hIFN-kappa) and study its biological activities.</p><p><b>METHODS</b>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.</p><p><b>RESULTS</b>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.</p><p><b>CONCLUSION</b>Antiviral activities of rhIFN-kappa on cell lines of different species are different, different viruses show different sensitivity to rhIFN-kappa.</p>
Sujets)
Animaux , Humains , Antiviraux , Pharmacologie , Lignée cellulaire , Prolifération cellulaire , Chlorocebus aethiops , Clonage moléculaire , ADN complémentaire , Génétique , Électrophorèse sur gel de polyacrylamide , Escherichia coli , Génétique , Expression des gènes , Interféron de type I , Génétique , Pharmacologie , Cellules K562 , Cellules tueuses naturelles , Biologie cellulaire , Allergie et immunologie , Tests de sensibilité microbienne , Plasmides , Génétique , Protéines recombinantes , Métabolisme , Pharmacologie , Cellules VeroRésumé
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.
Sujets)
Humains , Clonage moléculaire , Escherichia coli , Génétique , Métabolisme , Foetus , Facteur de croissance fibroblastique de type 10 , Génétique , Vecteurs génétiques , Génétique , Poumon , Chimie , Protéines recombinantes , GénétiqueRésumé
<p><b>BACKGROUND</b>To study the anti-SARS virus activities of different recombinant human interferons on the cell culture system.</p><p><b>METHODS</b>Anti-SARS virus activities of interferons were determined by using CPE inhibition test in human skeletal muscle sarcoma (Rda) cell culture.</p><p><b>RESULTS</b>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.</p><p><b>CONCLUSION</b>All the tested recombinant interferons showed anti-SARS virus activities on the Rda cell culture with different sensitivities.</p>