RESUMO
Thrombin-like enzymes (TLEs) are studied widely because of their therapeutic potential in myocardial infarction and thrombotic diseases. We synthesized the DNA fragment encoding thrombin-like enzyme calobin from Agkistrodon caliginosus (Korean Viper) venom by fusion PCR and expressed it in Pichia pastoris. After induction by 0.5% methanol for 48 h, the expression level of recombinant calobin reached 3.5 g/L in medium. The recombinant calobin was purified by Q-Sepharose Fast Flow ion-exchange chromatography and Sephacryl-S-100 gel filtration chromatography. Purified sample had a molecular weight of 32 kD shown in SDS-PAGE. It hydrolyzed fibrinogen and formed a light white hydrolysis circle in fibrinogen plate. SDS-PAGE analysis showed that recombinant calobin cleaved Aalpha-chain of fibrinogen specifically, and produced an appropriately 40 kD new band. However, we failed to find its fibrin-clot formation activity.
Assuntos
Animais , Agkistrodon , Pichia , Genética , Metabolismo , Proteínas Recombinantes , Genética , Serina Endopeptidases , Genética , Trombina , Genética , Venenos de VíborasRESUMO
The expression of a soluble protein on cell surface is often desirable for study of a functional protein, wide application of a protein or investigation of protein-protein interaction. The expression of a soluble protein on the surface of a cell is often achieved by genetically linking a protein to the extra-cellular fragment of a transmembrane partner. In this study, the myc epitope was linked with N terminal of transmembrane proteins either A2TM or deltaLNGFR amplified by overlapping PCR. The plasmids expressing fusion protein were transfected into 293FT cells and the expression of target proteins was evaluated by fluorescent microscope, flow cytometry and Western blotting. The results of flow cytometry revealed that both A2TM and deltaLNGFR were expressed on the cell surface, but A2TM could only be detected with high copy number. Western blotting showed that the expression level of deltaLNGFR was very high and protein was heavily glycosylated, by contrast the expression of A2TM was hardly detected. The results indicate that glycosylated deltaLNGFR is a good candidate partner for the expression of a soluble protein on the cell surface.
Assuntos
Humanos , Proteínas Reguladoras de Apoptose , Genética , Membrana Celular , Metabolismo , Genes MHC da Classe II , Genética , Antígeno HLA-A2 , Genética , Proteínas de Fusão de Membrana , Genética , Metabolismo , Proteínas de Membrana , Genética , Metabolismo , Proteínas Proto-Oncogênicas c-myc , MetabolismoRESUMO
Bacterial ghost is intact bacterial envelope which is lysised by the lysis geneE of PhiX174. It can be used as vaccine directly. Foreign antigen can be targeted into outer membrane, inner membrane or the periplasmic space of bacterial, as a result, a recombinant bacterial ghost is constructed. Bacterial ghost, as a novel drug delivery system, is becoming more and more concerned, which can deliver DNA and protein vaccine or other drugs in order to have a better immune responses and therapeutic effects.
RESUMO
Objective:To detection antibody against heat-stable enterotoxin by fusion protein.Methods:Mutant heat-stable enterotoxin precursor gene was ligated in vector pGEX-4T-2 to inductively express as a fusion protein GST/proST_m with glutathione S-transferase(GST).To investigate the antigenic action,serum and fecal antibodies against heat-stable enterotoxin was detected with this fusion protein.Results:The fusion protein was a about 32 kD protein.All the samples contain the antibody against ST.Conclusion:Such strategy was a promising method to detect antibody against heat-stable enterotoxin.
RESUMO
The paper reported two methods. a genetic probe and the solid phase radio-immunological assay, for the detection of the thermolabile toxin produced by Escherichia coli. The former is the application of a 32P-IabelIed probe of B subunit DNA prepared from Hind Ⅲ restriction fragment of LT-encoding DNA. and the latter is by using CNBr-activated paper as carrier and 125I-SPA to substitute the secondary antibody. Applying the methods to 160 strains of E. coli isolated from patients suffering from infantile diarrhea, the results showed that 42 out of 160 strains belong to the strain which produces the thormolabile toxin. These two methods not only have the advantages of being sensitive and specific, but also are beneficial favourable to the epidemiological survey and clinical diagnosis, because the testing bacteria are decomposed on the nitrocellulose membrane, so that hundreds of samples can be examined in one test.