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Zhang K.,Shaanxi Microbiology Institute
Chinese Journal of Biologicals | Year: 2014

Objective: To optimize the preparation procedure of PEGylated recombined mutant human Cu,Zn-SOD (rmhCu, Zn-SOD) so as to increase its half-life in viva Methods The rmhCu,Zn-SOD was modified chemically with monomethoxypoly (ethylene glycol) succinimidyl propionate (mPEG-SPA) with a relative molecular mass of 5 000, and the molar ratio of mPEG-SPA to rmhCu, Zn-SOD as well as reaction buffer, reaction temperature, reaction mode and reaction time were optimized. The PEGylated rmhCu,Zn-SOD was isolated and purified by ammonium sulfate precipitation and gel filtration chromatography, and determined for half-life in vivo in mice. Results: The optimal molar ratio of mPEG-SPA to mihCu, Zn-SOD was 1.5:1, while the optimal buffer was phosphate buffer at a pH value of 8.5. The reaction solutions were mixed up, allow to stand at 4°C for 1 h. The purity, residual enzyme activity and protein modification rate of PEGylated rmhCu,Zn-SOD were about 90%, (84.3 ± 2.3)% and (65.4 ± 1.9)% respectively, while the half-life increased by 7 folds compared with that unmodified. Conclusion: The preparation procedure of PEGylated rmhCu,Zn-SOD was optimized, and the half-life in vivo of PEGylated rmhCu,Zn-SOD increased significantly. Source


Wang L.-H.,Xian University of Arts and Science | Ke Y.,Shaanxi Microbiology Institute | Qiang Y.,Shaanxi Normal University | Ma Y.,Shaanxi Microbiology Institute
Chinese Journal of Applied Ecology | Year: 2014

Inhibition spectrum and antagonistic mechanism of an endophytic fungus Trichoderma harzianum LH-7, isolated from wild medicinal plant Aloe barbadensis, were investigated by in vitro culture methods against 9 kinds of plant pathogens. The results showed that nutrient competition and hyper-parasitism were the two primarily antagonist approaches that strain LH-7 adopted to inhibit the tested plant pathogens with a significant inhibition rate of 62.4%-88.4%. Moreover, the active compound from metabolites of LH-7 could cause pathogen mycelial deformities, cell wall rupture and conidial malformation, leading to the effective inhibition on pathogens growth and reproduction. Source


Wan Y.,Shaanxi Microbiology Institute | Zhang K.,Shaanxi Microbiology Institute | Zhang Y.-J.,Shaanxi Microbiology Institute | He L.-Q.,Shaanxi Microbiology Institute | And 2 more authors.
Chinese Journal of Biologicals | Year: 2013

Objective To express recombinant human interferon α2a protein (rhIFNα2a) in CBD (chitin-binding-domain)-intein / pTWIN 1 expression system and determine its biological activity. Methods The coding sequence of rhIFN-α2a was amplified by PCR using plasmid pBV220-IFNα2a as a template and inserted into expression vector pTWINl fused in frame with an upstream Ssp DnaB intein gene. The constructed recombinant plasmid pTWINl-rhIFNα2a was transformed into E. coli BL21 (DE3), and protein expression was induced with IPTG. The expressed product was identified by SDS-PAGE and Western blot, and the biological activity of lysis supernatant was determined. Results Both restriction analysis and sequencing proved that the fusion gene was cloned into expression vector pTWINl. The expressed rhIFNα2a, with a relative molecular mass of about 44 300, contained 30% of total somatic protein, and reached an expression level of 25% in lysis supernatant. TTie expressed product showed specific binding to mouse McAb against hlFN. The rh!FNα2a activity was 5. 57 x 107 IU/ml in lysis supernatant, indicating significantly antiviral activity of the recombinant protein. Conclusion Soluble fusion protein of rhIFNα2a and Ssp DnaB, with biological activity, was successfully expressed in E. coli BL2l(DE3), which provided a new idea for large-scale production of IFNα2a. Source

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