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秦浚川, Wan-Li Wei, *, Jun-Chuan Qin† and Man-Ji Sun*‡
BIOCHEM PHARMACOL 60; 1: 121-126,-0001,():
-1年11月30日
The human butyrylcholinesterase (BChE, EC 3.1.1.8) gene was highly expressed in Bombyx mori using baculovirus vector, and the biochemical-pharmacological properties of its product were studied. BChE cDNA was cloned into transfer vector pBn96 and co-transfected with wild-type Bombyx mori nucleopolyhedrovirus (BmNPV) DNA into BmN cells. The recombinant virus with the highest enzyme activity was sorted out and purified. Once the BmN cells or silkworm larvae had been infected with the recombinant virus, recombinant human BChE (rhBChE) could be secreted into the culture medium or the hemolymph of the larvae at levels of 1.5mg•L-1 and 35mg•L-1, respectively. Western blot and enzymatic staining of the electrophoresis gel of non-denatured protein showed that rhBChE manifested similar antigenicity and enzyme activity to native human BChE (nhBChE). The production of rhBChE in the hemolymph was 23-fold higher than that in BmN cells and about 280-fold that in Chinese hamster overy cells (125μmg•L-1). This is the first report of human BChE expression in silkworm with the highest level of yield so far. rhBChE was highly similar to nhBChE in respect to substrate affinity, inhibitor sensitivity, and reactivity of the inhibited enzyme. It is suggested that rhBChE functions as well as nhBChE and has potential practical value.
butyrylcholinesterase, gene expression, baculovirus, Bombyx mori, sarin
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【期刊论文】Expression of a novel recombinant dual human stem cell factor in insect cells
秦浚川, Junhai Han, a, Xiaohui Yan, Jie Zhu, Xiaoyong Zhi, Yuhui Zang, Beifen Shen, b and Junchuan Qin a, *
Protein Expression and Purification 31(2003)311-317,-0001,():
-1年11月30日
Stem cell factor (SCF) is a hematopoietic cytokine that promotes the survival, proliferation, and differentiation of hematopoietic cells. A dual human stem cell factor (dhSCF) cDNA was constructed, which consisted of a full-length human stem cell factor cDNA plus a truncated hSCF cDNA (1-145aa), linked by a peptide (GGGGSGGGGSGG) coding region. The dhSCF gene was cloned into baculovirus transfer vector pAcSecG2T under the control of polyhedrin promoter. The Sf9 cells infected with the recombinant virus expressed rdhSCF up to 6000 U/106 cell in flask and 8300 U/106 cell in spinner flask. The rdhSCF was purified by two-step chromatography. The molecular mass of rdhSCF was examined by western blotting and HPLC analysis. The specific activity of rdhSCF was up to 3.1
Dual human stem cell factor, Insect expression system, Sf9 cell, pAcSecG2T
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秦浚川, Tao Chen, Yuhui Zang, Jie Zhu, Haiqin Lu, Junhai Han, Junchuan Qin*
Protein Expression and PuriWcation 41(2005)402-408,-0001,():
-1年11月30日
A novel human stem cell factor (SCF)/macrophage colony-stimulating factor (M-CSF) fusion protein gene was constructed, in which the coding regions of human SCF cDNA (1-165aa) and the truncated M-CSF cDNA (1-149aa) were connected by a linker sequence encoding a short peptide GGGGSGGGGSGG. The SCF/M-CSF gene was cloned into baculovirus transfer vector pVL1392 under the control of polyhedrin promoter and expressed in the Sf9 cells (Spodoptera frugiperda). SDS–PAGE and Western blot analysis showed that the puriWed fusion protein was a homodimer with a molecular weight about 84kDa under non-reducing conditions or a monomer about 42kDa under reducing conditions. The speciWc activity of rhSCF/M-CSF was 17 times as high as that of monomeric rhSCF to stimulate the proliferation of TF-1 cell. The results of macrophages colony-forming (CFU-M) assay performed with human bone marrow mononuclear cells demonstrated that rhSCF/M-CSF was more potent in promoting CFU-M than the equimolar of SCF, M-CSF or that of two cytokines mixture.
SCF, M-CSF, Fusion protein, Expression in insect cell
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秦浚川, Haiqin Lu, Jie Zhu, Yuhui Zang, Yuguan Ze, Junchuan Qin*
Biochemical Pharmacology 70(2005)1019-1025,-0001,():
-1年11月30日
Human paraoxnase-3 (hPON3) (EC3.1.8.1) is a lipid-associated enzyme with antioxidant activity, and can inhibit the oxidation of lowdensity lipoprotein (LDL), thereby inhibiting early atherogenic process. In the present study, human PON3 gene was cloned from Human Fetal Liver Marathon-Ready cDNA and expressed in insect cells using baculovirus vector. Twenty-eight milligrams of purified recombinant hPON3 (rhPON3) was obtained from 1 L Sf9 cells culture. The Km and Vmax values of rhPON3, with respective to phenylacetate hydrolysis were 7.46
Human paraoxonase-3, Baculovirus-mediated expression system, Sf9 cell, Gene expression, LDL oxidation, Dihydrocoumarin
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【期刊论文】Expression, refolding, and characterization of a novel recombinant dual human stem cell factor
秦浚川, Haiqin Lu, Yuhui Zang, Yuguan Ze, Jie Zhu, Tao Chen, Junhai Han, Junchuan Qin*
Protein Expression and PuriWcation 43(2005)126-132,-0001,():
-1年11月30日
A novel recombinant dual human stem cell factor (rdhSCF) gene which consisted of a full-length hSCF(1-165 aa) cDNA and atruncated hSCF (1-145 aa) cDNA, linked by a peptide (GGGGSGGGGSGG) coding region, was constructed and cloned into Escherichia coli expression vector pET-22b. The rdhSCF was expressed at high level in E. coli BL21(DE3) and existed mainly as inclusion bodies. The inclusion bodies were solubilized in urea and refolded by ion-exchange chromatography. After renaturation, the purity of the yielded rdhSCF was up to 90%. Cell proliferation assay showed that the speciWc activity of the rdhSCF was 2.86
Dual human stem cell factor, pET-22b, Refolding, Escherichia coli expression system, BL21(, DE3),
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