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1.
Chinese Journal of Biotechnology ; (12): 540-543, 2004.
Artigo em Chinês | WPRIM | ID: wpr-270090

RESUMO

The expression of cDNA encoding Tachyleus auti-lipoposaccharide (LPS) factor, which is of interest for use as a potential inhibitor of the common core subunit of Gram-negative bacterial endotoxin. First, the TALF gene was inserted into expression vectors pGEX-4T-2, pET22b and pET28a to construct recombinant expression plasmids. The recombinant plasmids were transformed to E. coli BL21 (DE3) and the expression of TALF was examined. Results show that TALF in pET22b and pET28a vectors can't be expressed. Only the fusion protein GST-TALF was expressed in E. coli BL21 existing as inclusion bodies. From 1 liter of culture, about 4mg of fusion protein GST-TALF with 91% purity was finally obtained. No apparent bactericidal activity and LPS neutralizing activity of the fusion protein GST-TALF were found. After digested with thrombin, the fusion protein GST-TALF exhibited strong bactericidal activity and LPS neutralizing activity.


Assuntos
Peptídeos Catiônicos Antimicrobianos , Proteínas de Artrópodes , Escherichia coli , Genética , Glutationa Transferase , Genética , Hormônios de Invertebrado , Genética , Farmacologia , Lipopolissacarídeos , Plasmídeos , Proteínas Recombinantes de Fusão , Farmacologia
2.
Acta Academiae Medicinae Sinicae ; (6): 39-42, 2003.
Artigo em Chinês | WPRIM | ID: wpr-301903

RESUMO

<p><b>OBJECTIVE</b>To construct a reasonable substitute for the autograft bone in vitro and transplant it back into the rabbit models to induce the spine fusion.</p><p><b>METHODS</b>The bone marrow stem cell from the seven New Zealand rabbits were cultured. Recombinant human bone morphogenetic protein-4 (rhBMP-4) that has been proved to be bioactive was obtained by the way of genetic engineering. Using the vacuum freezing machine to mix a certain quantity of rhBMP-4 into type I collagen to form a new kind of carrier. Animal model of spine facet process fusion was used. Bone marrow stem cells combined with rhBMP-4 and I type collagen were implanted between the facet process to induce the spine fusion. type I collagen and bone marrow stem cell was used in the controlled group.</p><p><b>RESULTS</b>New bone formation was obvious in the test group. The facet joint was fused very well in this side. No bone formation was present on the other side.</p><p><b>CONCLUSIONS</b>The new composite: bone marrow stem cells, rhBMP-4 and type I collagen was an ideal kind of substitute for the autograft bone.</p>


Assuntos
Animais , Coelhos , Células da Medula Óssea , Biologia Celular , Proteína Morfogenética Óssea 4 , Proteínas Morfogenéticas Ósseas , Genética , Substitutos Ósseos , Usos Terapêuticos , Transplante Ósseo , Células Cultivadas , Colágeno Tipo I , Química , Implantes Experimentais , Proteínas Recombinantes , Genética , Fusão Vertebral , Métodos , Transplante de Células-Tronco , Células Estromais , Biologia Celular , Engenharia Tecidual
3.
Chinese Journal of Biotechnology ; (12): 112-115, 2003.
Artigo em Chinês | WPRIM | ID: wpr-259184

RESUMO

Phytase gene of Aspergillus niger 963 was cloned into baculovirus transfer vector. DNA of the recombinant vector was co-transfected with Bm-BacPAK6 DNA into BmN cells, and recombinant virus was selected by plaque assays. The recombinant virus was identified by Dot blot and Southern-blot with the specific probe for phytase gene. Phytase gene was expressed in silkworm larvae and pupae. The expression product was 1.43 g/L haemolymph for silkworm larvae and 1.90 g/L haemolymph for pupae, respectively. The enzymic characteristicses of phytase expressed in baculovirus-expression system were studied in this paper.


Assuntos
Animais , 6-Fitase , Genética , Metabolismo , Aspergillus niger , Genética , Southern Blotting , Bombyx , Genética , Metabolismo , Eletroforese em Gel de Poliacrilamida , Proteínas Fúngicas , Genética , Metabolismo , Vetores Genéticos , Genética , Concentração de Íons de Hidrogênio
4.
Chinese Journal of Biotechnology ; (12): 106-108, 2002.
Artigo em Chinês | WPRIM | ID: wpr-231387

RESUMO

According to the reported sequence of Buthus martensii Karsch scorpion toxin gene (BmK IT3), we synthesized two primers, which were complementary in a region. By the means of PCR, we got the gene. The gene was fused in expression vector pET-28a, which gave rise to a recombinant plasmid pET(IT3R). Then it was transformed into E. coli BL21 (DE3). With IPTG induction, the gene was efficiently expressed. And the fusion product was soluble.


Assuntos
Animais , Clonagem Molecular , Escherichia coli , Genética , Expressão Gênica , Isopropiltiogalactosídeo , Farmacologia , Proteínas Recombinantes , Genética , Venenos de Escorpião , Química , Genética , Escorpiões , Química , Genética
5.
Chinese Journal of Biotechnology ; (12): 20-24, 2002.
Artigo em Chinês | WPRIM | ID: wpr-231381

RESUMO

Human calcitonin (hCT) is a 32 amino acid peptide hormone that requires C-terminal amidation for full biological activity. Calcitonin has important physiological function in vivo. We describe the couple expression of a synthesized modified human calcitonin(hmCT) gene fused with glutathione-S-transferase and rat peptidylglycine alpha-amidation monooxygenase (PAM) in insect cells infected by recombinant baculovirus GSTCT/PAM. Using Western blotting against hmCT or rat PAM, the GSThmCT fusion protein had been identified as well as the PAM. Following affinity chromatography with glutathione agarose column, the GSThmCT fusion protein produced by insect cells was purified. The purified fusion protein was also interacted with antibody against hmCT. The couple expression of a modification enzyme and its substrate in eucaryotic expression system may be used for producing other biological activity peptides.


Assuntos
Animais , Humanos , Ratos , Baculoviridae , Genética , Calcitonina , Genética , Células Cultivadas , Expressão Gênica , Glutationa Transferase , Genética , Insetos , Biologia Celular , Oxigenases de Função Mista , Genética , Complexos Multienzimáticos , Genética , Proteínas Recombinantes de Fusão , Genética
6.
Chinese Journal of Biotechnology ; (12): 698-702, 2002.
Artigo em Chinês | WPRIM | ID: wpr-256122

RESUMO

A 558 bp cDNA fragment was amplified by RT-PCR from adult Schistosoma japonicum(Chinese strain) mRNA with a pair of primers that were designed according to published Sj21.7p gene encoding 21.7 kD protein of Schistosoma japonicum(Philippines strain). Sequence analysis indicated that this frame, named Sj21.7 (Ch), with 99% homology to Sj21.7 p, contained a complete open reading fragment (ORF) of 21.7 kD protein gene of Schistosoma japonicum(Chinese strain). The amino acid sequence shared 98% homology with 21.7 kD protein of Schistosoma japonicum. This fragment was cloned into the expression vector pET28a (+) and subsequently expressed in Escherichia coli with IPTG induction. SDS-PAGE analysis revealed that the molecular weight of this expressed product was 25.4 kD. Western blotting showed that the recombinant protein reacted well with the rabbit serum immunized with Sj worm antigen, indicating that this expressed product had good antigenicity.


Assuntos
Animais , Coelhos , Sequência de Aminoácidos , Clonagem Molecular , Proteínas de Helminto , Química , Genética , Dados de Sequência Molecular , Peso Molecular , Fases de Leitura Aberta , Proteínas Recombinantes , Química , Alergia e Imunologia , Schistosoma japonicum , Genética , Homologia de Sequência
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