Résumé
Objective: To investigate effect of Yanghe Huayan Tang and phosphatidylinositol 3-kinase (PI3K) inhibitor LY294002 in intervening expressions of human epidermal growth factor receptor human epidermal growth factor receptor-2(HER-2), PI3K and phosphorylated serine/threonine kinase (p-Akt)in PI3K/Akt pathway of breast cancer cell line SK-BR-3 (HER-2 high expression) in vitro, and study intervention mechanism of Yanghe Huayan Tang. Method: Yanghe Huayan Tang intestinal absorption fluid was prepared, breast cancer cell strain SK-BR-3 was divided into blank group, Yanghe Huayan Tang group, LY294002 group, LY294002+Yanghe Huayan Tang group. The concentration was respectively 125 g·L-1 for Yanghe Huayan Tang, 0.05 g·L-1 for LY294002, 125 g·L-1 for Yanghe Huayan Tang+LY294002.The expressions of HER-2, PI3K and p-Akt protein were detected by immunohistochemistry and Western blot after 24 h. The most appropriate and effective concentration was obtained through extensive experiments. The expressions of HER-2, PI3K and p-Akt were detected by reverse transcription polymerase chain reaction (RT-PCR). Result: Compared with blank group, LY294002 group, and LY294002+Yanghe Huayan Tang group could inhibit protein and mRNA expressions of HER-2, PI3K, p-Akt in breast cancer cell lines(PPPPConclusion: Yanghe Huayan Tang can inhibit angiogenesis and invasion of breast cancer. Its main mechanism is expressed by intervening PI3K/Akt pathway, reducing expressions of HER-2, PI3K and p-Akt in pathway.
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To optimize Chinese hamster ovary (CHO)expression system and establish a process of screening CHO cell lines with high productivity;neomycin-phosphotransferase (NPT)expressed by the resistance marker gene on the expression vector was mutated with amino acid D at 261 changed to G.After selection by culturing with G418;the survival rate of CHO cells bearing mutant-NPT was significantly lower than that of the cells bear-ing wide type NPT.An enhanced green fluorescent protein (EGFP)was genetically linked to the N terminus of the IgG1 Fc fragment part to generate an EGFP-Fc fusion protein regarded as a report gene;which verified that the resistance of mutant-NPT to G418 was weakened.By comparing fluorescence assay of EGFP intensity in stable transfections after selection with the same concentration of G418 for 3 weeks;mutant-selected pools expressed more exogenous protein than the WT-selected pools.Therefore;the ratio of high producers in a transfected cell population greatly increased.
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Objective To explore the effect of mouse proliferation-associated protein 2G4 (p38-2G4) high-expression on the proliferation and erythriod differentiation of murine erythroleukemia ( MEL ) cells.Methods To establish the recombinant lentivirus vector p 38-2G4-pLJM1, the p38-2G4-pLJM1 was cotransfected into HEK293T cells to obtain lentivirus with pCMV-VSV-G and pCMV-dR8.2.Lentivirus were infected into MEL cells to establish the stably p 38-2G4 high-expressed MEL cells.Western blotting was used to analyse the high-expression efficiency.MTT assay and benzidine staining were applied to detect the cell viability and hemoglobin synthesis of the stable cell line in presence /absence of inducers.Results Western blotting showed that the p38-2G4 high-expression stable cell strain had a higher expression of p38-2G4 as compared to the control group ( MEL) ( P 0.05), but the hemoglobin synthesis had been reduced as compared to the control group (P<0.05).Conclusion p38-2G4 high-expression does not affect the cell viability of MEL cells , but inhibits the erythriod differentiation of MEL cells in three independent experiments .
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E.coli single-stranded DNA-binding protein (SSB) plays an important role in replication, recombination and repair of DNA and is thus crucial for the survival of the bacteria.We described a high expression and efficient purification scheme and kinetic assay of interaction with its substrate, single-stranded DNA (ssDNA). A ssb gene (537 bp) for encoding SSB was obtained by PCR amplification from E.coli K-12 genome. The expression vector of the fusion protein SSB was constructed by attaching ssb gene to pQE30. SSB fusion protein was expressed in M15 E.coli strain induced by IPTG. SDS-PAGE analysis revealed that the expected protein with a molecular weight 20.6kDa was soluble and amounted to about 30% of the total bacterial protein. SSB protein was purified by immobilized metal (Ni2+) chelation affinity chromatography and the purity was about 90%. The resulting SSB protein was a correctly folded tetramer analyzed by gel filtration. It could bind ssDNA with equilibrium dissociation constant (KD) of 4.79×10-7 mol/L as determined by surface plasmon resonance.
Résumé
E.coli single-stranded DNA-binding protein (SSB) plays an important role in replication, recombination and repair of DNA and is thus crucial for the survival of the bacteria.We described a high expression and efficient purification scheme and kinetic assay of interaction with its substrate, single-stranded DNA (ssDNA). A ssb gene (537 bp) for encoding SSB was obtained by PCR amplification from E.coli K-12 genome. The expression vector of the fusion protein SSB was constructed by attaching ssb gene to pQE30. SSB fusion protein was expressed in M15 E.coli strain induced by IPTG. SDS-PAGE analysis revealed that the expected protein with a molecular weight 20.6kDa was soluble and amounted to about 30% of the total bacterial protein. SSB protein was purified by immobilized metal (Ni2+) chelation affinity chromatography and the purity was about 90%. The resulting SSB protein was a correctly folded tetramer analyzed by gel filtration. It could bind ssDNA with equilibrium dissociation constant (KD) of 4.79×10-7 mol/L as determined by surface plasmon resonance.
Résumé
E.coli single-stranded DNA-binding protein (SSB) plays an important role in replication, recombination and repair of DNA and is thus crucial for the survival of the bacteria.We described a high expression and efficient purification scheme and kinetic assay of interaction with its substrate, single-stranded DNA (ssDNA). A ssb gene (537 bp) for encoding SSB was obtained by PCR amplification from E.coli K-12 genome. The expression vector of the fusion protein SSB was constructed by attaching ssb gene to pQE30. SSB fusion protein was expressed in M15 E.coli strain induced by IPTG. SDS-PAGE analysis revealed that the expected protein with a molecular weight 20.6kDa was soluble and amounted to about 30% of the total bacterial protein. SSB protein was purified by immobilized metal (Ni2+) chelation affinity chromatography and the purity was about 90%. The resulting SSB protein was a correctly folded tetramer analyzed by gel filtration. It could bind ssDNA with equilibrium dissociation constant (KD) of 4.79×10-7 mol/L as determined by surface plasmon resonance.
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Retroviral expression system which consists of retroviral vector,envelop protein vector and packaging cell line is an efficient expression system for recombinant protein.It has great potential in gene therapy and biopharmacy.Transcriptional active genome regions are the preferred targets for retrovirus integration.Furthermore,VSV-G protein enables this system a broader host range and makes virus integration more efficient.After infection of high-titer virus,high production clones can be selected through simple screening.So far,the research on retrovirus expression system has developed into application in bio-pharmacy industry.Here the composition of this system and the mechanism of virus transduction and summarize the application and prospect of retroviral expression system are introduced.
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Objective To construct a highly expresses catalase of Helicobacter pylori (H.pylori) and to test it's activity. Methods The catalase DNA was amplified from H.pylori chromosomal DNA by PCR techniques, inserted into the prokaryotie expression vector pET 22b(+) and then transformed into the BL21(DE3) E.coli strain which expressed catalase recombinant protein. Then the activity of H.pylori catalase was assayed by the Beers and Sizers. Results DNA sequence analysis showed that the sequence of catalase DNA was the same as that published by GenBank. The catalase recombinant protein amounted to 24.4% of the total bacterial protein after inducing with IPTG for 3 hours at 37?C and the activity of H.pylori catalase was high in the BL21 (DE3) E.coli strain. Conclusions A clone of expressing high activity H.pylori catalase has been obtained, and thus a good foundation for further study has been laid.
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The temperature effect on the recombinant protein production formation was investigated in present study. The culture temperature of growth phase is 30℃, and the culture temperature of induction phase was arranged according to three modes. Hign cell-density and high expression culture of E.coli to product recombinant human apolipoprotein A-I Milano by two temperature-shifted induction . Two temperature-shifted induction was carried out high density and high expression recombinant human ApoA-1 Milano. The recombinant protein ApoA-I Milano reached 4.8 g?L -1 with the final cell density of OD 600 150. And the two temperature-shifted induction avoided the acetic acid successfully to the influence of the high density and high expression. Two temperature-shifted induction was viable in high density culture and high expression of heterogenous protein in recombination E.coli.The sduty provides a basic work for production of recombinant ApoA-I Milano in scale.