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AIM: To investigate the possible use of anti-FGF-2 nanobody for the treatment of pathological neovascularization. METHODS: SD rats were divided into a sham operation group, a control group (3 mm diameter circular filter paper soaked with 1 mol/L NaOH solution was applied to the central part of the cornea of rats for 30 s to prepare the rat model of alkali-burn angiogenesis) and a treatment group (treated with a drop of 3 mg/mL anti-FGF-2 nanobody 7 days after the operation. Repeat application 3x/day for 14 days). Corneal angiogenesis was measured by stereoscopic microscopy and CD31 immunohistochemical staining. The mRNA and protein expression levels of VEGF and FGF-2 were detected by quantitative fluorescence PCR (qPCR), enzyme-linked immunosorbent assay (ELISA) and immunohistochemistry.RESULTS: (1) Blood vessel: The area of the treatment group was significantly reduced compared with the model group, and the vascular lumen was narrower (P0.05); (3) Expression levels of VEGF mRNA and protein: The treatment group was significantly higher than the model group (P<0.05). In addition, the expression of VEGF also increased significantly in the continuous administration of the sham operation group. CONCLUSION: Anti-FGF-2 nanobody can be used for the treatment of angiogenesis. However, the expressions of VEGF will compensatorily increase after blocking FGF-2 in normal or pathological rats.
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Aim To explore whether the polypeptide vaccines CKL9 and YL20 can induce immune response and anti-tumor effect on HER-2 (+) tumors in vitro and in vivo, and to provide suggestions for clinical use.Methods The proliferation of specific lymphocytes and cytotoxic T lymphocyte activity(CTL) stimulated by CKL9 and YL20 were studied with CCK-8 assay and LDH assay, and the antitumor activity of CKL9 and YL20 was evaluated in vivo.Results The lymphocyte proliferation was promoted by incubation with CKL9 and YL20, and the relative increase of cells was 11.1% and 16.7% respectively at the concentration of 50 mg·L-1 of CKL9 and YL20.The LDH assay confirmed the CTL effect induced by CKL9 and YL20 on HER2-positive tumor cells, not on HER2-negtive tumor cells.With an effector-target ratio of 80 ∶1, the inhibition of tumor cell by cytotoxic T lymphocyte stimulated by CKL9 and YL20 could reach 89.8% and 84.3%, respectively.The HER2(+) tumor cell N87 transplanted in Babes mice was inhibited by pre-immune polypeptide CKL9 and YL20.Conclusion The HER2-specific polypeptide vaccines CKL9 and YL20 could induce persistent specific CD4 and CD8 T cell immune and inhibit the growth of HER2 positive tumor cells.
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Objective:In the present study,Bac-to-Bac baculovirus expression system was used to obtain recombinant human Cosmc extracellular domain protein,which can lay the foundation for the research about the structure and function of Cosmc protein in vitro,and simultaneously provide ideas for the research of O-glycosylation and related diseases. Methods: The Cosmc extracellular domain ( Cosmc-ED) gene was cloned into a transfer vector pFastBac1 to form the recombinant donor plasmid pFastBac1-Cosmc ED, which was transformed into competent cells DH10Bac. By using blue-white selection and PCR analysis,we could obtain recombinant shuttle vector rBacmid-Cosmc ED. Then, the recombinant gene DNA of rBacmid-Cosmc ED was used to transfect Sf-9 mediated by cationic lipid formulation,and the recombinant baculovirus bacmid was obtained,which was further used to infect the serum-free cell Sf-9 to express Cosmc-ED in the supernatant. Then the protein of interest was detected by SDS-PAGE and Western blot and purified with Ni-NTA affinity column. Results:SDS-PAGE and Western blot analysis showed a specific band about 33 kD,consistent with the interest protein. Mass spectrometry results further prove that the protein was Cosmc extracellular domain protein. Conclusion: Human Cosmc-ED protein can be successfully expressed in Sf-9 insect cells and laid basis for subsequent studies.
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To produce recombinant Maxadilan using gene engineering technology, the gene of recombinant Maxadilan which expressed in protocaryon were designed and synthesized according to the amino acid sequences of Maxadilan. The recombinant plasmid pKYB-MAX was constructed and transformed into host bacteria Escherichia coli strain ER2566. After the MAX-intein-CBD fusion protein was purified by chintin-affinity chromatography, the self-cleavage activity of the intein was induced by beta-mercaptoethanol and the recombinant Maxadilan was released from the chitin-bound intein tag. The molecular weight of peptides was determined by the laser flight mass spectrometry and the results was conformity with the theoretical value. The biological activity analysis showed that recombinant Maxadilan significantly enhanced the concentration of serum glucose.
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Animals , Base Sequence , Escherichia coli , Genetics , Metabolism , Insect Proteins , Genetics , Inteins , Genetics , Isopropyl Thiogalactoside , Pharmacology , Molecular Sequence Data , Recombinant Proteins , GeneticsABSTRACT
AIM: To obtain a high and stable expression analog of human basic fibroblast growth factor by genetic engineering. METHODS: The cysteins 78 and 96 of natural hbFGF polypeptide was substituted with serines by means of site-directed mutagenesis. Using pET- 3c as vector, the mutated polynucleotide was cloned and then transferred into BL21 (DE3)plysS. After induction by IPTG, the analog was obtained and analyzed by SDS - PAGE. RESULTS: After purification the form of soluble mutant increased remarkably but the forms of dimmer and higher multimer were reduced greatly to no more than 8% of the total recombinant protein. By MTT assay, the analog showed the same biological activity. This new analog represented a desirable complementation for native hbFGF to develop pharmaceutical drug in clinical use. CONCLUSION: Substitution of certain amino acids of polypeptide without altering native protein' s bioactivity to get the analog is an effective means to increase stability of foreign protein and its solubility in E. coli.
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AIM: To examine the effect of traditional chinese medicine recipe, Taoren Honghua(semen persicae-flos carthami) decoction, on hyperlipidemia without symptom. METHODS: The plasma TC, TG, LDL, HDL, apolipoprotein(Apo) A, Apo B of the patients with hyperlipidemia without symptom were measured using automatic analyzer (shimadiu CL-7200), the production of nitric oxide(NO) was detected by Greiss reaction, and SOD activity and MDA formation were examined using o-trihydroxy benzene and barbituric methotheds, respectively. RESULTS: After oral administration of Taoren Honghua decoction, the plasma levels of TC, TG, LDL, and MDA of the patients were markedly decreased, however, the plasma levels of ApoA, HDL, SOD and NO were significantly increased and almost no change was detected in the plasma level of Apo B. In control group, it was found that although the plasma level of TC, TG and LDL were decreased ( P
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AIM: To construct engineered E.coli strains which can express nattokinase with fibrinolysis activity using gene engineering technology. METHODS: The pro-nattokinase (pro-NK) gene was amplified by PCR and inserted into expression vector pET3c. The recombined plasmid pENK which expressed the fusion protein of pro-NK and 22 amino acid peptide was then transferred into lysogenic host strains BL21(DE3)pLysS - and BL21(DE3)pLysS +. Both SDS-PAGE and the fibrin plate assay were used to examine the expression and the activity of the target protein. RESULTS: SDS-PAGE assay showed the fused gene encoding 42 kD fusion protein was expressed in both expression strains pENK-(DE3)pLysS - and pENK-(DE3)pLysS +, and the fibrin plate assay indicated that the expression product had fibrinolysis activity. pENK-(DE3)pLysS - exhibited the basal expression of the target gene, while fusion protein was only induced by IPTG in pENK-(DE3)pLysS +. Basal expression of the fused toxic gene in pENK-(DE3)pLysS - led to bacteriolysis and hollow lawns. CONCLUSION: A pro-NK fusion protein with fibrinolysis activity is successfully expressed in E.coli , which lay a foundation for the exploitation of nattokinase.
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AIM: To construct a recombinant hEGF-hbFGF(78-154aa)fusion protein, which not only has the heparin-binding ability, but also promotes the growth of the cells, and to express the fusion protein in E. coli expression system with high expression level.METHODS: hEGF gene was joined with 231 bp fragment coding hbFGF(78-154aa) and expressed in E. coli. The fusion protein was purified using affinity chromatography of heparin-Hyper D and analyzed with western blot. The pI value and the biological activity were both assayed.RESULTS: The fusion protein was expressed in a high expression level of about 30% of the total cell protein, as estimated by SDS-PAGE. Western analysis results showed that the antigenicity of fusion protein was similar to hEGF. Fusion protein could not only bind heparin but also promote the growth of 3T3 cell. The pI value of fusion protein was 5.2.CONCLUSION: The recombinant hEGF-hbFGF(78-154aa) fusion protein possessed the characteristics of both hEGF and hbFGF. This new-designed protein would become a good object for the research on the relationship between the structure and the function of the growth factor.