Expression of kringle 1-3 domain fragment of human plasmihogen in E.coli and the anti-tumor activity of its product / 中国免疫学杂志

Objective:To study the expression of kringle 1-3 domain fragment of human plasmihogen in E.coli and the anti-tumor activity of its product.Methods:The K1-3 domain fragment was cloned in expression vector pBV220,the resulted recombinant plasmid pBV-K13 was transformed into E.coli DH5? and its product was purified and assayed its bioactivity.Results:K1-3 domain fragment was expressed in(E.coli) DH5?.The results showed the expressed product covered 20% of the total bacterial protein on SDS-PAGE and the Western blot analysis showed that the product had immunological specificity with the antiserum of human plasminogen and inhibits the growth of chorioallantoic membrane(CAM) angiogenesis and mouse B16 melanoma.Conclusion:Human plasminogen K1-3 domain fragment was expressed in E.coli;the expressed product has anti-angiogenesis and anti-tumor activity.

Cloning and Expression of the Signaling Protein 14-3-3 of Toxoplasma gondii / 中国寄生虫学与寄生虫病杂志

Objective To clone and express the cell signaling protein 14-3-3 gene from Toxoplasma gondii RH strain. Methods Toxoplasma RH strain tachyzoites, which maintained by mouse passage, were harvested from ascites of mice and genomic DNA was prepared. A pair of primers were designed and synthesized based on the sequence of Toxo 14-3-3 cDNA. A specific fragment of Toxo 14-3-3 gene was obtained by RT-PCR amplification from Toxoplasma genomic DNA. The PCR products were ligated to pGEM-T. The EcoRI / Xho I restricted fragments, confirmed by PCR and EcoRI / XhoI digestion, were cloned into expression vector pET28a and the recombinants were transformd into E.coli BL21. Fusion expression was induced by isopropyl-beta-D-thiogalactoside (IPTG) and confirmed by Western blotting with rabbit anti-Toxoplasma sera. Results The molecular size of Toxo 14-3-3 was 803 bp, which is highly homologous to the previous report cloned from the parasites of intestinal epithelial stage in cat. High expression was obtained in pET28a/ Toxo 14-3-3/E.coli BL21 when confirmed by Western blotting. Conclusion The recombinant construction of Toxo 14-3-3 was generated and expression was induced.

Expression of Human Interleukin-33 and studies on its biology function / 中国免疫学杂志

Objective:To clone human interleukin-33(hIL-33)and express it in E.coli efficiently.Methods:The primers were synthesized according to the hlL-33 cDNA sequence in GeneBank.The hIL-33 was amplified by RT-PCR from human fibroblast cell line(L929),the PCR product was inserted into pUC19 vector.The IL-33 cDNA confirmed by sequencing was inserted into expressing vector PQE30 and expressed in E.coli M15 strain.IL-33 protein expression was induced by IPTG and purified by Ni-NTA affinity chromatography.The recombinant IL-33 was identified by Immunoblot and its biological activity was analyzed.Results:DNA sequencing confirmed that the cloned cDNA was identical to the published sequence of hIL-33.The recombinant plasmid PQE/hIL-33 was transformed into M15.An expected 18KD protein of hIL-33 found mainly in the induced host strains about 25% of total bacteria lysis by SDS-PAGE and coomassie blue staining.The 18 KD protein could be recognized by anti-IL33 antibody in western blot.The recombinant protein was purified to more than 95% of total protein and induces the production of IL-4 and IL-5 in human peripheral blood mononuclear cells.Conclusion:We have successfully expressed hIL-33 protein in E.coli and the expressed product has IL-33 specific bioactivity.