Construction and identification of eukaryotic expression vector of human angiopoietin-related protein-1 angioarrestin / 第二军医大学学报

Objective: To clone human angio poietin- related protein-1 angioarrestin genes and construct their recombinant eukaryotic expression vector. Methods: Full length sequence of angioarrestin gene and its C-FD domain were amplified from human liver cDNA library by PCR and were subsequently inserted into the eukaryotic expression vector pcDNA3. 1/ His-Myc (-)B. Then angioarrestin and FD recombinant plasmids were stablely transfected in NCI-H460 cell line. The positive clones were identified by RT-PCR and Western blot. Results: Full length fragment of angioarrestin gene (1 473 bp) and C-FD domain (560 bp) were successfully amplified from human liver cDNA library by PCR. The pcDNA3. 1-ARP and pcDNA3. 1-FD recombinant plasmids were also constructed successfully as identified by PCR and enzyme digestion. RT-PCR and Western blot showed the expression of target mRNA and protein in NCI-1-1460 cells. Conclusion: The eukaryotic expression vectors of angioarrestin and C-FD gene have been successfully constructed and expressed in NCI-H460 cells, which pave a way for further study on the anti-angiogenesis function of angioarrestin in cancer.

Screening, Identifying and Function Analysis of Polyketide Synthase I Cluster from the Environmental Strain X-2 Which Produce Macrolactins / 微生物学通报

Macrolactins are 24-membered macrolides produced by unidentified marine bacterium, Actinomadura sp. and Bacillus sp., which exhibit both antibacterial and antitumor activities in vitro. The environmental strain X-2 which was isolated from the sediment of the East China Sea produce Macrolatin A, B and O. In this study, a set of degenerate oligonucleotide primers, designed for amplification ketosynthase(KS) domains, had been employed to identify KS gene fragments of the X-2 DNA samples. One 645 bp KS fragment(GenBank accession no. EF486351)had been cloned and used as a probe to screen the genome DNA fosmid library of X-2. Three positive clones were selected and sequenced, Homologous analysis and the function prediction of the obtained PKS gene fragments suggested that macrolactin is the Polyketide Biosynthesis Product of the gene cluster obtained in the environmental strain X-2.

Expression of recombined human endothelial monocyte-activating polypeptideⅡand determination of its activity / 中国肿瘤生物治疗杂志

Objective: To chine and express the recombinant human endothelial monocyte-activating polypeptide-Ⅱ(EMAP-Ⅱ)and identify its anti-tumor biological activities.Methods: EMAP-Ⅱ_(147-312)was expressed by the expression vector pMAL-p2x and E.coli BL-21 and the product was purified.The production of tissue factor(TF)in human umbili- cal vein endothelial cell ECV-304 mediated by the recombinant EMAP-Ⅱwas determined by chemiluminescence sub- strate.The promoting effect of recombinant EMAP-Ⅱon TNF?-induced ECV-304 cell.Apoptosis was determined by flow cytometry.Its inhibitory effect on human pancreaic cancer cell SW1990 proliferation was determined by MTT method. Results:DNA sequencing verified that EMAP-Ⅱwas correctly cloned.The molecular mass of the protein identified by SDS-PAGE was consistent with the theoretic value.The productivity of recombinant EMAP-Ⅱwas 500?g per 1 g bacteria (wet mass).The purified product induced expression of tissue factor(TF)in ECV-304 cells;it also enhanced the sensi- tivity of ECV-304 cells to the apoptotic effect of TNF?([16.6?2.5]% vs[25.6?2.3]%,P

Expression of Tumstatin_(183-230)-TRAIL fusion protein and identification of its biological functions / 第二军医大学学报

Objective:To express Tumstatin_(183-230)-TRAIL fusion protein and to observe its biological functions.Methods: SOE-ing PCR was employed to amplify the recombinant sequence of Tumstatin_(183-230)and TNF-related apoptosis-inducing ligand (TRAIL_(114-281)).An expression vector pMAL-Tu-T was constructed by inserting Tu-T sequence into pMAL-c_2;the vector was used to transfect E.coli BL21(DE3)and expression of MBP-Tu-T fusion protein was induced by IPTG.Amylose Resin columns were employed to purify the fusion protein.The biological functions of MBP-Tu-T protein was examined by inhibitory test of endothelial cell proliferation,standard tumor cell cytotoxic assay,in vitro tube formation inhibition,and electron microscopic observation(apoptosis).Results:The expression rate of MBP-Tu-T fusion protein in E.coli was about 20%. Purified recombinant protein obviously inhibited endothelial cell proliferation(IC_(50)12.5?g/ml),induced apoptosis of pancreatic cancer cells,and inhibited tube formation.Conclusion:Constructed MBP-Tu-T fusion protein is bifunctional,which lays a solid foundation for further investigation of antitumor effect of Tumstatin_(183-230)-TRAIL in vivo.