Expression of Aurora-B in non-small cell lung cancer and its clinical significance / 南方医科大学学报

<p><b>OBJECTIVE</b>To study the expression of Aurora-B in non-small cell lung cancer (NSCLC) tissues and NSCLC cell lines.</p><p><b>METHOD</b>Aurora-B expression was examined using immunohistochemical SP method in 91 stage I and 69 stage II-III NSCLC tissues and 40 adjacent tissues. The mRNA and protein expressions of Aurora-B in NSCLC cell lines (A549, H460 and H1299) were examined by RT-PCR and Western blotting, respectively.</p><p><b>RESULTS</b>The protein expression of Aurora-B was detected in 77.7% (94/121) of the tumor tissues and 9.8% (4/41) of the adjacent tissues, showing a significant difference between them (P<0.01). The positivity rate of Aurora-B protein was not related with the gender and age of NSCLC patients, but with lymph node metastasis, differentiation and histological type of NSCLC (P<0.05). Aurora-B was expressed in all the NSCLC cell lines (A549, H460 and H1299) at both mRNA and protein levels. A549 cells showed the highest expression of Aurora-B.</p><p><b>CONCLUSION</b>Aurora-B protein is highly expressed in NSCLC tissues and cell lines, and may play a crucial role in the invasion, metastasis and development of NSCLC. The mRNA and protein expression levels of Aurora-B differ significantly between different NSCLC cell lines.</p>

Inducible expression of Mac-1-FP in Chinese hamster ovary cells using Tet-On gene expression system / 第二军医大学学报

Objective: To construct eukaryotic inducible expression plasmids pTRE-Tight-CFP-CD11b and pTRE-Tight-YFP-CD18 using Tet-On gene expression system and co-transfect them into Chinese hamster ovary (CHO) cells, so as to achieve the inducible expression of Mac-1-FP. Methods: The eukaryotic expression vectors pTRE-Tight-CFP-CN11b and pTRE-Tight-YFP-CD18 were constructed by recombinant DNA technique. The 2 vectors were co-transfected into CHO cells using liposome to fuse CD11b and CD18: the 2 subunits of Mac-1. Fluorescence microscope was used to observe the cyan fluorescence and the yellow fluorescence of Mac-1-FP. The influence of different levels of Dox (0, 0.01, 0.1, 0.5, 1, 2 μg/ ml) on expression levels of CD11b and CD18 in CHO cells was analyzed by RT-PCR and fluorescence intensity analysis. The adhesive rate of CHO-Mac-1-FP cells with ligand ICAM-1 was analyzed before and after PMA (1 μg/ml) stimulation. Results: The recombinant plasmids of pTRE-Tight-CFP-CD11b and pTRE-Tight-YFP-CD18 were successfully constructed. The cyan and yellow fluorescences were observed in co-transfected CHO cells under fluorescence microscope. The fluorescence intensity of the cells was increased with the increase of Dox concentration. RT-PCR analysis demonstrated that the CD11b and CD18 mRNA increased with the increase of Dox concentration. The adhesive rate of CHO-Mac-1-FP cells with ICAM-1 was increased after PMA stimulation (peaked at 2 h and 4 h after stimulation and decreased afterwards). Conclusion: This study achieves the inducible expression of Mac-1-FP in CHO cells. And Mac-1-FP, like widetype Mac-1, exhibits adhesive activity with ligand ICAM-1, which lays a foundation for studying the consisting dimmer, clustering, conformation and affinity of the ligands of Mac-1 using single molecule spectroscopy and fluorescence resonance energy transfer technique in living cells.

Cloning and expression of human glucagon-like peptide-1 cDNA / 第二军医大学学报

Objective: To clone hGLP-1 cDNA in the pBS SK(+/-)vector and construct the expression vector of pGEX-4T-3/hGLP-1cDNA to express GST-hGLP-1 fusion protein. Methods: The hGLP-1 cDNA was constructed by 6 synthetic oligonucleotides fragments, followed by the procedure of annealing and ligation with oligonucleotides fragments. The hGLP-1 cDNA was cloned into the pBS SK(+/-) vector, and was selected by α-complementation. It was confirmed by DNA sequening, then inserted into the MCS of the fusion expression vector pGEX-4T-3. The recombinant vector was transformed into E. coli TG1. Results: The recombinant plasmid DNA was digested with restrictive endonuclease BamHⅠand XhoⅠ. The result demonstrated that the hGLP-1 cDNA was successfully inserted into the pGEX-4T-3 vector and fusion protein GST-hGLP-1 had been expressed in SDS-PAGE. Conclusion： Expression of GST-hGLP-1 fusion protein can provide foundation for obtaining a larger quantity of recombinant hGLP-1 for experimental and clinic studies.

Cloning and expression of human glucagon-like peptide-1 cDNA / 第二军医大学学报

Objective: To clone hGLP-1 cDNA in the pBS SK(+/-)vector and construct the expression vector of pGEX-4T-3/hGLP-1cDNA to express GST-hGLP-1 fusion protein. Methods: The hGLP-1 cDNA was constructed by 6 synthetic oligonucleotides fragments, followed by the procedure of annealing and ligation with oligonucleotides fragments. The hGLP-1 cDNA was cloned into the pBS SK(+/-) vector, and was selected by α-complementation. It was confirmed by DNA sequening, then inserted into the MCS of the fusion expression vector pGEX-4T-3. The recombinant vector was transformed into E. coli TG1. Results: The recombinant plasmid DNA was digested with restrictive endonuclease BamHⅠand XhoⅠ. The result demonstrated that the hGLP-1 cDNA was successfully inserted into the pGEX-4T-3 vector and fusion protein GST-hGLP-1 had been expressed in SDS-PAGE. Conclusion： Expression of GST-hGLP-1 fusion protein can provide foundation for obtaining a larger quantity of recombinant hGLP-1 for experimental and clinic studies.