Construction of human antimicrobial peptide LL-37 vector and its expression in Pichia pastoris / 第二军医大学学报

Objective: To construct a eukaryotic expression vector of human antimicrobial peptide LL-37 (pPIC9-LL-37) and to express it in P. pastoris. Methods: The full-length gene encoding antimicrobial peptide LL-37 was synthesized by overlap extension-PCR method using the sequence of LL-37 and P. pastoris biased codon. The full-length gene was cloned into pPIC9 vector and the product was transformed into E. coli DH5α to construct expression vector pPIC9-LL-37. After identification by PCR and sequencing, pPIC9-LL-37 was used to transfect P. pastoris. The expression of LL-37 was induced by methanol and the highest expressing strain was screened. The concentrated fermentation product was analyzed by Tricine-SDS-PAGE and Western-blot, and the antibacterial activity of the expression product on E. coli. DH5α was tested. Results: The eukaryotic expression vector pPIC9-LL-37 was successfully constructed. The fusion of LL-37 gene into P. Pastoris was confirmed by PCR. High expression of LL-37 was expressed by 0.5% methanol and the highest expressing strain was screened out. The fermentation supernatant contained 0.5 μg/ml LL-37 and had strong antibacterial activity against E. coli. Tricine-SDS-PAGE and Western-blot analysis confirmed that the product was LL-37. Conclusion: We have successfully constructed pPIC9-LL-37 for transfecting P. pastoris. Methanol can induce the high expression of LL-37 and the expressed LL-37 has strong antimicrobial activity.

Modification of 5′-UTR sequences of pPIC9 increases expression of antimicrobial peptide LL-37 / 第二军医大学学报

Objective: To study the influence of 5′-untranslated region modification of pPIC9 on expression of LL-37 in Pichia pastoris. Methods: The sequence GGATCCAA was deleted from 5′-UTR of pPIC9 and the modified product was transformed into E. coli DH5α to construct a modified eukaryotic vector pPIC9-EDIT. After PCR and sequencing, pPIC9-EDIT was ligated with LL-37 sequence coded by the biased codon of yeast, the product was then transformed into E. coli DH5α to construct the recombinant expression vector pPIC9-EDIT-LL-37, the latter was transformed into P. pastoris GS115 by spheroplasting and the insert was confirmed by PCR. The bacteriolytic activity to E. coli. DH5α was analyzed to screen the highest expressing strain and to determine the best inducing time and concentration of methanol. The fermentation product was analyzed by Tricine-SDS-PAGE and Western blotting. The antibacterial activities of expression products of pPIC9-LL-37 and pPIC9-EDIT-LL-37 were compared, and the changes of LL-37 protein expression were determined before and after modification. Results: pPIC9-EDIT and pPIC9-EDIT-LL-37 were successfully constructed. Expression of LL-37 gene was confirmed by PCR in P. pastoris after pPIC9-EDIT-LL-37 transformation. The highest expressing strain was identified; the best inducing time was 72 h and the best concentration of methanol was 0.5%. Tricine-SDS-PAGE and Western blotting analysis showed that the expression product was LL-37. The expression level of LL-37 protein increased by 35 times after modification. Conclusion: Modification of pPIC9 5′-UTR can obviously improve expression of LL-37 protein in P. pastoris; it is worth to be used in the research of other heterogenous protein.

Celecoxib induces apoptosis and inhibits angiogenesis in gastric cancer / 中华肿瘤杂志

<p><b>OBJECTIVE</b>The aim of this study was to explore the effect of celecoxib, a cyclooxygenase-2 inhibitor, on induction of apoptosis and inhibition of angiogenesis in gastric cancer.</p><p><b>METHODS</b>Fifty nine gastric cancer patients were randomly divided into 2 groups: celecoxib group (n = 37) and control group (n = 22). The patients in the celecoxib group were treated orally with celecoxib 200 mg twice daily for 7 days before resection. The patients in the control group received surgical resection alone. Another group of 20 healthy subjects were recruited as normal control. The number of apoptotic tumor cells was measured by terminal deoxynucleotidyl transferse-mediated dUTP nick end labeling (TUNEL). The expression of COX-2, VEGF and the microvessel density (MVD) were evaluated by immunohistochemistry.</p><p><b>RESULTS</b>The TUNEL results showed an increase of apoptosis in the tumor cells after celecoxib treatment in comparison with that in the control group (7.1% +/- 1.0% vs. 6.2% +/- 0.9%, P < 0.05). The expression level of COX-2 and VEGF in the gastric cancer tissues was significantly decreased in the celecoxib group compared with those in the control group (P < 0.05). Furthermore, MVD was also significantly lower in the celecoxib group when compared with that in the control group (30.48 +/- 5.02 vs. 38.98 +/- 4.58, P < 0.05).</p><p><b>CONCLUSION</b>Oral intake of celecoxib can induce apoptosis and suppress angiogenesis in gastric cancer. It may become an effective agent in the treatment of gastric cancer.</p>