Construction and identification of recombinant Lactococcus lactis highly expressing human granulocyte-macrophage colony stimulating factor / 南方医科大学学报

<p><b>OBJECTIVE</b>To transfer human granulocyte-macrophage colony stimulating factor (hGM-CSF) gene into Actococcus lactis and obtain recombinant Lactococcus lactis highly expressing hGM-CSF (LL-CSF).</p><p><b>METHODS</b>The optimized hGM-CSF gene sequence capable of expression in Lactococcus lactis was cloned into the vector pNBC1000, which contained P59 promoter, RBS, MCS, USP45 signal peptide and USP45 stop codon, to generate the recombinant plasmid pNCSF. pNCSF was subcloned into a shuttle vector pTR1001c to acquire the plasmid pTRCSF, which was transferred into Lactococcus lactis to obtain LL-CSF by means of electroporation. SDS-PAGE was used to verify the expression of hGM-CSF protein by the constructed LL-CSF.</p><p><b>RESULTS</b>DNA sequencing and restriction enzyme digestion indicated the successful construction of the recombinant plasmid pNCSF, pTRCSF and the recombinant bacterium LL-CSF that was capable of steady and efficient expression of hGM-CSF as shown by SDS-PAGE.</p><p><b>CONCLUSION</b>The recombinant Lactococcus lactis LL-CSF has been successfully constructed, which can be valuable for studying the biological activity of recombinant hGM-CSF and for evaluating the potential clinical application of the protein.</p>

Short hairpin RNA-mediated survivin gene silencing inhibits invasion and metastasis of human colon carcinoma cell line SW480 in vitro / 南方医科大学学报

<p><b>OBJECTIVE</b>To investigate the effect of short hairpin RNA (shRNA) targeting survivin on adhesion and invasion of human colon carcinoma cell line SW480 in vitro.</p><p><b>METHODS</b>According to the sequence of the coding region of survivin gene, two strings of 19 nucleotides of inverted sequence flanking the loop sequence of two complementary 9-base oligonucleotides were designed and synthesized to prepare the hairpin construct as the DNA templates for the target shRNA. The shRNA templates were cloned into shRNA expression vector pRNAT-U6.1/Neo, and the resulted vector pRNAT-U6.1/Neo-survivin was transfected into SW480 cells using Lipofectamine 2000. Western blotting was performed to evaluate survivin gene silencing induced by shRNA transfection at the protein level, and the biological behaviors of the SW480 cells were investigated by cell-matrix adhesion, invasion and gelatin-zymography assays.</p><p><b>RESULTS</b>Western blotting revealed significantly lowered survivin protein expression in transfected SW480 cells, and survivin gene silencing induced by shRNA significantly suppressed the metastatic potential of SW480 cells in association with suppressed MMPs activity.</p><p><b>CONCLUSIONS</b>Survivin may play an important role in modulating human colorectal carcinoma cell invasion and metastasis, and survivin gene silencing can inhibit human colorectal cancer cell invasion and the production of MMP-2 and MMP-9. Survivin may affect invasion and metastasis of human colorectal carcinoma cells via regulating the production of MMPs.</p>