Effects of epigallocatechin-3-gallate on the proliferation of colonic cancer cell line SW620 and PAK1 gene expression / 南方医科大学学报

<p><b>OBJECTIVE</b>To investigate the effects of epigallocatechin-3-gallate (EGCG) on the proliferation of SW620 cells and the expression of PAK1 gene.</p><p><b>METHODS</b>Human colonic cancer cell line SW620 was treated with EGCG at 40, 60 and 80 micromol/L and cultured in RPMI 1640 medium for 0, 24, 48 and 72 h. The proliferation of SW620 cells was observed by MTT assay before and after EGCG treatment, and the expression of PAK1 protein was observed by Western blotting.</p><p><b>RESULTS</b>SW620 cells treated with EGCG displayed a slowed growth in comparison with the control cells, and the growth rate decreased with the increase of EGCG concentration. PAK1 protein expression was lowered in SW620 cells after EGCG treatment for 48 h.</p><p><b>CONCLUSION</b>EGCG can inhibit the proliferation and partially reduce the expression of PAK1 protein in SW620 cells.</p>

PCR-based assembly of the DNA sequence coding for tetanus toxin C fragment / 南方医科大学学报

<p><b>OBJECTIVE</b>To develop a PCR-based method for gene assembly of tetanus toxin C fragment (TETC) DNA sequence from a large number of oligodeoxyribonucleotides (oligos).</p><p><b>METHODS</b>To allow for its cloning and expression in Lactococcus lactis, the TETC gene sequence was designed according to the known TETC gene sequence (GenBank accession number M12739, 367-1719) and the amino acid coding in Lactococcus lactis. The sequence contained 1383 nucleotides (nt) with Sal I site added to its 5' end and Xho I and Hind III sites to its 3' end. There were 209 synonymous codon substitutions in the designed gene sequence as compared with the sequence reported in GenBank for amino acid coding in Lactococcus lactis and elimination of the restriction site of EcoR I and Kpn I. The 1380 nt of the sequence was divided into 68 oligos designated as TETC 1 to TETC 68, each containing 40 nt. A 16 nt oligos designated as TETC 69 was designed as the downstream primer. The TETC 1-24 fragment was acquired using the oligos TETC 1 to TETC 24 by PCR-based gene assembly method, and the TETC 23-46 and TETC45-68 fragments were assembled similarly. The full-length TETC gene was assembled using TETC 1 and TETC 69 as the primers when the 3 fragments were mixed. The target gene was gel-purified and digested with Sal I and Hind III, followed by ligation to the pBluescript II SK(+) and digestion with the same enzymes. The positive clones were confirmed by restriction enzyme excision and sequencing.</p><p><b>RESULTS</b>Three 500-bp fragments were acquired by PCR-based gene assembly, and the full-length TETC gene was obtained from the 3 fragment mixed at a equal concentration by a second PCR-based gene assembly using TETC 1 and TETC 69 as the primers. The target gene was cloned to pBluescript II SK(+) vector, and sequence analysis of the positive clones indicated that the assembled sequence was identical to the designed coding sequence of TETC gene.</p><p><b>CONCLUSION</b>PCR-based assembly of the synthesized constitutive gene fragments into the complete sequence can be an effective strategy for synthesis of long DNA sequences in vitro.</p>

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>

Construction of Lactococcus lactis expression vector of recombinant human trefoil factor family 2 / 南方医科大学学报

<p><b>OBJECTIVE</b>To construct a Lactococcus lactis expression vector of c-myc-tagged human trefoil factor family 2 (hTFF2) fusion gene to prepare for genetic modification of Lactococcus lactis that can secrete bioactive c-myc-hTFF2 protein.</p><p><b>METHODS</b>Based on the amino sequence of hTFF2 and optimal Lactococcus lactis codon usage, the cDNA of hTFF2 was designed and extended at their 5' ends with a sequence encoding c-myc as the molecular tag. According to the restriction sites of pBluescript II sk (+), the SalI and BamHI sites were arranged at the 5' and 3' ends of the fusion gene respectively. The sequence of the fusion gene c-myc-hTFF2 was designed as 14 oligonucleotides that overlapped with each other, and by means of PCR, all the oligonucleotides were spliced to complete the construction of c-myc-hTFF2 fusion gene. The target gene of c-myc-hTFF2 was inserted into pBluescript II sk (+) to construct the cloning vector pBS-hTFF2 of c-myc-hTFF2 followed by verification by enzyme digestion and DNA sequencing. By digestion of pBS-TFF2 with BamHI/SalI and of pNBC1000 with BamHI/XhoI, we connected c-myc-hTFF2 with pNBC1000 to construct the expression vector c-myc-hTFF2 in E. coli named as pNTFF2. After digestion of pNTFF2 and pTRKH2 with XbaI, the target gene was subcloned into pTRKH2 and the construction of the expression vector pTRTFF2 in Lactococcus lactis was completed. The constructed vector was identified by restriction enzyme digestion.</p><p><b>RESULTS AND CONCLUSION</b>The expression vector pTRTFF2 of c-myc-hTFF2 fusion gene has been successfully constructed. Assembly of oligonucleotides in vitro is an effective means to synthesize the target fusion gene and this prepares the ground for constructing engineered bacterium of Lactococcus lactis.</p>