Résumé
Objective To study the expression of human platelet factor 4 (hPF4)mRNA and its relationship with angiogenesis and lymph node metastasis in papillary throid carcinoma(PTC). Methods RT-PCR was used to detect the expression of hPF4 mRNA in 40 cases of PTC and 10 cases of normal tissue surrounding carcinoma. Immunohistochemical stain was performed to detect microvessel density(MVD) whose endothelia were marked by antibody CD105. Results The positive-expression rate of hPF4 mRNA was 80 % respectively in normal tissue surrounding carcinoma. The positive-expression rate of hPF4 was 47.5 % in PTC cases. The MVD value of PTC cases with hPF4 mRNA positive expression was significantly lower than that of cases without hPF4 mRNA expression (P
Résumé
AIM:To synthesize the human platelet factor-4(hPF4) gene with a convenient and effective approach, and high express the hPF4 gene in E. coli BL21 (DE3). METHODS: According to the primary structure of hPF4, the nucleotide sequence was synthesized using touch-down PCR method. The resultant gene fragment containing EcoR Ⅰ and Xho Ⅰ overhangs at 5' and 3' ends was cloned into the expression vector pGEX-4T-1 to construct the recombinant plasmid pGEX-4T-1-hPF4,which was then transformed into the E. coli strain BL21 (DE3). RESULTS: hPF4 gene was successfully synthesized by touchdown PCR method. A fusion protein composed of glutathione S-transferase (GST) and the recombinant hPF4 was expressed in BL21(DE3) by IPTG induction. The expression level of the fusion protein in E. coli was about 30% of the total cellular protein. CONCLUSION: Touch-down PCR may provide a convenient and effective approach to obtain other target genes. The expressed fusion protein forms the inclusion bodies, providing sufficient material for further purification and biological activities process.