ETAR-C58多肽生物合成及其对黑色素瘤A375细胞迁移和侵袭的影响

Biosynthesis of ETAR-C58 peptide and its effects on migration and invasion of melanoma A375 cells

目的获得ETAR羧基末端58个氨基酸的多肽(ETAR-C58),探讨其对黑色素瘤A375细胞迁移和侵袭的影响。方法通过PCR方法获得ETAR-C58碱基序列,将其克隆到原核表达载体pWaldoGFPe,转化到大肠杆菌BL21(DE3),诱导表达后,经镍柱亲和层析和分子筛层析,得到重组融合多肽,SDSPAGE分析多肽表达量和纯度,免疫印迹做进一步的鉴定。最后采用Transwell法测定A375细胞的迁移和侵袭能力。结果成功构建了重组表达载体pWaldo-ETAR-C58,优化表达条件获得了约90%的可溶性蛋白表达量,纯化获得纯度高达95%的融合多肽,免疫印迹的曝光条带和重组多肽的大小相一致,加入2μmol/L ETAR-C58后,A375细胞的迁移和侵袭能力显著下降(P <0.05)。结论 ETAR-C58多肽具有抗黑色素瘤的活性,为下一步的临床应用奠定了良好的基础。

Objective To obtain the 58 amino acid peptide of the carboxyl terminal of ETAR(ETARC58)and investigate the effects on the migration and invasion of melanoma A375 cells. Methods ETAR-C58 sequence was amplified by PCR,inserted into the vector pWaldo-GFPe and then transformed into Escherichia coli BL21(DE3). After expression,the fusion peptide was purified by nickel column affinity chromatography and gel filtration chromatography,analyzed by SDS-PAGE and further identified by immunoblotting. Finally,Transwell assay was carried out to measure the migration and invasion of A375 cells. Results The recombinant expression vector pWaldo-ETAR-C58 was successfully constructed and the soluble fusion peptide was accounted for approximately 90%. SDS-PAGE and immunoblotting results verified the successful purification of the ETAR-C58 with nearly95% purity. ETAR-C58 peptide could inhibit A375 cell migration and invasion at 2 μmol/L. Conclusion The ETAR-C58 peptide have anti-melanoma activity,which allows a possibility for further clinical application.