CAJ | 학술논문

目的通过构建大肠杆菌表达系统获得重组人三叶因子2（hTFF2），并研究其对结肠癌细胞SW480的迁移活性和失巢增殖活性作用。方法通过RT-PCR获得hTFF2的cDNA，构建重组载体pET32a-hTFF2，转化大肠杆菌BL21-32a中诱导表达TrxA-hTFF2融合蛋白。产物经镍-亲和层析分离、透析纯化和Western-blot鉴定后，用48孔趋化小室和琼脂软胶法检测重组hTFF2的细胞迁移活性和失巢增殖作用。结果通过原核表达系统，获得高纯度的表达量达到220 mg/L的TrxA-hTFF2重组融合蛋白，重组hTFF2可促进结肠癌细胞SW480的迁移活性和在琼脂软胶中生长形成克隆。结论大肠杆菌可表达高产量和纯度的重组hTFF2，其细胞活性提示结肠癌中TFF2的表达可能促进结肠癌细胞浸润和转移。
목적통과구건대장간균표체계통획득중조인삼협인자2（hTFF2），병연구기대결장암세포SW480적천이활성화실소증식활성작용。방법통과RT-PCR획득hTFF2적cDNA，구건중조재체pET32a-hTFF2，전화대장간균BL21-32a중유도표체TrxA-hTFF2융합단백。산물경얼-친화층석분리、투석순화화Western-blot감정후，용48공추화소실화경지연효법검측중조hTFF2적세포천이활성화실소증식작용。결과통과원핵표체계통，획득고순도적표체량체도220 mg/L적TrxA-hTFF2중조융합단백，중조hTFF2가촉진결장암세포SW480적천이활성화재경지연효중생장형성극륭。결론대장간균가표체고산량화순도적중조hTFF2，기세포활성제시결장암중TFF2적표체가능촉진결장암세포침윤화전이。
Objective To over-express human trefoil factor 2 (hTFF2) by Escherichia coli system and an-alyze its activities in promoting migration and anchorage-independent growth in SW480 colonic cancer cells. Meth-ods hTFF2 gene encoding mature peptide was obtained by RT-PCR, and the recombinant expression vector pET32a-hTFF2 was constructed. Then pET32a-hTFF2 was transformed into E. coli BL21-32a and TrxA-hTFF2 fu-sion protein was induced to over-express. The expressed product was isolated by Ni-NTA affinity chromatography, purified by dialysis and identified by Western blotting. The activities of the recombinant hTFF2 in promoting SW480 cells migration and anchorage-independent growth were analyzed by MicroChemotaxis Chamber migration assay and Soft-agar assay,respectively. Results The TrxA-hTFF2 fusion protein was expressed to 220 mg/L at high purity. In vitro model demonstrated that recombinant hTFF2 obviously enhanced SW480 cell migration activity and anchor-age-independent growth. Conclusion The recombinant hTFF2 can be expressed in E. coli with high production, purity and biological activities. And its roles in cell migration and anchorage-independent growth suggest that up-regulation of TFF2 in colonic cancer might be involved in cancer invasion and metastases.