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目的在大肠埃希菌中表达重组毒素rCCK8PE38,并检测其对结肠癌细胞的杀伤活性。方法采用分子生物学技术将反向翻译的8肽胆囊收缩素(cholecystokinin8,CCK8)与绿脓杆菌外毒素(pseudomonas exotoxin,PE)38基因融合,构建重组表达质粒pET-rCCK8PE38,转化大肠埃希菌BL21(DE3),IPTG诱导表达。通过细胞杀伤试验检测表达的重组毒素rCCK8PE38对结肠癌细胞、其他癌细胞系和一些正常细胞的杀伤活性。结果重组表达质粒pET-rCCK8PE38经双酶切证实构建正确;表达的重组蛋白相对分子质量约为40 000,表达量约占全菌总蛋白的40%;重组毒素rCCK8PE38对结肠癌细胞HCT-8有明显的杀伤效果,对其他癌细胞系和正常细胞无杀伤活性。结论成功在大肠埃希菌BL21(DE3)中表达了重组毒素rCCK8PE38,其可高效特异地识别并杀伤结肠癌细胞HCT-8。
Objective To express the recombinant toxin rCCK8PE38 in Escherichia coli and test its killing activity against colon cancer cells. Methods Reverse translating cholecystokinin 8 (CCK8) and pseudomonas exotoxin (PE) 38 gene were fused by using molecular biology technique to construct recombinant expression plasmid pET-rCCK8PE38 and transformed into Escherichia coli BL21 (DE3), IPTG induced expression. The cytotoxic activity of the expressed recombinant toxin rCCK8PE38 on colon cancer cells, other cancer cell lines and some normal cells was tested by cell killing assay. Results The recombinant plasmid pET-rCCK8PE38 was confirmed by double enzyme digestion. The relative molecular mass of the expressed recombinant protein was about 40 000 and the expression level was about 40% of the total bacterial total protein. The recombinant toxin rCCK8PE38 had a positive effect on colon cancer cell HCT-8 Clearly killing effect on other cancer cell lines and normal cells without killing activity. Conclusion The recombinant toxin rCCK8PE38 was successfully expressed in Escherichia coli BL21 (DE3), which can efficiently and specifically recognize and kill colon cancer cells HCT-8.