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目的构建TAT-Apoptin的原核表达载体,表达并纯化凋亡素蛋白,检测其对多种肿瘤细胞的抑制作用。方法利用PCR合成TAT-Apoptin序列,并将序列连接到pET-28b(+)原核表达载体的多克隆位点上,构建pET-28b-Apoptin载体,然后将重组载体转入E.coli BL21宿主菌,IPTG诱导表达,利用Ni-NTA亲和柱进行纯化获得目的蛋白。将TAT-Apoptin加入到肿瘤细胞中,用CCK8法检测TAT-Apoptin对肿瘤细胞的抑制作用。结果成功构建pET-28b-Apoptin质粒,IPTG诱导宿主菌表达TAT-Apoptin,经SDS-PAGE分析,在15×10~3Mr处发现目的蛋白条带,与预期一致。镜检、CCK8法检测发现纯化的TAT-Apoptin对肿瘤细胞A549、MDA-MB-231、SKB-R3均具有一定的促凋亡作用。结论 TAT-Apoptin原核表达载体构建完成,表达的TAT-Apoptin对肿瘤细胞生长有促凋亡作用。
Objective To construct prokaryotic expression vector of TAT-Apoptin, express and purify apoptin protein and test its inhibitory effect on various tumor cells. Methods The TAT-Apoptin sequence was synthesized by PCR and ligated into the multiple cloning site of prokaryotic expression vector pET-28b (+) to construct pET-28b-Apoptin vector. The recombinant vector was then transformed into E. coli BL21 , IPTG induced expression, the use of Ni-NTA affinity column purification to obtain the desired protein. TAT-Apoptin was added to tumor cells, and the inhibitory effect of TAT-Apoptin on tumor cells was detected by CCK8 assay. Results The plasmid pET-28b-Apoptin was successfully constructed. IPTG induced the expression of TAT-Apoptin in host bacteria. SDS-PAGE analysis showed that the target protein band was found at 15 × 10 ~ 3Mr. Microscopically, CCK8 assay showed that purified TAT-Apoptin had certain apoptosis-inducing effect on tumor cells A549, MDA-MB-231 and SKB-R3. Conclusion The prokaryotic expression vector of TAT-Apoptin is constructed. The expressed TAT-Apoptin can promote the growth of tumor cells.