重组HIV-TATm-Survivin(T34A)蛋白制备及其促癌细胞凋亡活性研究

从人乳腺癌细胞系B-Cap-37中克隆出Survivin的cDNA,并对其中编码34位Thr的密码子定点突变为Ala的密码子后,经一系列基因操作方法将天然HIV-TAT转导肽的突变体基因HIV-TATm引入Survivin(T34A)基因的5′端,正确构建了表达载体pRSET-B-HIV-TATm-Survivin(T34A),经转化E.coliBL21(DE3)后诱导表达,目的蛋白质以包涵体形式表达,占菌体总蛋白的45%。经3.7L发酵罐分批发酵可收获650mg/L的包涵体,经阳离子交换、凝胶层析分离和柱上复性与透析,得到纯度达96%以上的可溶性目的蛋白HIV-TATm-Survivin(T34A)。目的蛋白对人乳腺癌细胞B-Cap-37、人胰腺癌细胞SW1990和人肝癌细胞SSMC-7721作用4h后有在细胞形态上呈现出显著的凋亡特征,而对人宫颈癌细胞系Hela不敏感,对正常的人内皮细胞系EVC-304未见明显影响。作用24h时MTT法检测显示,120μg/mL目的蛋白对SW1990、B-Cap-37、SSMC-7721和Hela细胞的抑制率分别达到89%、63%、59.5%和39%,且具有剂量依赖性。对最为敏感的SW1990和B-Cap-37以每孔60μg/mL终浓度的目的蛋白作用48h的流式细胞技术检测发现,凋亡率分别为25.6%和19.3%,与对照相比,实验组细胞出现明显的凋亡峰,细胞周期的分布发生明显的变化,65%以上的癌细胞被阻滞于G1期。体外实验结果显示重组目的蛋白具有较强的抑制癌细胞增殖和促进凋亡作用,预示着良好的抗癌前景。

Preparation of Recombinant HIV-TATm-survivin (T34A) Protein and its Pro-apoptosis Activity to Cancer Cells in vitro

As a novel member of the IAP (Inhibitor of apoptosis protein) family, survivin was observed to be expressed in most human cancerous cells. Fusion protein TATm-survivin (T34A) has drawn considerable attention because it is a potential anti-tumor protein that can be transduced into cancer cell with the help of HIV-TAT domain. In this study, the cDNA encoding survivin was cloned by RT-PCR from human breast cancer cell lines B-Cap-37. An expression vector of pRSET-B-HIV-tatm-survivin (T34A) was constructed by PCR after survivin (T34A) was mutated by site-directed mutagenesis. Subsequently, the resultant plasmid was transformed into E. coli BL21 (DE3). Recombinant HIV-TATm-Survivin (T34A) protein was expressed efficiently with 0.5mM IPTG as inducer, reaching a yield of 650mg/liter (as inclusion body) in fermentation culture. The inclusion bodies were solubilized, refolded and purified to a purity of 96% by ion exchange chromatograghy and size-exclusion chromatography. Remarkable effects of the purified recombinant HIV-TATm-Survivin (T34A) on the morphology of cell line SW1990 and B-Cap-37 were observed after being administrated for 4h. MTT assay showed recombinant HIV-TATm-survivin (T34A) protein could inhibit significantly cell proliferation of SW1990 and B-Cap-37 and SSMC-7721 in vitro. Apoptosis rate and cell circle of SW1990 and B-Cap-37 that had been treated with target protein (final concentration 30 microg/mL) were detected with flow cytometry. Results revealed that more than 65% cancer cells were arrested at G1 phase. The study suggested that TATm-survivin (T34A) protein was a hopeful protein drug in the treatment of cancers by facilitating apoptosis of cancer cells. Key words recombinant HIV-TATm-Survivin (T34A), expression and purification, pro-apoptosis bioactivity, SW1990 and B-Cap-37 cancer cell lines