截短型rBTI的表达、纯化及其对EC9706细胞生长的抑制作用

依据先前获得的重组荞麦胰蛋白酶抑制剂（rBTI）氨基酸序列及三维分子构像，分别构建了rBTI C末端缺失VVM、TPVVM或VDTPVVM的截短型pExsecI BTI t1，pExsecI BTI t2和pExsecI BTI t3重组质粒.转入大肠杆菌BL21中进行表达，并通过Resource Q阴离子交换层析分离.实验结果显示，3个工程菌均以可溶方式表达，目的蛋白在SDS PAGE图谱中显示单一条带，其纯度达98％以上. 理化性质分析表明，截短型rBTI与野生型rBTI具有相似的胰蛋白酶抑制活性，并具有很好的热稳定性及酸碱稳定性. 将野生型和截短型rBTI分别作用于人食管癌EC9706细胞.MTT检测发现，C末端截短不同数目的氨基酸后,与野生型rBTI相比，在相同浓度下截短型rBTI仍具有一定的抑制肿瘤细胞生长作用，其抑制作用范围是截短前的50%左右. 这些结果提示, rBTI的 C末端氨基酸残基缺失未引起活性区域或功能部位的较大改变，从而保留了其对胰蛋白酶的抑制作用和部分生物学功能.

Expression,Purification of Truncated rBTI and Its Inhibitory Effect on EC9706 Cell Proliferation

On the basis of the amino acid sequence analysis and 3 D structure of the recombinant buckwheat trypsin inhibitor (rBTI), pExsecI BTI t1，pExsecI BTI t2 and pExsecI BTI t3 recombinant plasmids were constructed by deleting VVM，TPVVM or VDTPVVM in the C–terminal region of rBTI， respectively. The constructed vectors were transformed into E. coli BL21 (DE3).The fusion protein was purified by anion exchange chromatography on Resource Q column. The purity was above 98%. Assayed of the inhibitory activity to trypsin suggested that truncated rBTI was similar to wild type rBTI in inhibitory activity. Truncated rBTI showed remarkable stability to heat and pH. The possible effects of wild type rBTI and truncated rBTI on the proliferation of human EC9706 cell line were detected by MTT assay.The results showed that truncated rBTI still could inhibit the growth of EC9706 cells compared with wild type rBTI at the same concentration. And the inhibitory activity of truncated rBTI was about 50% of wild type rBTI. These results implied that the absence of part amino acids of C terminal in rBTI did not influence its activity, and the truncated rBTI still reserved inhibitory activity to trypsin and some biological functions.