α-苦瓜素基因的克隆和原核表达

Cloning and Prokaryotic Expression of Alpha-momorcharin Gene

α-苦瓜素(Alpha-momorcharin)是一种多功能核糖体失活蛋白,具有抗肿瘤、抗HIV-1和抗菌活性,并同时具有免疫抑制活性。利用PCR(polymerase chain reaction)技术从苦瓜基因组中扩增出了成熟的α-苦瓜素蛋白基因,并亚克隆到表达载体pET28a(+)上,然后分别在BL21(DE3)和RosettaTM(DE3)pLysS中进行表达。结果表明只在RosettaTM(DE3)pLysS中,重组α-苦瓜素基因能成功地表达出33kD大小的重组蛋白,而且在18℃和22℃的温度下,经IPTG诱导后,能成功地表达出大量可溶性重组蛋白。利用重组蛋白的N末端带有的6×His标签,通过Ni-NTA柱纯化获得了α-苦瓜素重组蛋白。Western杂交实验表明,纯化后的重组蛋白可与鼠抗His-Tag单克隆抗体发生特异性反应。α-苦瓜素重组蛋白的获得为进一步系统研究和改造其功能与活性奠定了基础。

Momordicin （Alpha-momorcharin） is a kind of multifunctional ribosome inactivating protein （RIP） with anti-tumor, anti-bacterial, anti-HIV and immunosuppressive activity. In this study, the gene encoding mature Alpha-momorcharin protein was amplified from the genomic DNA of Momordica charantia L. by using PCR（ polymerase chain reaction） method. The amplified product was cloned into expression vector pET28a（ ＋ ） ,and then expressed in the BL21 （DE3） and Rosetta^TM （DE3）pLysS. Only in the Rosetta^TM （DE3）pLysS strain, the 33-kD-weight recombinant protein of Alpha-momorcharin was successfully expressed and the soluble recombinant protein was occurred when the strain was induced with IPTG at 18℃ and 22℃ temperature. The soluble recombinant protein was purified by the Ni-NTA resin and followed by Western-blotting analysis. This result revealed that the purified recombinant protein could be recognized by His-Tag antibody. It lays a foundation for further studies of the function and activity of the Alpha-momorcharin recombinant protein.