重组α-银环蛇毒素P22-A31的原核表达及功能分析

Expression and functional analysis of recombinant α-bungarotoxin (P22-A31)

α-银环蛇毒素在神经科学研究以及在临床和制药上有重要作用,为获得大量的α-银环蛇毒素,我们用已构建的pGEX-BgTX(P22-A31)质粒在大肠杆菌BL21(DE3)中表达,并用谷胱甘肽Sepharose FF纯化GST-α-银环蛇毒素融合蛋白,再用凝血酶切掉融合标签谷胱苷肽转移酶(Glutathione S-transferase,GST),得到了较纯的重组α-银环蛇毒素同工毒素,得率约为1.225mg/L。用重组α-银环蛇毒素制备多克隆抗体,经ELISA和Western杂交鉴定后可知重组α-银环蛇毒素与天然α-银环蛇毒素的抗原性一致。小鼠毒性试验表明,重组α-银环蛇毒素同工毒素的半致死剂量LD50为1.598mg/kg,约为天然α-银环蛇毒素的1/5。小鼠化学法镇痛试验显示,重组α-银环蛇毒素有一定的镇痛药效,1/4LD50剂量的镇痛百分率为55.2%,1/8LD50剂量的镇痛百分率为20.5%,在外周镇痛作用中呈一定的量效关系。

α-bungarotoxin plays very important role in neuroscience research, clinical application and the pharmaceutical industry. In order to acquire quantitites of α-bungarotoxin, we expressed GST-α-bungarotoxin fusion protein using constructed plasmid pGEX-BgTX （P22-A31） in E. coli BL21 （DE3） cell, purified GST-α-bungarotoxin using glutathione- Sepharose FF, cleavaged Glutathione S-transferase （GST） tag by thrombin in the column, and finally, we obtained recombinant α-bungarotoxin with yields of about 1.225 mg/L. In order to compare the immunogenicity of recombinant protein and crude α-bungarotoxin, we prepared multi-antibody using recombinant α-bungarotoxin. The results of both ELISA and Western blot showed that recombinant α-bungarotoxin has the same antigenicity as natural α-bungarotoxin. In vivo toxicity tests showed that the LD50 of recombinant α-bungarotoxin was 1.598 mg/kg, about 1/5 that of natural α-bungarotoxin. Analgesis percentages with doses of 1/4 LD50 and 1/8 LD50 were 55.2 % and 20.5 % respectively, indicating that recombinant α-bungarotoxin possesses analgesic efficiency [ Acta Zoologica Sinica 51 （6）： 1102- 1108, 2005 ].