蜱抗凝血肽与RGDS肽重组基因在毕赤酵母中的表达及活性分析

目的利用毕赤酵母表达系统进行蜱抗凝血肽-RGDS肽双功能分子重组基因的真核表达研究。方法首先将蜱抗凝血肽-RGDS肽双功能分子的重组基因的表达质粒pPIC9K/TAP经限制性内切酶SalⅠ酶切线性化后,电转化酵母菌株GS115感受态细胞,并经MD平板与MM平板、G418抗性筛选,PCR鉴定,得到重组酵母工程菌GS115/pPIC9K-TAP,经甲醇诱导分泌表达并对表达产物进行初步鉴定。结果蜱抗凝血肽-RGDS肽双功能分子重组基因在毕赤酵母中获得成功表达,表达产物有抗凝血因子活性和抗血小板的作用。结论蜱抗凝血肽-RGDS肽双功能分子重组基因在毕赤酵母中获得分泌性表达,表达产物显示抗凝血因子活性和抗血小板的作用。

Expression of recombined gene of tick anticoagulant peptide and RGDS peptide in yeast Pichia pastoris

To express the recombined gene of tick anticoagulant peptide and RGDS peptide in yeast Pichia pastoris.The reconstructed expression plasmids pPIC9K/TAP of tick anticoagulant peptide and RGDS peptide gene were linearized by the digestion of restriction enzyme SalⅠ,and transformed into the susceptible yeast Pichia pastoris GSl15 cell by using electroporation transformation method.The reconstructed yeast engineering yeast strains GS 115/pPIC9K-TAP were identified by MD and MM and G418 plate selecting and PCR.Meanwhile the induced protein was analyzed by SDS-PAGE,and was determinded by blood clotting experiment and the platelet aggregation test.It was demonstrated that the recombined gene of tick anticoagulant peptide and RGDS peptide was expressed exactly.The expressed product possessed anticoagulant activity.It is evident that the recombined gene of tick anticoagulant peptide and RGDS peptide can be expressed in the yeast Pichia pastoris,and the product is active in the test of blood clotting experiment and the platelet aggregation test.