华游蛇PLIγ的毕赤酵母表达及其抗蛇毒sPLA_2活性

Expression of Sinonatrix annularis PLIγin Pichia pastoris and its Application in Neutralization of Snake Venom sPLA_2s

抗蛇毒血清作为抗蛇伤中毒特效药,其生产和临床应用上的局限导致很多伤者得不到及时救治。PLI(phospholipase A_2 inhibitors)是蛇自身表达的解毒蛋白,它可以抑制蛇毒主要毒素PLA_2进行自我保护。我们实验室前期在华游蛇中分离到一个PLIγ,它对蛇毒PLA_2酶活性和出血毒均有强抑制效果。本研究通过酵母对华游蛇PLIγ进行表达。将PLIγ连接到pPICZαA表达载体,重组质粒通过电转化方式导入毕赤酵母GS115。在30℃、250r·min-1条件下,使用终浓度0.5%甲醇对筛选的转化子进行诱导,连续培养120h,每隔24h收集发酵液上清,用Western Bolt检测PLIγ表达水平,确定72h为最佳诱导时间。采用镍柱亲和层析分离纯化PLIγ,SDSPAGE检测纯度。活性实验表明:重组PLIγ对五步蛇蛇毒PLA2酶活的抑制效率为84%,并可以有效抑制其出血毒性。体外免疫共沉淀表明重组PLIγ与蛇毒sPLA_2IB和IIA型均存在直接相互作用。

Anti-venom is the sole specific drug for treatment of snakebite envenomation. However, the limitations in production and clinical application hamper their timely administration on patients. Phospholipase A2 inhibitors （PLI） are antitoxic proteins that commonly expressed in liver of snake, exert self-protection by blocking PLA2s,the main toxin of snake venom. We had demonstratedthat the PLIγ,which we isolated from serum ,was highly effective to attenuate enzymatic activity and hemorrhage toxicity of snake venom PLA2. This study described the eukaryotic expression of PLIγ in yeast. The PLIγ gene was cloned and linked to vector pPICZ2A. The recombinant plasmid was introduced in Pichia pastoris GS115 by electroporation. Positive yeast clones were cultivated at 30℃ and 250prm for 120h,and subjected to 0.5% methanothel of final concentration to induce PLI7 expression. Fermentation broth was collected at 24 hours interval for the detection of PLI7 level using western blot. As a result, 72h was consideredasthe optimum inducement time. The His6 tagged PLIγ was purified on Ni-NTA column with imidazole elution,and examined by SDS-PAGE. The recombinant PLIγ showed a good inhibition to D. acutus venom PLA2 ＇s enzymatic activity at a rate of 84% and also significant relieve to hemorrhage symptom of the crude venom. In addition,the PLIγ interacts directly with both Ⅰ B and Ⅱ A venom PLA2s using eo-immunoprecipitation in vitro.