蜂毒PLA2原核表达及溶血作用研究  

Bee venom PLA2 Prokaryotic expression and hemolytic activity study

作　　者：刘苗苗 王兆宇 陈龙龙 吕建华[1] 赵慧婷[1] Liu Miaomiao;Wang Zhaoyu;Chen Longlong;LüJianhua;Zhao Huiting(College of Life Sciences,Shanxi Agricultural University,Jinzhong 030800,China;Institute of Chinese Materia Medica China Academy Of Chinese Medical Sciences,Beijing 100700,China)

机构地区：[1]山西农业大学生命科学学院,山西晋中030800 [2]中国中医科学院中药研究所,北京100700

出　　处：《山西农业大学学报（自然科学版）》2024年第2期63-70,共8页Journal of Shanxi Agricultural University(Natural Science Edition)

基　　金：山西省自然科学青年基金项目(201901D211356)。

摘　　要：[目的]蜂毒(Bee_Venom,BV)是天然的动物药,药理效应广泛,成分较为复杂。磷脂酶A2(phospholipase A2,PLA2)是蜂毒的重要活性成分和主要过敏原,可与蜂毒肽发挥协同作用,诱发溶血。本研究旨在通过原核系统表达并纯化出蜂毒磷脂酶A2(BvPLA2)蛋白,并通过溶血试验对比蜂毒(bee venom,BV)、蜂毒肽(melittin)以及BvP⁃LA2的溶血效果。[方法]将已构建好的pET28a-BvPLA2融合表达载体转化到大肠杆菌BL21菌株中并通过IPTG低温诱导蛋白表达,对表达产物进行鉴定并纯化获得重组BvPLA2蛋白。采集小鼠眼球全血与生理盐水等体积混合配制成2%的红细胞混悬液,加入不同浓度的BvPLA2蛋白液、蜂毒液及蜂毒肽,通过酶标仪测定溶血率。[结果]经IPTG诱导后的表达产物在约15 kDa的分子量处呈现清晰的条带,与理论推算值一致。超声破碎后在上清及沉淀中均表达出特异性条带,且上清中的表达量与沉淀表达量基本一致。不同浓度的BvPLA2蛋白液均具有溶血效果,溶血率均超过5%,且低浓度BvPLA2溶血率较高。经对比得出,BvPLA2溶血率显著低于蜂毒和蜂毒肽(P<0.05)。[结论]通过原核表达系统成功诱导并纯化出可溶性目标蛋白BvPLA2,且BvPLA2具有一定的溶血效果,但溶血效果显著低于蜂毒和蜂毒肽。[Objective]Bee venom is a natural animal medicine with broad pharmacological effects and complex composition.Phospholipase A2(PLA2)is an important active ingredient and major allergen of bee venom,which can synergize with melittin to induce hemolysis.This study aimed to express and purify BvPLA2 protein in prokaryotic system and compare the hemolytic effects of bee venom(BV),melittin,and BvPLA2 through hemolysis tests.[Method]The constructed pET28a-BvPLA2 fusion expression vector was transformed into E.coli BL21 strain,and the protein expression was induced by IPTG at low temperature.The expression product was identified and purified to obtain the recombinant BvPLA2 protein.Whole blood from mice was collected and mixed with physiological saline to prepare a 2%red blood cell suspension,and different concentrations of BvPLA2 protein solution,bee venom,and melittin were added.The hemolysis rate was determined by enzyme-linked immunosorbent assay.[Results]The expression product induced by IPTG showed a clear band at approximately 15 kDa,consistent with the theoretical calculation value.Specific bands were expressed in both the supernatant and sediment after ultrasonication,and the expression levels in the supernatant were basically consistent with those in the sediment.BvPLA2 protein solutions at different concentrations exhibited hemolytic effects,with hemolytic rates exceeding 5%,and the hemolysis rate was higher at low concentration of BvPLA2.By comparison,the hemolysis rate of BvPLA2 was significantly lower than that of bee venom and melittin(P<0.05).[Conclusion]Soluble target protein BvPLA2 was successfully induced and purified through the prokaryotic expression system,and BvPLA2 exhibited certain hemolytic effects,but the hemolytic effect was significantly lower than that of bee venom and melittin.

关 键 词：蜂毒 磷脂酶A2 原核表达 溶血性 

分 类 号：Q965.9[生物学—昆虫学]