拟穴青蟹精氨酸激酶的重组表达及致敏性分析  

Recombinant Expression and Allergenicity Analysis of Arginine Kinase from Scylla paramamosain

作　　者：杨阳 何欣蓉[2] 何少贵 陈锦莉 刘萌 费丹霞 毛海燕 刘光明[2] YANG Yang;HE Xinrong;HE Shaogui;CHEN Jinli;LIU Meng;FEI Danxia;MAO Haiyan;LIU Guangming(College of Environment and Public Health,Xiamen Huaxia University,Xiamen 361024,China;College of Ocean Food and Biological Engineering,Jimei University,Xiamen 361021,China;Xiang An Biomedicine Laboratory,Xiamen 361104,China;College of Marine Biology,Xiamen Ocean Vocational College,Xiamen 361102,China)

机构地区：[1]厦门华厦学院环境与公共健康学院,福建厦门361024 [2]集美大学海洋食品与生物工程学院,福建厦门361021 [3]翔安创新实验室,福建厦门361104 [4]厦门海洋职业技术学院海洋生物学院,福建厦门361102

出　　处：《食品科学》2024年第7期19-27,共9页Food Science

基　　金：福建省自然科学基金面上项目(2023J011666);国家自然科学基金青年科学基金项目(31901811,32001695);国家自然科学基金面上项目(32072336)。

摘　　要：为比较拟穴青蟹(Scylla paramamosain)重组精氨酸激酶(recombinant arginine kinase,rAK)和天然AK(native AK,nAK)的致敏性,并鉴定AK分子中的致敏优势区域,首先基于AK的抗原表位分布与空间结构,将AK分子分为AK-E1(氨基酸(amino acids,AA)1~92)、AK-E2(AA 87~187)、AK-E3(AA 172~265)和AK-E4(AA 276~357)4个片段,采用大肠杆菌原核表达系统对其进行分段表达,并分别分离纯化nAK、rAK及AK的4个分段表达产物。用BALB/c小鼠模型评价重组蛋白的致敏性,结果显示,rAK致敏小鼠血清中特异性抗体水平、脾脏淋巴细胞的Th2型细胞因子释放水平均显著升高,表明rAK可使机体致敏,但其免疫原性较nAK弱;AK的4个分段表达产物中AK-E2的免疫原性最强。同时,rAK能够刺激RBL-2H3细胞释放β-己糖苷酶,但rAK对效应细胞的刺激作用低于nAK;4个分段表达产物中AK-E2和AK-E4对效应细胞的刺激作用较强。综上,通过原核表达系统获得的rAK免疫原性较nAK弱,而AK分子中免疫原性较强的区域为AA 87~187,免疫反应性较强的区域为AA 276~357。To compare the allergenicity of native arginine kinase(nAK)and recombinant AK(rAK)from Scylla paramamosain and to identify the predominant allergenic domain of AK,AK was divided into 4 fragments:AK-E1(amino acid(AA)1–92),AK-E2(AA 87–187),AK-E3(AA 172–265),and AK-E4(AA 276–357)based on the distribution of epitopes and the spatial structure of the AK molecule.The four recombinant fragments were expressed in the prokaryotic system Escherichia coli,and then nAK,rAK,and the recombinant fragments were purified.The allergenicity of recombinant proteins were evaluated using BALB/c mice.The results showed that the levels of specific antibodies in the serum and the secretion of Th2 type cytokines by the splenocytes of mice sensitized with rAK significantly increased,but the immunogenicity of rAK was weaker than that of nAK.Among the 4 fragments of AK,AK-E2 had the strongest immunogenicity.Meanwhile,rAK could stimulate RBL-2H3 cells to releaseβ-hexokinase,but it was less effective than nAK.Among the 4 expressed fragments,AK-E2 and AK-E4 had a stronger stimulating effect on effector cells.In conclusion,the rAK expressed in the prokaryotic system showed weaker immunogenicity than nAK,and among the 4 fragments of AK,AA 87–187 has the strongest immunogenicity while AA 276–357 has the strongest immunoreactivity.

关 键 词：拟穴青蟹 精氨酸激酶 重组蛋白 致敏性 BALB/C小鼠 

分 类 号：Q789[生物学—分子生物学]