转化体qXY中外源基因表达重组蛋白PA的食用安全性评价

Food safety evaluation of recombinant protein PA expressed by exogenous genes in transformant-qXY

目的 从毒性和过敏性2个方面对转化体qXY中外源基因表达重组蛋白PA进行食用安全性评价。方法 对大肠杆菌表达重组蛋白PA和转化体qXY中植物提取蛋白PA进行等同性分析后,分别采用毒性蛋白数据库和过敏原数据库对PA蛋白氨基酸序列进行毒性和过敏性生物信息学分析,对重组PA蛋白进行模拟胃/肠液消化稳定性试验,并且采用限量法对重组蛋白PA进行小鼠急性经口毒性试验。结果 大肠杆菌表达重组蛋白PA和转化体qXY中植物表达蛋白PA的表观分子量均为55 kD,且二者均可与PA蛋白特异性抗体结合,二者均无翻译后糖基化修饰,飞行质谱分析重组PA蛋白N端1-10氨基酸残基与理论序列一致,因此认为二者具有等同性。毒性生物信息学分析未发现PA蛋白与毒性蛋白数据库中已知毒蛋白具有序列相似性,CD-1小鼠急性经口毒性试验结果显示,5 000 mg/kg·BW剂量下未产生毒性效应。过敏性生物信息学发现,PA蛋白与已知过敏原高峰淀粉酶A(Asp o 21)存在较高的序列同源性,但模拟胃/肠液消化稳定性试验结果显示,重组蛋白PA在模拟胃液和肠液中0~15 s内均迅速消化,属于极易消化,不具有潜在过敏性。结论 未发现转化体qXY中外源基因表达重组蛋白PA具有潜在毒性和过敏性,验证了该蛋白的食用安全性。

Objective To evaluate the food safety of recombinant protein PA expressed by exogenous genes in transformant-qXY from the toxicity and allergenicity. Methods After equivalence analysis of the recombinant protein PA expressed by Escherichia coli and PA protein extracted from transformant-qXY,bioinformatics comparison of protein sequences with known allergenic and toxic proteins was performed. The digestive stability test of the recombinant PA protein was simulated by gastric/intestinal fluid.Acute oral toxicity test of recombinant protein PA was performed in mice by limited dose method. Results The apparent molecular weight of the recombinant protein PA expressed in Escherichia coli and the protein PA in transformant-qXY was 55 kD and both proteins could bind to specific antibodies of the PA protein. There was no post-translational glycosylation modification in both of them. The 1-10 amino acid residues at the N-terminal of recombinant PA protein by flight mass spectrometry were consistent with the theoretical sequence,so they were considered to be isotropy. Bioinformatics analysis of toxicity showed no sequence similarity between PA protein and known toxic protein,and acute oral toxicity test of CD-1 mice showed no toxic effect at 5 000 mg/kg·BW dose. Allergenic bioinformatics found that PA protein had a high sequence homology with the known allergen Asp o 21,but the results of simulated gastric/intestinal fluid digestion stability test showed that the recombinant protein PA was rapidly digested in simulated gastric and intestinal fluid within 0-15 s,which was extremely easy to digest,so it was considered not potentially allergic. Conclusion The recombinant protein PA expressed by exogenous genes in the transformed qXY is not found to be potentially toxic and allergic,and the food safety of the protein is verified.