肺炎链球菌蛋白PLYd和PsaA的制备与鉴定

发布时间：2018-06-08 21:02

本文选题：肺炎链球菌 + 肺炎链球菌脱毒溶血素　；参考：《中国新药杂志》2017年11期

【摘要】：目的:对2种肺炎链球菌蛋白肺炎链球菌脱毒溶血素(detoxified pneumolysin,PLYd)和肺炎链球菌表面黏附素(pneumococcal surface adhesin A,Psa A)进行全基因合成、重组表达、制备和鉴定。方法:根据Gen Bank中PLYd,Psa A的基因序列和氨基酸序列,通过密码子优化和全基因合成的方式获得2种蛋白的基因,在PLY基因中引入多位点突变保证表达的蛋白无毒性,构建无标签、高效重组表达菌株,建立2种重组蛋白的纯化制备方法,并进行抗原鉴定及免疫原性检测。结果:人工合成的PLYd,Psa A基因序列经鉴定与预期一致。2种蛋白在大肠杆菌中均获得了高效表达,表达量在40%以上,经2步纯化其纯度均达到95%。2种蛋白均能与阳性血清产生特异性抗原抗体反应,免疫小鼠后均可诱导产生较高水平的蛋白特异性抗体。结论:成功制备2种无标签肺炎链球菌蛋白PLYd,Psa A,且具备与天然抗原相似的抗原性和免疫原性,为开展应用研究奠定基础。
[Abstract]:Objective: to synthesize, express, prepare and identify two strains of Streptococcus pneumoniae protein detoxified pneumolysin plyd and pneumococcal surface adhesin PSAA. Methods: according to the gene sequence and amino acid sequence of PLYdSSA A in GenBank, two kinds of protein genes were obtained by codon optimization and gene synthesis. Two methods of purification and preparation of recombinant protein were established, and antigen identification and immunogenicity detection were carried out. Results: the synthetic PLYdfen Psa A gene sequence was confirmed to be in accordance with the expectation. 2 proteins were highly expressed in Escherichia coli, and the expression level was over 40%. After two steps of purification, the purity of 95. 2 proteins could react with the positive serum to produce specific antigens and antibodies, which could be induced to produce a higher level of protein specific antibodies after immunizing mice. Conclusion: two kinds of unlabeled streptococcus pneumoniae protein PLYdN Psa A were successfully prepared with antigenicity and immunogenicity similar to those of natural antigens, which laid the foundation for application research.
【作者单位】：中国食品药品检定研究院;
【基金】：国家高技术研究发展计划(863计划)(2012AA02A402)
【分类号】：R945

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