肺炎链球菌蛋白PLYd和PsaA的制备与鉴定

Preparation and identification of recombinant Streptococcus pneumoniae proteins PLYd and PsaA

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。

Objective： To perform the gene synthesis, recombinant expression, preparation and identification of the two kinds of Streptococcus pneumoniae proteins PLYd and PsaA. Methods： The genes of PLYd and PsaA were obtained by chemical synthesis after codons optimization according to the gene sequences and amino acid sequences in the GenBank, which additionally ensuring the expression of the protein is non-toxic by the introduction of multiple points mutations in the PLY gene. The label-free and efficient recombinant expression strains were constructed by a series of molecular biological methods. The purification methods of the two recombinant proteins were established. And its antigen identification and immunogenicity testing were performed. Results： The chemically synthesized gene sequences were identified as the same with that of expectation. The two proteins had been highly expressed in E. coli, which account for above 40% of total bacterial proteins. After purification by two steps of chromatography respectively, the purity of them reached 95%. The two proteins could react with human serum of clinically diagnosed pneumonia, which indicated the proteins have good antigenicity. And high level of specific antibodies could be induced after immunization with them in mice. Conclusion： The recombinant Streptococcus pneumoniae proteins PLYd and PsaA are prepared successfully without label, which have the same antigenicity and immunogenicity with that of natural proteins. This research provides a foundation for further study and the application of PLYd and PsaA.