Preparation and identification of recombinant Streptococcus pneumoniae proteins PLYd and PsaA

TAN Ya-jun; WANG Li-chan; WEI Chen; ZHANG Hua-jie; HOU Qi-ming; ZHANG Shu-min; MA Xiao

Abstract：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.