| Toxoplasma gondii(T.gongdii),an intracellular Apicomplexan protozoan,can infect almost all mammals and birds.A large number of studies have shown that the virulence of different T.gondii strains varies.At present,the classification of T.gondii mainly relies on molecular biological methods.But serological typing is a more convenient and rapid method for the detection of large-scale T.gondii,and the previous studies showed that three clonal strain types(I,II,and III)of T.gondii can be distinguished using serotyping based on a series of polymorphic proteins.However,to establish a systematic serotyping method that can distinguish T.gondii strains belonging to the same type,more specific peptide markers derived from polymorphic proteins are needed.The objective of the present study was to determine the possibility of the polymorphic dense granule protein 15(GRA15)for T.gondii serotyping.Firstly,the amino acid sequences of GRA15 of T.gondii GT1,PRU and RH strains were retrieved from Toxo DB database,and sequence alignment was performed..Then,the amino acid sequences of T.gondii GT1 strain GRA15 was analyzed by bioinformatics analysis software to find out the peptides that are most likely to have good antigenicity.According to the amino acid sequence corresponding to the selected peptide,three pairs of specific primers with restriction sites were designed.Three different T.gondii GT1 strain GRA15 gene fragments encoding a 584-residue peptide,a 199-residue peptide and a84-residue peptide were amplified by PCR and were ligated into the cloning vector(p MD19T),respectively.The amplified gene fragments were digested by Bam HⅠ and Eco RⅠ respectively,and then cloned into prokaryotic expression vector.The expression of GRA15584,GRA15199 and GRA1584 was induced by IPTG.Then,the proteins were purified by chromatography column with Protein Iso? GST Resin and Protein Iso?Ni-NTA Resin respectively.Western blot was used to analyze the reaction of anti-T.gondii GT1 strain antibodies,anti-T.gondii RH strain antibodies and anti-T.gondii PRU strain antibodies with these three proteins,respectively.The results showed that GRA15584 protein might be a potential candidate protein for serological diagnosis of T.gondii.RH strain from GT1 strain could be distinguished by the GRA15199 or GRA1584,and T.gondii GT1 strain could be distinguished from PRU strain by the GRA1584.When there is on information about the serum to be examined,RH strain infection and non-RH strain infection can be distinguished by the GRA15199.To sum up,this study revealed,for the first time,the potential role of GRA15 derived peptides in the diagnosis and serological identification of T.gondii infection.This findings provides new specific peptide markers for establishing a systematic serotyping method with higher resolution even being equal to that of genotyping. |
