Cloning, Expression And Identification Of Surface Antigen SAG4 Of Toxoplasma Gondii

Objective.To analyze the features and predict the vaccination potential of the protein encoded by SAG4 gene,to constructe recombinant expression plasmid pET28a(+)-SAG4,to express the recombinant protein in E.coli and investigate its immunoreactivity in order to lay the foundation of immunization diagnosis and vaccine development for toxoplasmosis.Methods:Genomic DNA of T.gondii strain RH was extracted,and specific primers of SAG4 were designed according to the DNA sequence of T.gondii strain RH released in GenBank.These primers were used to amplify the SAG4 from the genomic DNA of T.gondii strain RH by PCR.After identification with sequencing and purification,the PCR fragment was cloned into pMD19-T in order to constructe recombinant cloning plasmid pMD19-T-SAG4.Then,the target fragment which was double digested from pMD19-T-SAG4 with Nco I and Xho I,was subcloned into pET28a(+).The recombinant expression plasmid pET28a(+)-SAG4 was constructed and also identified by double digesting and sequencing.Sequence alignment,prediction of secondary structure and epitope location for the coding sequence of recombinant SAG4 were performed by bioinformatics software.The recombinant SAG4 gene was finally expressed in E.coli BL21 after IPTG induction,and its immunoreactivity was then analyzed by Western blotting.Results:The target gene was amplified with the length of 537 bp,and the BLAST analysis showed that the sequence is highly homologous to SAG4 of T.gondii strain RH,and lower to other species.According to double digested reaction and sequencing analysis,SAG4 gene had been properly connected into the recombinant cloning plasmid pMD19-T-SAG4 and recombinant expression plasmid pET28a(+)-SAG4.BLAST and hydrophobicity analysis showed that the recombinant could code for SAG4 as a membrane protein in T.gondii RH strain;a number region of random coil andβ-turn could be formed by prediction of secondary structure;aggregate analysis of hydrophilicity,accessibility, flexibility,polarity parameters and epitope forecast that,the higher antigenicity region were near the site of 40th,80th,120th,130th amino acid.After IPTG inducing,the recombinant SAG4 was expressed in an inclusion body form in E.coli BL21.A specific protein band at 18.74 ku position was detected by SDS-PAGE,and could be also recognized after reaction with serum from mice which were chronically infected by T.gondii RH strain.Conclusions:SAG4 genes of T.gondii RH strain were cloned and identified. Recombinant cloning plasmid pMD19-T-SAG4 and recombinant expression plasmid pET28a(+)-SAG4 were successfully constructed,and also be analyzed by bioinformatics software with the results of significant antigenicity.The recombinant SAG4 was expressed highly in an inclusion body form in E.coli BL21,and displayed specific immunoreactivity.Recombinant SAG4 may be used as a potential vaccine candidate of T.gondii and a novel diagnostic antigen to detect chronic T.gondii infection.