Cloning, Expression, Purification And Identification Of Surface Antigen SAG3, SAG1 Of Toxoplasma Gondii

Objective: To obtain Toxoplasma gondii SAG3 and SAG1 gene by PCR, and construct recombinant prokaryotic expression plasmids pET29a(+)-SAG3 and pET29a(+)-SAG1. To express the recombinant proteins in Escherichia coli,respectively. Using Ni-Resin HP separated and purified recombinant proteins SAG3 and SAG1,and identify theirs antigenicities,in order to lay the foundation of vaccine for toxoplasmosis.Methods: To collecte trophozoites of T.gondii RH strain from mice ascites, with a small amount of tissue/cell genomic DNA Extraction Kit extracted genomic DNA. Using Primer5.0 designed specific primers of SAG3 and SAG1 according to the standard gene sequence of T.gondii RH strain registered in GenBank, and amplified the SAG3 and SAG1 gene by PCR. The products of PCR were identified by agarose gel electrophoresis, DNA sequence analysis and BLAST. The SAG3 gene fragment was cloned into pUCm-T vector in order to constructed cloned plasmid pUCm-T-SAG3, which identified by colony PCR, restriction enzymes and sequencing.Using restriction enzymes Ndeâ… and EcoRâ… from pUCm-T-SAG3 cut SAG3 gene,and subcloned into pET29a(+). The purified SAG1 gene fragment was cloned into pET29a(+) vector which all double digested with restriction enzyme Ndeâ… and Xhoâ… ,directly. The recombinant expression plasmids pET29a(+)-SAG3 and pET29a(+)-SAG1 were constructed and the positive clones were identified by colony PCR, restriction enzyme and sequencing analysis. The recombinant expression plasmids transforred into E.coli BL21,respectively. The expressions induced by IPTG, and using SDS-PAGE analyzed the recombinant proteins.Then using SPSS software calculated the molecular weight of recombinant proteins.Using Anti-His antibody checked up His tag by Western blotting. Recombinant proteins were purified by Ni-Resin HP, and analyzed theirs immune responsivenesses with T.gondii positive serum by Western blotting.Results: Using specific primers amplified SAG3 and SAG1 genes in vitro, and the products analyzed by gel electrophoresis and sequencing, 1059bp and 799bp long fragment obtained,respectively. The BLAST analysis indicated that the two fragments corresponded with T.gondii RH strain SAG3 and SAG1 cDNA gene homology 100%.Cloned plasmid pUCm-T-SAG3 was identified by agarose gel electrophoresis displayed a specific band at 1 000bp, and sequencing results also showed that SAG3 gene fragment had been correctly inserted downstream of cloning plasmid T7 promoter,containing Ndeâ… and EcoRâ… restriction site. Recombinant expression plasmids pET29a(+)-SAG3 and pET29a(+)-SAG1 identified by colony PCR and double restriction enzyme digestion,there were specific bands displayed at 1 000bp and 800bp by agarose gel electrophoresis,respectively,which expected SAG3 and SAG1 gene fragment size. After sequencing revealed two fragments were inserted expression plasmid pET29a(+) downstream sequence of T7 promoter at ribosome binding site(AAGGAG sequence). Through the reading analysis recombinant proteins can be expressed the end of the His tag. After IPTG induction, the recombinant proteins SAG3 and SAG1 were stably expressed. The results of SDS-PAGE indicated that recombinant proteins SAG3 and SAG1 had specific protein bands, and the relative molecular weight were 41 366.2 and 29 991.6 by linear correlation analysis. Two kinds of recombinant proteins by Western blotting with Anti-His antibodies had specific reaction bands. After purification by Ni-Resin HP, two kinds of purified proteins showed clear background protein bands by SDS-PAGE. By means of Western blotting analysis both two purification proteins were recognized by serum of T.gondii infection.Conclusions: Successfully amplified gene length 1059bp of SAG3 and length 799bp of SAG1 by PCR,and constructed recombinant expression plasmid pET29a(+)-SAG3 and pET29a(+)-SAG1. The recombinant proteins SAG3 and SAG1 was expressed in vitro, and they had higher activities. Successfully using Ni-Resin HP purified recombinant proteins, the purification proteins SAG3 and SAG1 also displayed good antigenicities. Recombinant SAG3 and SAG1 genes may be used as potential molecular vaccine candidate of T.gondii. The two purification proteins could be settled foundation of polyvalent protein vaccine.