Construction Of Toxoplasma PET28a-SAG1 And PET28a-SAG4 Recombinant Gene And Expression And Immune Protection

Objective Construction of Toxoplasma pET28a-SAG 1 and pET28a-SAG4 recombinant gene and prokaryotic expression to evaluate the immune effect of immunizing animals pET28a-SAG1 and pET28a-SAG4 recombinant protein. The gene toxoplasmosis vaccine applications provide a scientific basis.Methods According to T. gondii RH strain in GenBank sequence SAG1 and SAG4, designing two pairs of specific primers. Toxoplasma gondii extract genomic DNA, as a template, PCR amplified the two antigen genes, and build on the pMD19 (Simple) cloning vectors.After colony PCR, double digestion and sequenced properly, and then digested product was purified, cloning them into pET28a prokaryotic expression system, the recombinant plasmids were transformed into BL21 E.coli,Identification and Purification of Recombinant Protein, immunized mice and got serum, indirect ELISA assay in mouse serum anti-Toxoplasma IgG, and mouse to observe the incidence of insect attack and death by these two tests to evaluate recombinant protein immune protection for recombinant protein vaccine foundation.Results Get SAG1 and SAG4 genes successfully, and construct into pMD19 (Simple) vector and Prokaryotic expression vector plasmid pET28a, successfully obtained pET28a-SAG4 and pET28a-SAGl. After prokaryotic expression were acquired SAG4 and SAG1 target recombinant protein. OD value of the difference between groups was statistically significant serum (p <0.01) after the purified recombinant protein immunized mice,and the highest OD value was the double protein-immunized group; during challenge experiments, the double protein-immunized group lived the longest survival time, pET28-SAG1 group survival time was statistically significant (p<0.01) than pET28-SAG4.Conclusions 1. Successfully constructed pET28a-SAG1 and pET28a-SAG4 recombinant gene and expression and purification of the target recombinant protein.2. The first time made with mice Toxoplasma attack experiments show that mice susceptible to Toxoplasma gondii.3. Double-tapping experiments show that protein immunized mice survived longer than the survival time of a single protein immunization, pET28a-SAG1 mice survived longer than pET28a-SAG4 group.Immune protection pET28a-SAGl protein is better than pET28a-SAG4 protein.4. Protein expression of these two genes have immune protection.