Study Of Recombinant GRA5 Protein On Immuno-diagnosis Of Toxoplasma Infection

Objective:Prokaryotic expression and identification of Toxoplasma gondii dense granule protein 5(GRA5)was used to obtain the recombinant GRA5 protein of Toxoplasma gondii antigen and antigen activity similar to natural.The purified recombinant GRA5 protein was used as antigen coated ELISA plate to establish Toxoplasma ELISA detection method,applied to the immunological diagnosis of toxoplasmosis.Methods: A couple of primers were designed according to the known sequence of Toxoplasma gondii GRA5 and the GRA5 gene was synthesized in vitro.By cloning GRA5 gene into a prokaryotic expression vector,p ET28 a,a recombinant p ET28a-GRA5 was constructed and transferred into E.coli BL21/DE3.The positive recombinant p ET28a-GRA5 was screened and identified by endonuclease digestion.The BL21/DE3 which was transformed with p ET28a-GRA5 was induced by IPTG,and the expressed protein was analyzed by SDS-PAGE and Western-blotting.Purification of recombinant protein is applied to antigen coated enzyme labeled plate to establish Toxoplasma gondii ELISA detection method.Results: The products of GRA5 gene of 363 bp in length was successfully amplified by PCR,the prokaryotic expression vector p ET28a-GRA5 was constructed successfully,the result of double digestion showed that the Insert fragment size was correct,and the result of DNA sequencing was compared with that in Gen Bank for homology which was 100%.Also the expression of GRA5 protein was successfully detected in BL21 by Western blot(about 14 ku).Using purified recombinant GRA5 protein as antigen coated ELISA plate,established Toxoplasma gondii serum ELISA detection method,the optimal coating concentration of antigen ELISA method was the best condition 10ug/ml,37 DEG 2h after 4 DEG C coated overnight,the best serum dilution was 1:25,closed conditions for 5% skim milk 37 ? 2H two anti optimal concentration of 1:20000,TMB,the best color time is 20 min.ELISA method was used to detect 100 cases ofpatients.The result indicated that 73 cases is positive and the positive diagnosis rate was 73%.Among them,the positive detection rate of Ig G positive samples was 72.5% in 40 cases,the positive detection rate of Ig M positive samples was 53.3% in 30 cases,the positive detection rate of Ig G and Ig M positive samples was 93.3% in 30 cases.