Study On The Diagnosis Of Toxoplasmosis With Recombinant T.gondii GRA7

Objective:To clone,express and purify the recombinant dense granule protein-7(rGRA7)of Toxoplasma gondii(T.gondii)and analyze its value for diagnosing of toxoplasmosis so as to provide a new experimental basis for the serological diagnosis of toxoplasmosis.Methods:(1)To design and synthesize the primers of GRA7 based on its sequences in GenBank.The GRA7 gene was amplified by polymerase chain reaction(PCR).Then the GRA7 gene was cloned into pMD-18T.Clones containing the GRA7 gene were identified by colony PCR,plasmid digestion and DNA sequencing.(2)The GRA7 gene was subcloned into the prokaryotic expression vector pET-28a(+)and identified by PCR and double enzyme digestion.BL21 containing pET-28a-GRA7 was induced by β-D-thiogalactoside(IPTG)to express the rGRA7.The bacteria were lysed by sonication and the rGRA7 were purified by nickel chelate chromatography.We used western-blot to detect the reactogenicity of GRA7 and enzyme-linked immunosorbent assay(ELISA)to analyze the value of GRA7 in diagnosing of Toxoplasma gondii infection.Results:(1)The amplified gene GRA7 was about 630bp.The recombinant expression plasmid pET-28a-GRA7 was successfully constructed;(2)The fusion protein GRA7 with a molecular weight of 33 kDa was successfully expressed and purified,which could be specifically recognized by the serum of patients with toxoplasmosis.The sensitivity,specificity,positive predictive value,negative predictive value and diagnostic efficiency of rGRA7-ELISA for detection of Toxoplasma gondii infection were 81.54%,95.38%,94.64%,83.78%and 88.46%respectively.Conclusions:(1)Recombinant protein GRA7 was obtained by recombinant DNA technology;(2)The recombinant GRA7 has good reactivity and is highly sensitive and specific for the detection of Toxoplasma gondii infection.It has potential value in the diagnosis of Toxoplasma gondii infection.