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Expression and Purification of P30 Outer Membrane Recombinant Protein of Toxoplasma Gondii and Evaluation of Its Diagnostic Potential

Author: CaiLiang
Tutor: YangQiuLin
School: Nanhua University
Course: Pathogen Biology
Keywords: Toxoplasma gondii P3O Expression Protein Purification ELISA
CLC: R383
Type: Master's thesis
Year: 2006
Downloads: 147
Quote: 1
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Abstract


Objective To construct containing Toxoplasma gondii ( Toxoplasma gondii ) Table membrane protein P30 gene expression vector induced expression in E. coli , purified expression products P30 and antigenic analysis of P30 in the serological diagnosis of toxoplasmosis . ( 1 ) P30 protein expression , purification and refolding macrophages cultured Toxoplasma gondii Toxoplasma gondii DNA was extracted as a template , PCR amplified fragment The fragment was cloned into the pUCm-T Vector , digestion and PCR amplification , and then subcloned into the prokaryotic expression vector pET28b ( ) , the recombinant plasmid pET28b ( ) / P30 , transformed into the expression host strain BL21 (DE3) induced after digestion, PCR and sequencing , SDS - PAGE and Western - Blot identification expression product ; inclusion bodies were 8M urea-denatured Ni - NTA affinity chromatography and purification of recombinant proteins , denatured proteins diluted dialysis , SDS - PAGE and Western - blot analysis and identification of the P30 protein refolding , determination of the BCA method refolding protein concentration for ELISA package board . (2 ) mice Toxoplasma gondii antiserum and clinical Toxoplasma gondii in patients with positive serum collection Toxoplasma gondii frozen by -80 ° C repeated three times, after attenuated infection Kunming mice , preparation mice Toxoplasma gondii positive serum , to collect clinical Toxoplasma gondii serum . (3) patients with Toxoplasma gondii IgG antibody ELISA detection method to the purified refolded P30 board package , anti- Toxoplasma gondii the indirect ELISA clinical Toxoplasma gondii serum antibodies to Toxoplasma gondii positive serum in mice as control , comparison with the test results of the imported kit evaluation purified P30 in the serological diagnosis of toxoplasmosis . Results PCR amplified the purpose of long - 800bp fragment sequencing results Genbank logon sequence ; containing pET28b ( ) / P30 recombinant bacteria by IPTG induction , SDS - PAGE electrophoresis results showed that a molecular weight of approximately 30 kDa ( M_r ) restructuring protein , the target protein in the bacterial cells form of inclusion bodies ; 8M urea dissolved by Ni-NTA affinity purified recombinant protein purity greater than 95% ;

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