Cloning,prokaryotic expression and immunological identification of Toxo-plasma surface antigen IMP1
10.16250/j.32.1374.2015025
- VernacularTitle:弓形虫表面抗原IMP1的克隆原核表达及免疫学鉴定
- Author:
Jingxuan KOU
;
Guihua ZHAO
;
Qingkuan WEI
;
Chao XU
;
Song ZHU
;
Kun YIN
- Publication Type:Journal Article
- Keywords:
Toxoplasma gondii;
IMP1 gene;
Surface antigen;
Prokaryotic expression;
Purification
- From:
Chinese Journal of Schistosomiasis Control
2015;(3):285-289
- CountryChina
- Language:Chinese
-
Abstract:
Objective To subclone express and identify the immune mapped protein 1 IMP1 which encodes a surface an?tigen of Toxoplasma gondii. Methods The cDNA of T. gondii RH strain was synthesized by reverse transcription PCR the IMP1 open reading frame ORF was amplified by PCR using the T. gondii RH strain cDNA as template the PCR products were identified by TA?cloning and sequencing then the IMP1 ORF was subcloned into the NdeⅠand Xho I sites of the vector pET28b and the positive recombinant pET28b?IMP1 was identified by double?digesting and sequencing. The protein of 6 × His tagged IMP1 was inducibly expressed in E. coli strain BL21 DE3 with isopropylβ?D?1?thiogalactopyranoside IPTG and the induction time concentration of IPTG and temperature gradients to optimize protein expression conditions were determined. After the cells carried IMP1 were induced by the optimized conditions and harvested the resulting bacteria were suspended in resuspension buffer and lysed by sonication and the supernatants were loaded onto the Ni2+Chelating Sepharose Fast Flow col?umn for affinity chromatography of the N?terminal 6 × His tagged IMP1 protein. Finally the fusion IMP1 proteins were identified by Western blotting. Results The ORF sequence of IMP1 was successfully subcloned from the cDNA of Toxoplasma Gondii RH strain and the amplified product was sequenced and identified based on which the IMP1 ORF gene was inserted into the prokaryotic expression vector pET28b and the recombinant pET28b?IMP1 was constructed successfully. The double?digesting and sequencing results indicated the validity of the recombinant vector. And the optimized conditions for the expression of IMP 1 was determined namely 0.3 mmol/L IPTG induction for 9 h at 20℃. Furthermore IMP1 protein was expressed solubly and che?lated on Ni2+sepharose beads with high affinity thus this protein could be purified efficiently by affinity chromatography. The pure fusion protein was confirmed with fine immunocompetence by SDS?PAGE and Western blotting. Conclusions IMP1 pro? tein can be high efficiently expressed by the E. coli prokaryotic expression systems the protein of IMP1 is soluble and has stable characters. The study may lay a useful foundation for the following works including in vivo expression of IMP1 crystal structure study of IMP1 and anti?toxoplasmosis subunit vaccine development.
