Abstract: To clone the open reading frame 1(ORF1)gene of porcine torque teno virus type 2 and construct a prokaryotic expression vector. TTV2 ORF1 recombinant protein was expressed in Escherichia coli and optimal ex-pression was performed.The ORF1 gene of TTV2 was amplified from the TTV2 positive samples and sub-cloned in a prokaryotic expression vector pcoldI. The constructed recombinant plasmid pcold-ORF1 was transformed to E.coli BL21 for expression the ORF1 fusion protein with six histidine-tag under induction of IPTG. The induction time,in-duction in different temperature and IPTG concentrations were optimized. The recombinant fusion protein had high expression level in BL21(DE3),and SDS-PAGE analysis showed the exogenous gene was mainly expressed as in-clusion bodies and its molecular weight was about 39 kDa.The induction time were positively related trends with re-combinant protein expression,the best induction temperature was 15 ℃,whereas IPTG concentration had no signifi-To clone the open reading frame 1(ORF1)gene of porcine torque teno virus type 2 and construct a prokaryotic expression vector. TTV2 ORF1 recombinant protein was expressed in Escherichia coli and optimal ex-pression was performed.The ORF1 gene of TTV2 was amplified from the TTV2 positive samples and sub-cloned in a prokaryotic expression vector pcoldI. The constructed recombinant plasmid pcold-ORF1 was transformed to E.coli BL21 for expression the ORF1 fusion protein with six histidine-tag under induction of IPTG. The induction time,in-duction in different temperature and IPTG concentrations were optimized. The recombinant fusion protein had high expression level in BL21(DE3),and SDS-PAGE analysis showed the exogenous gene was mainly expressed as in-clusion bodies and its molecular weight was about 39 kDa.The induction time were positively related trends with re-combinant protein expression,the best induction temperature was 15 ℃,whereas IPTG concentration had no signifi- cant impact on protein expression. Western Blotting revealed that the recombinant protein reacted positively with TTV2 positive serum and had a good immunogenicity. The ORF1 gene of TTV2 was successfully cloned and ex-pressed in prokaryotic cells, which provides the immunogen for ELISA. Adding IPTG to a final concentration of 0. 2 mmol/L and shaking the culture at 15 ℃,and then the optimal expression of ORF1 fusion protein were accu-mulated after 24 h.

Key words: Torque teno virus type 2( TTV2), Gene cloning, Prokaryotic expression

CLC Number: 

• S852.65