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《中国生物化学与分子生物学会第十二届全国会员代表大会暨2018年全国学术会议摘要集》2018年
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Expression and purification of proteins related to Recombinase polymerase amplification

Suyun Liu  Yong Qi  Jiameng Li  Wenwen zeng  Jixain Rao  Wanpeng Shen  Shulong Zheng  Yuexi Li  
【摘要】:Recombinase polymerase amplification(RPA) is a novel nucleic acid amplification technology that does not require repeated control of temperature and precision instruments.It has the characteristics of high sensitivity,strong specificity and fast response.The RPA system consists of three interacting enzymes,a recombinase(UvsX),a single-stranded binding protein(Gp32),and a polymerase(Bsu),and an accessory protein(UvsY).In this study,three recombinant enzymes,two accessory proteins and a single-stranded binding protein sequence were screened,and the His tag was fused to the C-terminus of UvsX,the N-terminus of UvsY and the C-terminus of Gp32,respectively,and cloned into PET-28(+) vector.The constructed recombinant plasmid was transformed into E.coli BL21(DE3),induced by IPTG,and the strain expressing the fusion protein was screened by SDS-PAGE.The cultured high-expression strain of the fusion protein was expanded,and the expressed recombinant protein was purified to detect the purity and concentration of the protein.The results showed that the expression of UvsX,UvsY and Gp32 were soluble when induced at 25 °C,and could be purified by nickel column to obtain protein with high purity and concentration.It has good application value in the field of recombinase polymerase amplification technology.

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