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| Expression and Purification of HPT gene in Escherichia coli and Preparation of Its Polyclonal Antibodies |
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Abstract Hpt gene was cloned from genetically modified plants’ genome and digested by BamHⅠ和Hind III. pET30a vector was digested by the same enzymes and ligated to hpt gene to construct plasmid pET30a-hpt. HPT protein was expressed by E.coli BL21 and purified firstly by ammonium sulfate, then by Ni+-NTA column. Hygromycin B phosphotransferase (HPT) was highly expressed in Escherichia coli BL21(DE3) in the presence of isopropyl-β-D-thiogalactopyranoside (IPTG) and most products existed in soluble form. After cleavage of His•Tag fusion protein by enterokinase, intact、biologically active HPT was released from the fusion protein and was purified to homogeneity with Ni2+ affinity chromatography. Polyclonal antibodies against were raised in three rabbits and the purified polyclonal anti-RCT antiserum appears a very high degree of sensitivity and specificity.
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Received: 22 October 2007
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