PCR technique was used for amplifying THP gene in an unknown vector with primer AFP1 and AFP2.Then THP gene was ligated to pGEM T-Vector to be the plasmid pGTHP4.The plasmid pCAMBIA1301 was digested with restriction enzyme BstEⅡ and NcoⅠ,and digestion product was separated with 1% of agarose gel,then big fragment containing promoter was isolated and purified with the Agarose Gel DNA Extraction Kit.At the same way,the plasmid pGTHP4 was digested with restriction enzyme BstZⅠ and NcoⅠ,and the small fragment containing THP gene was purified from 1% agarose gel with the Agarose Gel DNA Extraction Kit.The big fragment and the small fragment were ligated at Nco Ⅰ digested cohesive-end.The ligation product was re-ligated to be cyclic plsmid by addition to a specific adapter,resulting in the pCTH823,a expression vectorof V.volvacea.
