Construction of the plant vector carrying fused gene

Objective: To construct the plant expression vector of fused gene of pacP and ctxB. Methods: pBPC55 and pCAMBIA2301 were digested with double restriction endonuclease Hind III and EcoR±respectively, then the two digestive products were linked each other. The ligated products transformed E.coli JM109, and the recombinants were selected and were detected by restriction endonuclease. The new constructed vector were named as p2355. Designed the primers to amplify the fused gene through polymerase chain reaction(PCR) from plasmid pEPC10 and pEPC15, which carried pacP-ctxB10 and pacP-ctxB15 genes respectively. Both the PCR products and the plasmid p2355 were recombined through double restriction endonuclease Xba±and Kpn±respectively and linked each other. The recombinants were transformed E.coliJM109 and were detected by restriction endonuclease and PCR. p2355-EPC10 and p2355-EPC15 were transformed into Agrobacterium tumefaciens EHA105 respectively by Electroporation. Results: Restriction endonuclease comfirmed that plant binary vector p2355 was constructed successfully. Restriction endonuclease and PCR tests comfirmed that plasmid p2355-EPC10 and p2355-EPC15 were constructed successfully and the fused gene were integrated correctly betweent CaMV35S promoter and Nos3' terminator, which would ensure the fused gene could express in plant. Conclusion:The plant expression vector carrying fused gene of antigen epitope P region in surface protein of streptococcus mutans and cholera toxin B subunit were constructed successfully