The Design Of New Antibacterial Peptide Gene Synthesis And Expression Study

Cecropin AD is a hybrid peptide that exhibits greater antibacterial activity than the natural Cecropins. It is composed of 1-11 amino acid residues of Cecropin A and 12-37 amino acid residues of Cecropin D. The hydrophobicity analysis and secondary structure prediction by Chou-Fasman method showed that the Cecropin AD possessed the typical structural characteristics of Cecropins, which was two a-helical regions linked by a hinge region with Ala-Gly-Pro. The NH₂-terminus of Cecropin AD is more perfect amphipathic helix than Cecropin D, while the COOH-terminus is more hydrophobic than Cecropin A.Based on the amino acid sequence of Cecropin AD ,a novel Cecropin AD gene was designed. The coden selection of the designed Cecropin AD gene was carried out according to the coden usage of yeast. The designed gene is 140 bp in length. It contains the encoded sequence ,the start and stop codons, and the restriction sites of BamH I ,EcoR I and Sal I at both ends.The Cecropin AD gene was synthesized by the combination of oligo-deoxynucletides synthesis and PCR method. At First, two oligodeoxy-nucletides , one of which is 82 bases and another is 78 bases in length, were synthesized. Both oligodeoxynucletides located at 5' terminus of the single strand of gene respectively, and their 3' terminus have 20 paired bases. Then, the Cecropin AD gene was synthesized by PCR, while the two oligodeoxy-nucletides act as primers each other. The PCR product was cloned into the pCR^TM2.1 vector by means of T-A paring direct molecular cloning method. The recombinant plasmid pCR-CAD was transformed into E.coli JM103. The synthetic gene was proved to be correct by the restriction pattern and the DNA sequence analysis.Using BamH I and Sal I to digest the recombinant plasmid pCR-CAD ,the Cecropin AD gene of 134bp was obtained and it was inserted into...