Study on expression and activity of combinant antimicrobial peptide in Pichia pastoris / 国际检验医学杂志

ABSTRACT

Objective To construct cecropin A-thanatin combinant gene engineering antimicrobial peptide gene CA(1-7)-T(4-19) for expression in Pichia pastoris.Methods The combinant antimicrobial peptide gene was artificially synthesized via gene splicing by overlap extension (SOE).The gene was cloned into the pPICZαA vector and transformed into Pichia pastoris X-33 by electroporation.The positive clones obtained by the screening of bleomycin resistance were induced by methanol ,and the antibacterial activity of the products was detected and the antimicrobial spectrum was established.Results The combinant peptide gene CA (1-7)-T (4-19) was successfully cloned on the carrier pPICZαA.The identification results were consistent with the pre-designed gene sequence.The combinant peptide gene was expressed under the induction of methanol ,and the minimum inhibitory concentration of 76 strains of Gram-egative and Gram-positive pathogenic bacteria isolated from the clinic was obtained ,and the minimum inhibitory concentration was up to 5 μg/mL.Conclusion A combinant genetic engineering antimicrobial peptide with antibacterial activity was obtained successfully and it had obvious inhibition effect on clinical common multidrug-resistant strains.