Expression Of Resistance Associated Serine Proteinase Genes NYD-Tr And NYD-Ch From Culex Pipiens Pallens For Metabolism Of Deltamethrin

1 Expression and identification of NYD-Tr and NYD-Ch proteinIn order to study the function of NYD-Tr and NYD-Ch to the metabolism of deltemathrin, we constructed the recombinant plasmid pET32a(+)/NYD-Tr and pET32a(+)/NYD-Ch. Because the initial recombinant plasmid pET32a(+)/NYD-Tr' did not generate significant amount of stable protein in E.coli BL21 (DE3), this sequence was re-engineered for expression by inserting deleted signal peptides (19 peptides) coding sequences into the pET-32a(+) vector. Recombinant plasmid of NYD-Ch gene contained the whole open reading frame (ORF) of the gene. The two genes were respectively cloned into pET32a(+) vector, resulting in the recombinant plasmids of pET32a(+)/NYD-Tr and pET32a(+)/NYD-Ch. The positive clone was verified by PCR and sequencing. The recombinant plasmids were transformed in the E.coli BL21 (DE3).The pET-32a(+)/Tr and pET-32a(+)/Ch were expressed successfully after induction in 1mM IPTG. SDS-PAGE and Western blot analysis of the recombinant proteins revealed that the molecular weight of NYD-Tr and NYD-Ch was 42kDa and 50kDa, which correspond with the theoretical molecular weight of NYD-Tr and NYD-Ch. After the...