Expression And Identification Of Resistance Associated NYD-Ch And NYD-Tr Genes Of The Mosquito, Culex Pipiens Pallens

The resistance to insecticide played a serious role in the control of pests. All the research gives more evidence that the resistance to insecticide in pest is determined by gene. NYD-Ch gene and NYD-Tr gene were isolated and identified from the mosquito, Cx. pipiens pallens, by suppression subtractive hybridization (SSH) combined with cDNA chip, which were highly expressed in the deltamethrin-resistant strain compared in the susceptible strain. And this study is to construct the prokaryotic and eukaryotic expression plasmids of gene NYD-Ch and NYD-Tr, to obtain the polyclonal antibody of mouse anti-bovine CHY and TRY, and to learn prokaryotic expressions of the two genes.Part one: Construction and identification of expression plasmids of NYD-Ch gene and NYD-Tr geneThe complete open reading frames (ORFs) of NYD-Ch gene and NYD-Tr gene were amplified from the full-length cDNA express library of the deltamethrin-resistant strain of Cx.pipiens pallens by PCR according to the reported sequences of our laboratory. The enzyme restriction sites were added at the head of ATG of the two genes considering the express of the prokaryotic plasmid pET-28a(+) and the eukaryotic plasmids pcDNA3.1(+) and pEGFP-C3 when designing the primers of the two genes. The PCR fragments were cloned into pGEM-T easy vector, and then identified by PCR, enzyme cutting and gene sequencing. Cut from the re-construction plasmid pGEM-T/NYD-Ch andpGEM-T/NYD-Tr, and from the super coil plasmids pET-28a(+), pcDNA3.1(+) and pEGFP-C3, the fragments NYD-Ch , NYD-Tr, pET-28a(+), pcDNA3.1(+) and pEGFP-C3 were obtained with the enzyme restriction site ends. The gene and plasmid fragments were re-combined in the condition of T4 DNA ligase on the basis of some ratio. And then the re-construction plasmids were identified using PCR, double enzyme cutting and gene sequencing. The results showed that NYD-Ch gene and NYD-Tr gene sequences were the same as the reported, indicating that the express plasmids pET-28a(+)/NYD-Ch,pET-28a(+)/NYD-Tr, pcDNA3.1(+)/NYD-Ch, pcDNA3.1(+)/NYD-Tr, pEGFP-C3/NYD-Ch and pEGFP-C3/NYD-Tr were re-constructed successfully.Part two: Preparation of polyclonal antibody of mouse anti-CFTY and anti-TRY, and preliminary detection of resistance in mosquitoThe bovine CFFY and TRY mixed equally with the Freund's complete adjuvant were used to immunize the mice separately. 15 days later, the antigen mixed equally with the Freund's incomplete adjuvant was injected under the dermis of mice on the back to enhance the effect of immune. And then 10 days later, the serum of antibody titer was determined primarily using ELISA, and 5 days latter again, the complete antigen alone was used to immunize the mice. After 45 days from the first immune, the serum of mice was obtained and the titer of antibody was determined by ELISA. During the course of the immune, the dose of antigen was equal. The antibody was used to study the resistance in the mosquito elementarily by Western blot. The results show that high titer of the polyclonal antibody of mouse anti-CHY and anti-TRY were prepared and the titer was more than 1:10000. And a tape of protein about 28kD was observed on the NC membrane of resistance strain when the firstantibody was anti-TRY polyclonal antibody. The protein was coincident with the protein of NYD-Tr. But in the group of anti-CHY, none was appeared in the resistant strain.Part three: Prokaryotic expression and identification of resistance associated NYD-Ch and NYD-Tr geneThe plasmids of pET-28a(+)/NYD-Ch and pET-28a(+)/NYD-Tr were transfected into the E.coil BL21(DE3) by CaCb, and then induced by ImM IPTG to obtain the protein in different time. Mixed with the 1 X Loading Buffer and degenerated in the condition of 100癈 water , the sediment of bacterium were separated by SDS-PAGE and then dyed with Coomassie Light blue to learn the molecular weight of the express protein. And then the positive sample was identified by the Western blot using the polyclonal antibody of mouse anti-CHY and anti-TRY a...