棉蚜P450 CYP6CY3的克隆、原核表达及多克隆抗体的制备

doi:10.3864/j.issn.0578-1752.2017.07.018

Abstract

Abstract: 【Objective】The objective of this study is to clone and express CYP6CY3 in Escherichia coli to obtain the recombinant protein, and then to immunize mice for the preparation of polyclonal anti-CYP6CY3 antibody, which provides a basis for further analysis of the role of CYP6CY3 in different tissues from insecticide-resistance Aphis gossypii. 【Method】The open reading frame (ORF) of CYP6CY3 from A. gossypii was cloned by RT-PCR and 3′-RACE, and analyzed by the bioinformatics and phylogenetic relationship. And then it was expressed in E. coli (DE3). The recombinant protein was purified by gel extraction. The polyclonal antibody of CYP6CY3 was prepared by immunizing Kunming white mice with the purified protein. The antibody titer was detected by ELISA. The specificity of the antibody was monitored by Western blot and immunohistochemistry. 【Result】The ORF of CYP6CY3 contained 1 266 bp, encoding 421 amino acids, with the predicted molecular weight of 48.8 kD, the theoretical isoelectric point of 8.99, and there was no signal peptide sequence. As a kind of hydrophilic protein, the amino acid sequence included W×××R, AG××T, E××R, P××F×PE/DRT and F××G×××C×G five characteristic motifs of P450 family. The analysis of homologous sequences by NCBI Blastx, cotton aphid CYP6CY3 amino acid sequence shared 81% identity with the pea aphid (Acyrthosiphon pisum) (XP_001948581.1) , 79% amino acid identity with the wheat aphid (Diuraphis noxia) (XP_015379193.1) , 76%, green peach aphid (Myzus persicae) (AHB52749.1), respectively. The amino acid sequences of four species of aphids have completely conserved sequence E××R in the spiral K and heme binding site consensus sequence F××G×××C×G. The phylogenetic tree was constructed using MEGA5 software, the results showed that A. gossypii, A. pisum, D. noxia and M. persicae were clustered into one group, suggesting that they likely developed from a common ancestral gene. The relative molecular weight of the CYP6CY3 fusion protein was about 66 kD. The antibody titer was determined as high as 1﹕409 600 dilution ratio by ELISA. Western blot and immunohistochemistry analysis showed that the antibody could specifically bind both the recombined expression protein and the endogenous CYP6CY3 from the A. gossypii.【Conclusion】The ORF of CYP6CY3 from A. gossypii was cloned, and the polyclonal antibody against CYP6CY3 was prepared by protein immunoassay, which will lay a foundation for further study on the function of CYP6CY3, and the role in the resistance of A. gossypii.

Key words: Aphis gossypii, CYP6CY3, prokaryotic expression, protein purification, polyclonal antibody

WEI YuanJie, WANG YaMei, HUANG LiNa, LIU Ning, ZHAO Jie, AI XinYu, LIU XiaoNing. Cloning, Prokaryotic Expression and Preparation of the Polyclonal Antibody Against CYP6CY3 from Aphis gossypii[J].Scientia Agricultura Sinica, 2017, 50(7): 1351-1360.

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Cloning, Prokaryotic Expression and Preparation of the Polyclonal Antibody Against CYP6CY3 from Aphis gossypii

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