棉蚜P450 CYP6CY3的克隆、原核表达及多克隆抗体的制备

【目的】克隆得到棉蚜(Aphis gossypii)P450 CYP6CY3,将其开放阅读框(ORF)在原核细胞中进行表达,纯化融合蛋白,多次免疫小鼠获得CYP6CY3多克隆抗体,为进一步分析CYP6CY3在棉蚜不同组织部位的分布及其在抗性中的作用打下基础。【方法】利用RT-PCR及3′-RACE技术克隆获得CYP6CY3的ORF,并对其进行生物信息学及系统进化分析。构建重组质粒pET32a-CYP6CY3,转化大肠杆菌transetta(DE3)进行原核表达,利用切胶回收法获得纯化的融合蛋白,继而用纯化得到的融合蛋白免疫昆明白小鼠制备鼠抗棉蚜CYP6CY3多克隆抗体;ELISA检测鼠抗CYP6CY3蛋白多克隆抗体的效价,并通过Western blot及免疫组化检测该抗体特异性。【结果】棉蚜CYP6CY3 ORF长1 266 bp,编码421个氨基酸,预测蛋白分子量为48.8 k D,理论等电点为8.99,不含有信号肽序列,属亲水性蛋白;氨基酸序列包括W×××R、AG××T、E××R、P××F×PE/DRT和F××G×××C×G 5个P450家族的特征基序。通过NCBI Blastx进行同源序列的比对分析,棉蚜CYP6CY3氨基酸序列与豌豆长管蚜(Acyrthosiphon pisum)(XP_001948581.1)氨基酸一致性最高,达到81%,与麦双尾蚜(Diuraphis noxia)(XP_015379193.1)一致性为79%,与桃蚜(myzus persicae)(AHB52749.1)氨基酸序列一致性为76%,4种蚜虫的氨基酸序列都含有位于螺旋K中参与稳定核心结构的E××R完全保守序列及P450基因标志性的F××G×××C×G(358—367)血红素结合位点的共有序列。使用MEGA5软件构建系统进化树,显示棉蚜、豌豆长管蚜、麦双尾蚜及桃蚜聚为一类,亲缘关系较近。通过原核表达得到的CYP6CY3融合蛋白的相对分子量约为66 k D,基于纯化的CYP6CY3重组蛋白多次免疫昆明白小鼠制备多克隆抗体,ELISA检测获得的鼠抗CYP6CY3抗体效价达到1﹕409 600。Western blot和免疫组化进一步证实,该抗体既能与异源表达的CYP6CY3蛋白特异性结合,也能与棉蚜组织中存在的天然CYP6CY3蛋白特异性结合,具有较好的免疫反应特异性。【结论】利用3′-RACE技术克隆获得CYP6CY3的开放阅读框,并用异源表达的蛋白免疫昆明白小鼠制备了高滴度、特异性强的鼠抗棉蚜CYP6CY3的多克隆抗体,可用于进一步研究CYP6CY3功能及其在棉蚜抗药性方面的作用。

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

【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.