棉铃虫Hsp70的多克隆抗体制备及鉴定
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摘要
HSPs(热休克蛋白)是机体在不利环境条件刺激下合成的一类蛋白质,在进化上高度保守,普遍存在于生物体,其中Hsp70是最为保守的成员。研究发现,昆虫滞育过程中普遍存在Hsp70表达上调的现象。然而,现有的研究均是在基因水平的检测结果。为了能从蛋白水平检测棉铃虫中Hsp70的表达,本研究制备了棉铃虫Hsp70的多克隆抗体。构建了hsp70的原核表达载体并在大肠杆菌BL21(DE3)中成功表达,重组蛋白经镍柱纯化后免疫兔子,制备了棉铃虫Hsp70的多克隆抗体。抗体的效价较高,达到了1∶256 000,此外Western结果表明,制备的抗体能检测出热诱导的Hsp70蛋白。本研究制备了高效价且较为特异的Hsp70对克隆抗体,该抗体为后续从蛋白水平研究棉铃虫滞育过程中Hsp70的表达及其分子机制奠定了基础。
HSPs(Hot shock protein), induced proteins under adverse environmental conditions, are ubiquitous in all sorts of organism with highly conservative in evolution. Hsp70 is the most conservative member of HSPs. The common up-regulation of hsp70 is reported in insect diapause. However, these studies are all based on the levels of gene. To detect the expression of Hsp70 in Helicoverpa armigera from protein level, polyclonal antibody against Hsp70 from H. armigera was prepared. Firstly, hsp70 was cloned into prokaryotic expression vector pET32 a and the recombinant protein was expressed in Escherichia coli BL21(DE3). After purified by Ni column, the recombinant Hsp70 protein was used to immune rabbit. The titer of antibody against β-catenin was estimated as high as 1 ∶256 000 dilution ration detected by ELISA. In addition, induced Hsp70 of H. armigera can be detected by our antibody using Western Blotting. In conclusion, high titer and specificity antibody against Hsp70 from H. armigera was obtained, which lays a foundation for further investigating the expression and molecular regulation mechanism of Hsp70 in H. armigera diapause.
HSPs(Hot shock protein), induced proteins under adverse environmental conditions, are ubiquitous in all sorts of organism with highly conservative in evolution. Hsp70 is the most conservative member of HSPs. The common up-regulation of hsp70 is reported in insect diapause. However, these studies are all based on the levels of gene. To detect the expression of Hsp70 in Helicoverpa armigera from protein level, polyclonal antibody against Hsp70 from H. armigera was prepared. Firstly, hsp70 was cloned into prokaryotic expression vector pET32 a and the recombinant protein was expressed in Escherichia coli BL21(DE3). After purified by Ni column, the recombinant Hsp70 protein was used to immune rabbit. The titer of antibody against β-catenin was estimated as high as 1 ∶256 000 dilution ration detected by ELISA. In addition, induced Hsp70 of H. armigera can be detected by our antibody using Western Blotting. In conclusion, high titer and specificity antibody against Hsp70 from H. armigera was obtained, which lays a foundation for further investigating the expression and molecular regulation mechanism of Hsp70 in H. armigera diapause.
引文
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Xiao HJ, Wei ZJ, Xue FS. Advances in studies on insect diapause associated heat shock proteins [J].Journal of Environmental Entomology, 2011,54(9): 1068-1075. [肖海军, 魏兆军, 薛芳森. 昆虫滞育关联热休克蛋白的研究进展 [J]. 昆虫学报, 2011,54(9): 1068-1075]
Xia QW. Why diapause education? [J].Jiangxi Plant Protection, 2011, 1(1): 1-5. [夏勤雯. 为什么研究滞育? [J]. 江西植保, 2011, 1(1): 1-5]
Hahn DA, Denlinger DL. Energetics of insect diapause [J].Annual Review of Entomology, 2011, 56(1): 103-121.
Hao YJ, Li WS, He ZB,et al. Differential gene expression between summer and winter diapause pupae of the onion maggot Delia antiqua, detected by suppressive subtractive hybridization [J]. Journal of Insect Physiology, 2012, 58(11): 1444-1449.
Lyupina YV, Zatsepina, Timokhova AV et al. New insights into the induction of the heat shock proteins in baculovirus infected insect cells [J]. Virology, 2011, 421(1): 34-41.
Rinehart JP, Li A, Yocum, GD,et al. Up-regulation of heat shock proteins is essential for cold survival during insect diapause [J]. Proceedings of the National Academy of Sciences, 2007, 104(27): 11130-11137.
Spiess C, Beil A, Ehrmann M. A temperature-dependent switch from chaperone to protease in a widely conserved heat shock protein [J].Cell, 1999, 97(3): 339-347.
Wang L, Yang S, Han L,et al. Phenotypic plasticity of HSP70s gene expression during diapause: Signs of evolutionary responses to cold stress among soybean pod borer populations (Leguminivora glycinivorella) in Northeast China [J]. PLoS ONE, 2014, 9(10): 1-10.
Zhang Q, Denlinger DL. Molecular characterization of heat shock protein 90, 70 and 70 cognate cDNAs and their expression patterns during thermal stress and pupal diapause in the corn earworm [J].Journal of Insect Physiology, 2010, 56(2): 138-150.
Zhang Q, Lu YX, Xu WH. Proteomic and metabolomic profiles of larval hemolymph associated with diapause in the cotton bollworm,Helicoverpa armigera [J]. BMC Genomics, 2013, 14(1): 751.
Zhang Q, Nachman RJ, Kaczmarek K,et al. Disruption of insect diapause using agonists and an antagonist of diapause hormone [J]. Proceedings of the National Academy of Sciences, 2011, 108(41): 16922-16926.
Wang ZB, Liao SQ. Effects of several pesticides on cotton bollworm and natural enemy ladybugs [J].Botanical Doctor, 2013,1(1): 32-33. [王志斌, 廖舜乾. 几种药剂对棉铃虫及天敌瓢虫的影响 [J]. 植物医生, 2013,1(1): 32-33]
Xiao HJ, Wei ZJ, Xue FS. Advances in studies on insect diapause associated heat shock proteins [J].Journal of Environmental Entomology, 2011,54(9): 1068-1075. [肖海军, 魏兆军, 薛芳森. 昆虫滞育关联热休克蛋白的研究进展 [J]. 昆虫学报, 2011,54(9): 1068-1075]
Xia QW. Why diapause education? [J].Jiangxi Plant Protection, 2011, 1(1): 1-5. [夏勤雯. 为什么研究滞育? [J]. 江西植保, 2011, 1(1): 1-5]