飞蝗双链RNA降解酶的抗体制备及组织定位

doi:10.3864/j.issn.0578-1752.2018.19.008

摘要/Abstract

摘要： 【目的】双链RNA降解酶（dsRNA degrading enzyme, dsRNase）是制约RNA干扰（RNAi）技术在害虫防治中应用的关键因素之一。本研究旨在利用原核表达系统获得飞蝗LmdsRNase2和LmdsRNase3的特异抗原，进而制备抗体，对其进行组织定位和蛋白表达量检测，为进一步解析飞蝗中肠dsRNA降解酶基因的功能分化提供蛋白水平的证据。【方法】通过对LmdsRNase2和LmdsRNase3的氨基酸序列（GenBank: ARW74135.1和ARW74134.1）进行比对，分别选取特异的LmdsRNase2和LmdsRNase3抗原序列（R2’、R3’）进行后续的研究。根据LmdsRNase2和LmdsRNase3的cDNA全长序列，设计包含酶切位点BamHI、HindIII的引物，采用PCR技术扩增目标片段，双酶切后连接至pET-32a载体。将重组质粒转化至大肠杆菌BL21（DE3）感受态细胞中，加入IPTG至终浓度为0.5 mmol·L^-1，37℃下诱导4 h后提取蛋白，采用SDS-PAGE电泳方法检测蛋白表达。大量培养带重组质粒的大肠杆菌，诱导目的蛋白大量表达，用镍柱亲和层析法分离R2’和R3’蛋白，Bradford法检测蛋白浓度。经两次免疫新西兰大白兔后，最终获得LmdsRNase2和LmdsRNase3的多克隆抗体。利用ELISA法测定多克隆抗体的效价，通过western blot检测抗体的特异性。提取飞蝗5龄第3天的中肠组织蛋白和中肠液，分别用LmdsRNase2和LmdsRNase3的抗体检测其表达量。最后制备飞蝗5龄第3天的中肠石蜡切片，通过免疫组化对LmdsRNase2和LmdsRNase3蛋白进行亚细胞定位。【结果】通过氨基酸序列比对发现，LmdsRNase2和LmdsRNase3具有37%的序列一致度，选取特异的序列R2’和R3’设计引物，分别包含157和153个氨基酸残基，理论分子量分别为17.0和16.8 kD。在IPTG浓度为0.5 mmol·L^-1条件下，37℃诱导4 h后，目的蛋白在包涵体中大量表达。扩大培养重组菌株后提取蛋白，用镍亲和层析柱分离得到纯度为85%的R2’蛋白，可直接用于免疫；而R3’蛋白的纯度低于85%，利用电泳切胶纯化后免疫新西兰大白兔，36 d后取抗血清进行检测，效价均达到1﹕102 400，表明抗体效果良好。用多克隆抗体杂交实验室保存的His-LmdsRNase2和His-LmdsRNase3融合蛋白，对抗体进行特异性检测，结果表明R2和R3的抗体分别特异性识别LmdsRNase2和LmdsRNase3，无交叉杂交现象，且条带与His抗体杂交到的条带大小一致。采用western blot方法检测，发现LmdsRNase2蛋白在中肠组织中高表达，但在中肠液中未检测到可见表达，LmdsRNase3蛋白在中肠组织和中肠液中均未检测到表达。进一步采用免疫组化方法对两个蛋白进行组织定位，发现LmdsRNase2和LmdsRNase3均在中肠细胞质中表达，但LmdsRNae3的表达量较低。【结论】成功制备了特异性良好的飞蝗LmdsRNase2和LmdsRNase3抗体，western blot和免疫组化分析表明LmdsRNase2在中肠细胞质中高表达，而LmdsRNase3的表达量很低。研究结果为飞蝗中肠两个dsRNA降解酶LmdsRNase2和LmdsRNase3的功能分化提供了蛋白水平的证据。

关键词: 飞蝗, dsRNA降解酶, 多克隆抗体, 组织定位

Abstract: 【Objective】The dsRNA degrading enzyme (dsRNase) is an important obstacle of using RNAi technology for pest control. The objective of this study is to obtain specific antigens of LmdsRNase2 and LmdsRNase3 by using prokaryotic expression system, and then to prepare antibodies, and to detect the protein expression level of midgut and subcelluar localization. The results will provide strong evidence from protein level to further analysis of functional differentiation of LmdsRNase2 and LmdsRNase3 in the midgut of Locusta migratoria.【Method】The specific antigen sequences (R2’ and R3’) of LmdsRNase2 and LmdsRNase3 (GenBank: ARW74135.1, ARW74134.1) were chosen by alignment analysis. The primers with BamHI, HindIII restriction sites were designed according to the complete cDNA sequences. The target sequences were amplified by PCR and ligated to pET-32a vector through double enzyme digestion. The recombinant plasmids were transformed into E. coli BL21 (DE3) competent cells, and then the cells were induced with 0.5 mmol·L^-1 IPTG for 4 hours at 37℃. SDS-PAGE electrophoresis was performed to examine the target proteins. After that, large amounts of E. coli cells were cultured for protein extraction. The target proteins R2’ and R3’ were purified through Ni-NTA agarose chromatography, and the protein concentration was determined according to the method of Bradford. The anti-LmdsRNase2 and 3 polyclonal antibodies were obtained after immunizing New Zealand white rabbit. The specificity of antibody was detected by western blot analysis, the recombinant LmdsRNase2 and LmdsRNase3 proteins were used as antigens. The titers of two antibodies were determined by ELISA. Furthermore, the total proteins were extracted from L. migratoria midgut and midgut fluid on day 3 of 5th instar nymph, and western blot was used to test the expression level of LmdsRNase2 and LmdsRNase3. Finally, the paraffin sections of the midgut on day 3 of 5th instar nymph were prepared, the subcellular localization of LmdsRNase2 and LmdsRNase3 proteins in the midgut of L. migratoria was conducted by immunofluorescence.【Result】LmdsRNase2 and LmdsRNase3 showed 37% sequence identity by full-length amino acid sequence alignment, and the variant sequence regions (named as R2’ and R3’ for LmdsRNase2 and LmdsRNase3, respectively) were selected for PCR primer design. The length of R2’ and R3’ is 157 and 153 a.a., respectively, and the calculated molecular mass of them is 17.0 and 16.8 kD, respectively. The protein expression was induced with 0.5 mmol·L^-1 IPTG for 4 hours at 37℃, finally the expressed proteins were only detected in inclusion bodies. The recombinant proteins R2’ and R3’ were purified using Ni-NTA agarose chromatography, and the purity of R2’ is 85%, it is suitable to directly immune New Zealand white rabbit. But the purity of R3’ is under 85%. To further obtain highly purified R3’, R3’was loaded on a gel and the expected band was cut for protein extraction and following immunization. After 36 days, ELISA results indicated that the titer of LmdsRNase2 and LmdsRNase3 antibodies was 1﹕102 400. Western blot demonstrated that anti-LmsRNase2 and 3 polyclonal antibodies could specifically detect LmdsRNase2 or LmdsRNase3 proteins, respectively, no cross hybridization was observed. The molecular weight was consistent with the predicted size. LmdsRNase2 protein was highly expressed in L. migratoria midgut by western blot, nevertheless, there was no detectable signal in midgut fluid. However, LmdsRNase3 could not be detected in both midgut and midgut fluid. Immunofluorescence results showed that both LmdsRNase2 and LmdsRNase3 were expressed in cytoplasm of L. migratoria midgut cells, but the expression level of LmdsRNase3 protein was much lower than that of LmdsRNase2.【Conclusion】Anti-LmdsRNase2 and 3 specific polyclonal antibodies were successfully prepared, western blot and immunofluorescence showed that LmdsRNase2 protein was highly expressed in midgut, whereas the expression of LmdsRNase3 was very low. These results provide evidence of protein level for the functional differentiation of LmdsRNases in the midgut of L. migratoria.

Key words: Locusta migratoria, dsRNA degrading enzyme (dsRNase), polyclonal antibody, subcelluar localization

宋慧芳，张建琴，范云鹤，李涛，马恩波，张建珍. 飞蝗双链RNA降解酶的抗体制备及组织定位[J]. 中国农业科学, 2018, 51(19): 3704-3713.

SONG HuiFang, ZHANG JianQin, FAN YunHe, LI Tao, MA EnBo, ZHANG JianZhen. Antibody Preparation and Subcelluar Localization of dsRNA Degrading Enzyme in Locusta migratoria[J]. Scientia Agricultura Sinica, 2018, 51(19): 3704-3713.

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参考文献

[1] BETTENCORT R, TERENIUS O, FAYE I. Hemolin gene silencing by ds-RNA injected into Cecropia pupae is lethal to next generation embryos. Insect Molecular Biology, 2002, 11(3): 267-271.

[2] TIAN H G, PENG H, YAO Q, CHEN H X, XIE Q, TANG B, ZHANG W Q. Developmental control of a Lepidopteran pest, Spodoptera exigua, by ingestion of bacteria expressing dsRNA of a non-midgut gene. PLoS One, 2009, 4(7): e6225.

[3] YU R R, LIU W M, LI D Q, ZHAO X M, DING G W, ZHANG M, MA E B, ZHU K Y, LI S, MOUSSIAN B, ZHANG J Z. Helicoidal organization of chitin in the cuticle of the migratory locust requires the function of the chitin deacetylase2 enzyme (LmCDA2). The Journal of Biological Chemistry, 2016, 291(47): 24352-24363.

[4] ZHANG J, KHAN S A, HECKEL D G, BOCK R. Next-generation insect-resistant plants: RNAi-mediated crop protection. Trends in Biotechnology, 2017, 35(9): 871-882.

[5] ZHU K Y, MERZENDORFER H, ZHANG W Q, ZHANG J Z, Muthukrishnan S. Biosynthesis, turnover, and functions of chitin in insects. Annual Review of Entomology, 2016, 61(1): 177-196.

[6] SONG H F, ZHANG J Q, LI D Q, COOPER A M W, SILVER K, LI T, LIU X J, MA E B, ZHU K Y, ZHANG J Z. A double-stranded RNA degrading enzyme reduces the efficiency of oral RNA interference in migratory locust. Insect Biochemistry and Molecular Biology, 2017, 86: 68-80.

[7] LIU J S, SWEVERS L, IATROU K, HUVENNE H, SMAGGHE G. Bombyx mori DNA/RNA non-specific nuclease: expression of isoforms in insect culture cells, subcellular localization and functional assays. Journal of Insect Physiology, 2012, 58(8): 1166-1176.

[8] WANG K X, PENG Y C, PU J, FU W X, WANG J L, HAN Z J. Variation in RNAi efficacy among insect species is attributable to dsRNA degradation in vivo. Insect Biochemistry and Molecular Biology, 2016, 77: 1-9.

[9] CAO M, GATEHOUSE J A, FICHES E C. A systematic study of RNAi effects and dsRNA stability in Tribolium castaneum and Acyrthosiphon pisum, following injection and ingestion of analogous dsRNAs. International journal of molecular sciences, 2018, 19(4): 1079.

[10] ARIMATSU Y, FURUNO T, SUGIMURA Y, TOGOH M, ISHIHARA R, TOKIZANE M, KOTANI E, HAYASHI Y, FURUSAWA T. Purification and properties of double-stranded RNA-degrading nuclease, dsRNase, from the digestive juice of the silkworm, Bombyx mori. Journal of Insect Biotechnology and Sericology, 2007, 76(1): 57-62.

[11] GARBUTT J S, BELLES X, RICHARDS E H, REYNOLDS S E. Persistence of double-stranded RNA in insect hemolymph as a potential determiner of RNA interference success: evidence from Manduca sexta and Blattella germanica. Journal of Insect Physiology, 2013, 59(2): 171-178.

[12] WYNANT N, SANTOS D, VERDONCK R, SPIT J, WIELENDAELE P V, BROECK J V. Identification, functional characterization and phylogenetic analysis of double stranded RNA degrading enzymes present in the gut of the desert locust, Schistocerca gregaria. Insect Biochemistry and Molecular Biology, 2014, 46: 1-8.

[13] SPIT J, PHILIPS A, WYNANT N, SANTOS D, PLAETINCK G, VANDEN BROECK J. Knockdown of nuclease activity in the gut enhances RNAi efficiency in the Colorado potato beetle, Leptinotarsa decemlineata, but not in the desert locust, Schistocerca gregaria. Insect Biochemistry and Molecular Biology, 2017, 81: 103-116.

[14] LUO Y, CHEN Q G, LUAN J B, CHUNG S H, VAN ECK J, TURGEON R, DOUGLAS A E. Towards an understanding of the molecular basis of effective RNAi against a global insect pest, the whitefly Bemisia tabaci. Insect Biochemistry and Molecular Biology, 2017, 88: 21-29.

[15] ARIMATSU Y, KOTANI E, SUGMURA Y, FURUSAWA T. Molecular characterization of a cDNA encoding extracellular dsRNase and its expression in the silkworm, Bombyx mori. Insect Biochemistry and Molecular Biology, 2007, 37: 176-183.

[16] 赵静, 孙洋, 谭永安, 肖留斌, 姜义平, 柏立新. 甜菜夜蛾卵黄原蛋白多克隆抗体制备及其在不同发育时期蛋白表达. 中国农业科学, 2017, 50(22): 4316-4324.

ZHAO J, SUN Y, TAN Y A, XIAO L B, JIANG Y P, BAI L X. Polyclonal antibody preparation of Spodoptera exigua vitellogenin and its protein expression at different developmental stages. Scientia Agricultura Sinica, 2017, 50(22): 4316-4324. (in Chinese)

[17] 魏原杰, 王亚美, 黄丽娜, 刘宁, 赵洁, 艾新宇, 刘小宁. 棉蚜P450 CYP6CY3的克隆、原核表达及多克隆抗体的制备. 中国农业科学, 2017, 50(7): 1351-1360.

WEI Y J, WANG Y M, HUANG L N, LIU N, ZHAO J, AI X Y, LIU X N. Cloning, prokaryotic expression and preparation of the polyclonal antibody against CYP6CY3 from Aphis gossypii. Scientia Agricultura Sinica, 2017, 50(7): 1351-1360. (in Chinese)

[18] BRADFORD M M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Analytical Biochemistry, 1976, 72: 248-254.

[19] 宋慧芳, 李应龙, 马恩波, 张建珍. 飞蝗β-N-乙酰氨基葡萄糖苷酶基因的表达及酶学特性分析. 中国农业科学, 2016, 49(21): 4140-4148.

SONG H F, LI Y L, MA E B, ZHANG J Z. The heterogenous expression and enzymatic characteristics of β-N-acetylglucosaminidase from Locusta migratoria. Scientia Agricultura Sinica, 2016, 49(21): 4140-4148. (in Chinese)

[20] LIU X, ZHANG H, LI S, ZHU K Y, MA E, ZHANG J. Characterization of a midgut-specific chitin synthase gene (LmCHS2) responsible for biosynthesis of chitin of peritrophic matrix in Locusta migratoria. Insect Biochemistry and Molecular Biology, 2012, 42(12): 902-910.

[21] FIRE A, XU S Q, MONTGOMERY M K, KOSTAS S A, DRIVER S E, MELLO C C. Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans. Nature, 1998, 391(6669): 806-811.

[22] WANG Y B, ZHANG H, LI H C, MIAO X X. Second-generation sequencing supply an effective way to screen RNAi targets in large scale for potential application in pest insect control. PLoS One, 2011, 6(4): e18644.

[23] ZHOU X G, WHEELER M M, OI F M, SCHARF M E. RNA interference in the termite Reticulitermes flavipes through ingestion of double-stranded RNA. Insect Biochemistry and Molecular Biology, 2008, 38: 805-815.

[24] BAUM J A, BOGAERT T, CLINTON W, HECK G R, FELDMANN P, ILAGAN O, JOHNSON S, PLAETINCK G, MUNYIKWA T, PLFEAU M, VAUGHN T, ROBERTS J. Control of coleopteran insect pests through RNA interference. Nature Biotechnology, 2007, 25(11): 1322-1326.

[25] ZHA W J, PENG X X, CHEN R Z, DU B, ZHU L L, HE G C. Knockdown of midgut genes by dsRNA-transgenic plant-mediated RNA interference in the hemipteran insect Nilaparvata lugens. PLoS one, 2011, 6(5): e20504.

[26] MAO Y B, TAO X Y, XUE X Y, WANG L J, CHEN X Y. Cotton plants expressing CYP6AE14 double-stranded RNA show enhanced resistance to bollworms. Transgenic Research, 2011, 20(3): 665-673.

[27] ZHANG J, KHAN S A, HASSE C, RUF S, HECKEL D G, BOCK R. Full crop protection from an insect pest by expression of long double-stranded RNAs in plastids. Science, 2015, 347(6225): 991-994.

[28] LI D Q, ZHANG J Q, WANG Y, LIU X J, MA E B, SUN Y, LI S, ZHU K Y, ZHANG J Z. Two chitinase 5 genes from locusta migratoria: Molecular characteristics and functional differentiation. Insect Biochemistry and Molecular Biology, 2015, 58: 46-54.

[29] LUO Y, WANG X, WANG X, YU D, CHEN B, KANG L. Differential responses of migratory locusts to systemic RNA interference via double-stranded RNA injection and feeding. Insect Molecular Biology, 2013, 22(5): 574-583.

[30] ARIMATSU Y, SUGIMURA Y, FURUSAWA T. In Vitro processing of the dsRNase precursor isolated from silkworm midgut tissue, Bombyx mori. Journal of Insect Biotechnology and Sericology, 2011, 79(3): 125-127.