飞蝗几丁质酶5-1抗体制备及表达特性分析

doi:10.3864/j.issn.0578-1752.2018.12.018

摘要/Abstract

摘要： 【目的】通过制备飞蝗（Locusta migratoria）几丁质酶5-1（LmCht5-1）的多克隆抗体，建立飞蝗体内LmCht5-1蛋白水平检测方法，分析LmCht5-1在4龄飞蝗的表达特性及组织定位。【方法】采用MEGA 软件对LmCht5-1和LmCht5-2氨基酸序列进行比对，利用Expression网站对LmCht5-1氨基酸序列进行抗原表位预测分析，获得LmCht5-1抗原结构区域；以飞蝗cDNA为模板，通过PCR扩增LmCht5-1的抗原片段；同时通过酶切连接构建含有鸡血清白蛋白OVA的载体pET32a-OVA；然后通过酶切连接将LmCht5-1插入到pET32a-OVA载体构建pET32a-OVA-LmCht5-1；将pET32a-OVA-LmCht5-1转入表达菌株BL21（DE3）中，IPTG诱导表达重组蛋白OVA-LmCht5-1；利用Ni-NTA纯化后免疫新西兰白兔，制备多克隆抗体。通过ELISA方法检测抗体效价；利用Western blot检测抗体特异性。提取4龄飞蝗不同日龄表皮，采用Western blot分析LmCht5-1蛋白表达模式。对4龄飞蝗注射dsLmCht5-1，检测蛋白水平LmCht5-1表达量，并通过免疫组化方法对LmCht5-1进行定位及功能分析。【结果】通过LmCht5-1和LmCht5-2序列比对和抗原表位预测分析，获得LmCht5-1的471—533AA可作为抗原区域；通过酶切连接分别获得pET32a-OVA和pET32a-OVA-LmCht5-1。经诱导表达和纯化后获得重组蛋白OVA-LmCht5-1，分子量为71.34 kD；免疫后制备多克隆抗体OVA-LmCht5-1，ELISA检测效价为1﹕102 400。多克隆抗体OVA-LmCht5-1用于Western blot检测时可特异性识别LmCht5-1，与LmCht5-2蛋白无交叉反应。利用Western blot方法检测4龄若虫各日龄表皮中LmCht5-1蛋白的表达，发现LmCht5-1蛋白随发育日龄其表达量逐渐增加，在蜕皮当天达到峰值，蜕皮后快速降至最低点。对飞蝗若虫N4D2注射dsLmCht5-1，检测到在N4D5时，LmCht5-1的转录水平和蛋白水平均被显著抑制，抑制率分别为70.0%和73.6%。选取注射dsLmCht5-1和dsGFP的4龄飞蝗表皮，进行免疫组化实验显示LmCht5-1定位于表皮细胞和旧表皮中，其功能失活引起旧表皮中几丁质不降解。【结论】获得飞蝗LmCht5-1多克隆抗体可特异性识别LmCht5-1，可用于Western blot和免疫组化检测。明确LmCht5-1在飞蝗4龄若虫蜕皮当天表达最高，定位于表皮细胞和旧表皮中。dsLmCht5-1可减少表皮细胞和旧表皮中LmCht5-1的表达，阻碍旧表皮中几丁质的降解。

关键词: 飞蝗, 几丁质酶5-1, 抗体制备, 表达分析, Western blot

Abstract: 【Objective】 The objective to this study is to prepare polyclonal antibody of Locusta migratoria chitinase5-1 (LmCht5-1), establish method for detection of LmCht5-1 protein, understand the expression pattern of LmCht5-1 protein in the 4thinstar nymph and finally confirm its location and function in vivo.【Method】LmCht5-1 antigen structure area, which was selected by sequence alignment between LmCht5-1 and LmCht5-2 with MEGA software and antigen epitope prediction analysis with the website of Expression, was amplified using cDNA of L. migratoria as the template. The LmCht5-1 antigen sequence was cloned into expression vector pET32a-OVA, which contains ovalbumin to improve immunogenicity, to generate the antigen expression recombinant plasmid pET32a-OVA-LmCht5-1. pET32a-OVA-LmCht5-1 was then transformed into E. coli strain BL21 (DE3). The IPTG induced OVA-LmCht5-1 was purified through Ni-NTA system and used as the antigen to generate polyclone antibody by rabbit immunization. The titer and specificity of OVA-LmCht5-1 were detected by ELISA and Western blot analysis, respectively. Then the expression pattern of LmCht5-1 in cuticle of the 4th instar nymph was analyzed in parallel by Western blot. Finally, the intracellular localization and function of LmCht5-1 in the 4thinstar nymph cuticle were detected by immunohistochemical analysis after the dsLmCht5-1 injection. 【Result】The 5′ fragment (471-533AA) of LmCht5-1, which was selected as the antigen area with the sequence alignment between LmCht5-1 and LmCht5-2 and the epitope prediction analysis, was inserted into pET32a-OVA to obtain recombinant plasmid pET32a-OVA-LmCht5-1. The 71.34 kD recombinant protein OVA-LmCht5-1 was expressed, purified and immunized with rabbit to generate polyclone antibody. The titer of anti-serum of OVA-LmCht5-1 was 1﹕102 400 by ELISA detection. Antibody OVA-LmCht5-1 could accurately distinguish the LmCht5-1 but not LmCht5-2 in Western blot analysis. Further, it was found that the protein expression level of LmCht5-1 increased gradually with the age in the 4th instar nymph of L. migratoria and reached to the highest at the same day of molting, while it declined to the lowest after molting. Injection dsLmCht5-1 into the 4th instar nymph led to the expression of LmCht5-1 significantly declined to 70.0% in mRNA level and 73.6% in protein level. The immunohistochemical analysis with the epidermis of L. migratoria after dsLmCht5-1 injection, showed that LmCht5-1 was located in old cuticle and epidermis cell. Finally, it was found that the inhibition of protein expression of LmCht5-1 led to the chitin degradation blocking in old cuticle. 【Conclusion】 High titer and specificity of LmCht5-1 antibodies, which could be used for Western blot and immunohistochemistry analysis were obtained. The protein expression of LmCht5-1 reached to the highest level at the day of molting in 4th instar nymph. LmCht5-1 was located in the epidermis cell and old cuticle in the 4th instar nymph. Moreover, the injection of dsLmCht5-1 to L. migratoria could inhibit the protein expression of LmCht5-1 in epidermis cells and old cuticle, and finally caused the chitin degradation deficiency in old cuticle.

Key words: Locusta migratoria, LmCht5-1, polyclonal antibody preparation, expression pattern analysis, Western blot

张婷婷，柳伟伟，高璐，李任建，付穗业，刘晓健，李大琪，刘卫敏，董卿，张建珍. 飞蝗几丁质酶5-1抗体制备及表达特性分析[J]. 中国农业科学, 2018, 51(12): 2418-2428.

ZHANG TingTing, LIU WeiWei, GAO Lu, LI RenJian, FU SuiYe, LIU XiaoJian, LI DaQi, LIU WeiMin, DONG Qing, ZHANG JianZhen. The antibody preparation and expression analysis of Chitinase 5-1 in Locusta migratoria [J]. Scientia Agricultura Sinica, 2018, 51(12): 2418-2428.

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