飞蝗表皮蛋白LmKnk3-5′的抗体制备及组织定位

doi:10.3864/j.issn.0578-1752.210559

摘要/Abstract

摘要： 【目的】LmKnickkopf3-5′(LmKnk3-5′)是飞蝗（Locusta migratoria）蜕皮发育中重要的表皮蛋白。本文旨在制备LmKnk3-5′特异抗体并对其进行组织定位，为LmKnk3-5′生物学功能研究提供蛋白水平的证据，同时为进一步深入探究LmKnk3-5′与其他表皮蛋白在飞蝗表皮形成过程中的协同作用打下基础。【方法】首先比对飞蝗Knickkopf(Knk)家族4个基因LmKnk、LmKnk2、LmKnk3-FL和LmKnk3-5′的氨基酸序列，选取LmKnk3-5′的3段特异抗原序列R1、R2、R3，设计包含BamH I、Hind III酶切位点的引物，以LmKnk3-5′全长cDNA为模板，PCR获得LmKnk3-5′的特异抗原片段；然后将特异抗原片段与pET-32a经双酶切、T4连接酶连接、测序验证、成功构建重组质粒后，转入BL21（DE3）表达菌株中，加入IPTG（终浓度0.5 mmol·L^-1）低温（16℃）诱导重组蛋白表达，SDS-PAGE检测重组蛋白表达情况；接着扩大培养可在菌体裂解液的上清中表达重组融合蛋白的大肠杆菌，提取蛋白并用Ni-NTA对其进行纯化，之后再用纯化后的重组特异抗原免疫小鼠4次，获得anti-LmKnk3-5′多克隆抗体，ELISA法对其进行效价检测，并用western blot法检测其特异性。取飞蝗5龄第8天表皮，制备石蜡切片，通过免疫组化法对LmKnk3-5′蛋白进行组织定位。【结果】通过氨基酸序列比对，选取LmKnk3-5′的特异抗原区域R1、R2和R3，分别包含208、147和131个氨基酸残基，蛋白理论分子量分别为24.0、17.0和14.8 kD。酶切连接后，成功获得 pET-32a-R1、pET-32a-R2 和pET-32a-R3重组质粒。诱导表达检测，发现只有含pET-32a-R2的表达菌在菌体裂解液的上清液中大量表达重组蛋白。将其扩大培养纯化后获得 R2重组融合蛋白，免疫小鼠取抗血清进行效价检测，抗体效价高达1﹕512 000，可满足抗体使用需求。Western blot结果表明，注射dsLmKnk3-5′后飞蝗体壁LmKnk3-5′的蛋白表达显著减少，表明anti-LmKnk3-5′抗体特异性良好。免疫组化试验结果表明，表皮蛋白LmKnk3-5′在飞蝗腹部体壁皮细胞及新表皮中均有分布，特别是新合成的外表皮顶端。【结论】成功获得飞蝗LmKnk3-5′多克隆抗体，证明其特异性良好。LmKnk3-5′在飞蝗腹部体壁新合成的外表皮顶端分布较多。研究结果为飞蝗表皮蛋白LmKnk3-5′功能研究提供了蛋白水平的证据。

关键词: 飞蝗, LmKnk3-5′, 抗体制备, 组织定位

Abstract: 【Objective】LmKnickkopf3-5′ (LmKnk3-5′) is an important cuticular protein involved in the development of Locusta migratoria. The purpose of this paper is to get the anti-LmKnk3-5′ polyclonal antibody and confirm the localization of LmKnk3-5′ in L. migratoria. The results will be beneficial to the biological functional analysis of LmKnk3-5′ from protein level, meanwhile, it can lay the foundation for further study of the interaction of cuticular proteins in cuticle formation in L. migratoria.【Method】Three specific antigen sequences (R1, R2 and R3) of LmKnk3-5′ were chosen after amino acid sequences alignment of Knickkopf (Knk) family genes in L. migratoria, including LmKnk, LmKnk2, LmKnk3-FL and LmKnk3-5′. The target antigen sequences were amplified by PCR used primers with BamH I, Hind III restriction sites and full-length cDNA sequences of LmKnk3-5′ as template. The antigen sequences and pET-32a vector were all digested by BamH I, Hind III and ligated to each other by T4 ligase enzyme to make recombinant plasmids, then the recombinant plasmids were transformed into BL21 (DE3) competent cells. The cells were incubated with 0.5 mmol·L^-1 IPTG at 16℃ for 20 h to produce the recombinant fusion protein, SDS-PAGE was used to analysis the expression of target proteins. After that, E. coli cells that can express target proteins in dissolved state were expanded cultured for protein extraction. Ni-NTA agarose were used for target proteins purification and BCA method were used to determine the protein concentration. The LmKnk3-5′ polyclonal antibodies were obtained after immunizing BALB/c mouse four times. ELISA and western blot were used to analysis the antibody titer and specificity respectively. Finally, the paraffin sections were prepared used the integument of 8-day-old fifth-instar nymphs after dsLmKnk3-5′ and dsGFP injection, immunofluorescence were conducted to confirm the subcellular localization of LmKnk3-5′ in L. migratoria.【Result】R1, R2 and R3 were selected as specific antigen regions through amino acid sequences alignment. R1, R2, R3 consist of 208, 147 and 131 aa. and the predicted molecular weight of them are 24.0, 17.0 and 14.8 kD, respectively. Three recombinant plasmids (pET-32a-R1, pET-32a-R2, pET-32a-R3) were obtained successfully after enzyme digestion and ligation. SDS-PAGE analysis showed that only the cells consist of pET-32a-R2 plasmids can express target proteins in dissolved state after induced by IPTG. R2 recombinant fusion protein were purified and used to obtain anti-LmKnk3-5′ polyclonal antibody successfully after immunizing mice. ELISA analysis indicated that the titer of LmKnk3-5’ antibody was up to 1﹕512 000. The results of western blot demonstrated that after dsLmKnk3-5′ injection, the expression of LmKnk3-5′ was significantly decreased in comparison of the dsGFP injection group. The results of immunofluorescence showed that LmKnk3-5′ was located in the epidermal cells and new cuticle, especially the apical site of exocuticle in L. migratoria. 【Conclusion】Anti-LmKnk3-5′ polyclonal antibody were obtained successfully, it has high titer and specificity. LmKnk3-5′ was mainly located in the apical site of exocuticle newly synthesized in L. migratoria. The results will provide protein level evidence for the functional research of LmKnk3-5′ in the cuticle formation of L. migratoria.

Key words: Locusta migratoria, LmKnk3-5’, Antibody preparation, Subcellular localization

张睿, 张学尧, 赵小明, 马恩波, 张建珍. 飞蝗表皮蛋白LmKnk3-5′的抗体制备及组织定位[J]. 中国农业科学, 0, (): 1-.

ZHANG Rui, ZHANG XueYao, ZHAO XiaoMing, MA EnBo, ZHANG JianZhen. Antibody Preparation and Subcellular Localization of LmKnk3-5′ in Locusta migratoria[J]. Scientia Agricultura Sinica, 0, (): 1-.

[1]	张睿,张学尧,赵小明,马恩波,张建珍. 飞蝗表皮蛋白LmKnk3-5′的抗体制备及组织定位[J]. 中国农业科学, 2022, 55(2): 329-338.
[2]	刘晓健,郭俊,张学尧,马恩波,张建珍. 飞蝗核受体基因LmE75的分子特性和功能分析[J]. 中国农业科学, 2020, 53(11): 2219-2231.
[3]	马雯,刘娇,张学尧,申国华,秦雪梅,张建琴. 飞蝗LmGSTS2的酶学特性及其对马拉硫磷、 p,p’-DDT的代谢分析[J]. 中国农业科学, 2019, 52(8): 1389-1399.
[4]	贾盼,张晶,杨洋,刘卫敏,张建珍,赵小明. 飞蝗内表皮结构糖蛋白基因LmAbd-2的表达与功能分析[J]. 中国农业科学, 2019, 52(4): 651-660.
[5]	杨亚亭，赵小明，秦忠玉，刘卫敏，马恩波，张建珍. 飞蝗表皮蛋白基因LmNCP1的分子特性及功能分析[J]. 中国农业科学, 2018, 51(7): 1303-1314.
[6]	张奎，潘光照，苏晶晶，谈娟，徐曼，李钰添，崔红娟. 家蚕glial cell missing (BmGcm)基因鉴定、表达、亚细胞定位和功能[J]. 中国农业科学, 2018, 51(7): 1401-1411.
[7]	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.
[8]	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.
[9]	赵盼，张学尧，刘晓健，赵小明，于荣荣，董玮，马恩波，张建珍，张敏. 飞蝗几丁质脱乙酰基酶的真核表达、亲和纯化及酶活性[J]. 中国农业科学, 2017, 50(6): 1057-1066.
[10]	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[J]. 中国农业科学, 2017, 50(22): 4316-4324.
[11]	于荣荣，丁国伟，刘卫敏，张敏，赵小明，韩鹏飞，马恩波，张建珍. 飞蝗几丁质脱乙酰基酶基因的分子特性和生物学功能[J]. 中国农业科学, 2017, 50(13): 2498-2507.
[12]	赵小明，贾盼，勾昕，刘卫敏，马恩波，张建珍. 飞蝗内表皮蛋白基因LmAbd-5的表达与功能分析[J]. 中国农业科学, 2017, 50(10): 1817-1826.
[13]	刘晓健，孙亚文，崔淼，马恩波，张建珍. 飞蝗海藻糖酶基因的分子特性及功能[J]. 中国农业科学, 2016, 49(22): 4375-4386.
[14]	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[J]. 中国农业科学, 2016, 49(21): 4140-4148.
[15]	王艳丽，杨美玲，宋天琪，马恩波，张建珍. 飞蝗Argonaute1的分子特性及生物学功能[J]. 中国农业科学, 2016, 49(18): 3569-3577.

飞蝗表皮蛋白LmKnk3-5′的抗体制备及组织定位

Antibody Preparation and Subcellular Localization of LmKnk3-5′ in Locusta migratoria

赞

可视化

摘要/Abstract

引用本文

使用本文

参考文献

相关文章 15

Metrics

本文评价

推荐阅读 0