动物营养学报 >

2013 , Vol. 25 >Issue 5: 1025 - 1036

DOI: https://doi.org/10.3969/j.issn.1006-267x.2013.05.018

分子营养 Molecular Nutrition

兔抗鸡碱性氨基酸转运载体b0,+AT多克隆抗体的制备及其效价和特异性分析

  • 孙育平 ,
  • 甘鲁飞 ,
  • 夏伟光 ,
  • 冯定远 ,
  • 左建军
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  • 华南农业大学动物科学学院,广州 510642

收稿日期: 2013-01-30

  网络出版日期: 2013-04-12

基金资助

国家973资金资助(2012CB124701)

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Rabbit Anti-Chicken Polyclonal Antibodies against b0,+AT, A Basic Amino Acid Transporter: Preparation and Specificity Analysis

  • SUN Yuping ,
  • GAN Lufei ,
  • XIA Weiguang ,
  • FENG Dingyuan ,
  • ZUO Jianjun
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  • College of Animal Science, South China Agricultural University, Guangzhou 510642, China

Received date: 2013-01-30

  Online published: 2013-04-12

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摘要

为了获得兔抗鸡碱性氨基酸转运载体b0,+AT多克隆抗体,并对多克隆抗体进行特异性分析,本试验利用鸡b0,+AT cDNA全序列(GenBank登录号HQ338713)进行生物信息学分析,预测其蛋白质理化性质、跨膜区域、二级结构及抗原表位,并设计出1段抗原多肽;构建鸡pET-32a(+)-b0,+AT质粒,原核表达融合蛋白经纯化后,注射于新西兰大白兔,经3次加强免疫后,制备兔抗鸡b0,+AT多克隆抗体,并采用酶联免疫分析(ELISA)和蛋白质免疫印迹(Western blot)技术分析其效价和特异性。结果如下:1)鸡肠道b0,+AT分子质量为53.8 ku,等电点为8.27,编码493个氨基酸,其中含33个强碱性氨基酸残基(K、R)、28个强酸性氨基酸残基(D、E)、235个疏水性氨基酸残基(A、I、L、F、W、V)、122个极性氨基酸残基(N、C、Q、S、T、Y);经推测,b0,+AT有12个跨膜螺旋结构,由32.05% α-螺旋、25.96%延伸链和41.99%无规则卷曲组成。2)成功构建了鸡pET-32a(+)-b0,+AT质粒并获得兔抗鸡b0,+AT多克隆抗体,抗体效价可达到1:204 800,且特异性较好。3)利用制备的兔抗鸡b0,+AT多克隆抗体作为一抗,肠道组织膜蛋白为抗原,采用Western blot验证获得预期的特异性条带,一抗稀释比例为1:4 000。结果提示,本试验成功制备了与膜蛋白b0,+AT特异性较好的多克隆抗体,同时其在鸡肠道组织中具有较好的应用效果。

关键词： 鸡; 碱性氨基酸转运载体; b0,+AT; 多克隆抗体

本文引用格式

孙育平 , 甘鲁飞 , 夏伟光 , 冯定远 , 左建军 . 兔抗鸡碱性氨基酸转运载体b0,+AT多克隆抗体的制备及其效价和特异性分析[J]. 动物营养学报, 2013 , 25(5) : 1025 -1036 . DOI: 10.3969/j.issn.1006-267x.2013.05.018

Abstract

To generate rabbit anti-chicken polyclonal antibodies against b0,+AT (a basic amino acid transporter), and identify its specificity, an antigen peptide was designed after bioinformatics analysis and forecast of physical and chemical properties, transmembrane regions, secondary structure and antigen epitope of b0,+AT according to its cDNA complete sequence (GenBank No. HQ338713). Then, chicken b0,+AT gene was cloned into pET-32a(+) vector to construct a plasmid of pET-32a(+)-b0,+AT, and transformed into E. coli BL21, in which the fusion protein was induced to express. Rabbits were immunized with purified fusion protein for 3 times, and the antiserum was collected. Titer and specificity of b0,+AT antibody were detected by ELISA and Western blot. The results showed as follows: 1) chicken intestinal b0,+AT with a molecular weight 53.8 ku and isoelectric point 8.27 encodes 493 amino acids, including 33 alkaline amino acid residues (K, R), 28 acidic amino acid residues (D, E), 235 hydrophobic amino acid residues (A, I, L, F, W, V), and 122 polar amino acid residues (N, C, Q, S, T, Y). It was inferred that b0,+AT had 12 transmembrane helices, which was composed of 32.05% of alpha screw, 25.96% extension chain and 41.99% random coil. 2) Expressive plasmid of pET-32a-b0,+AT was successfully constructed, and rabbit anti-chicken polyclonal antibody against b0,+AT with good specificity was obtained. The titer of purified polyclonal antibody was 1:204 800. 3) The anticipated specificity band was presented by Western blot with reaction of purified rabbit anti-chicken polyclonal antibody against b0,+AT and intestinal tissue membrane protein, and antibody appropriate dilution ratio was 1:4 000. The results suggest that the rabbit anti-chicken polyclonal antibody against b0,+AT with good specificity to membrane protein a successfully prepared and presents a good reaction to intestinal b0,+AT protein.

Key words： chicken; basic amino acid transporter; b0,+AT; polyclonal antibody

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