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(Westernblot)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。
To generate rabbit anti-chicken polyclonal antibodies against bo＇＋AT （ a basic amino acid transport- er）, 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 bo＇＋AT according to its cDNA complete sequence （GenBank No. HQ338713 ）. Then, chicken bo＇＋AT gene was cloned into pET-32a（ ＋ ） vector to construct a plasmid of pET-32a（ ＋ ）-bo＇＋AT, and transformed into E. coli BL21, in which the fusion protein was induced to express. Rabbits were immunized with purified fusion pro- tein for 3 times, and the antiserum was collected. Titer and specificity of bo＇＋AT antibody were detected by ELISA and Western blot. The results showed as follows ： 1 ） chicken intestinal bo＇＋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 bo＇＋AT had 12 transmembrane heli- ces, which was composed of 32.05% of alpha screw, 25.96% extension chain and 41.99% random coil. 2） Expressive plasmid of pET-32abo＇＋AT was successfully constructed, and rabbit anti-chicken polyclonal anti- body against bo＇＋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 bo＇＋AT and intestinal tissue membrane protein, and antibody appropri- ate dilution ratio was 1：4 000. The results suggest that the rabbit anti-chicken polyclonal antibody against bo＇＋AT with good specificity to membrane protein a successfully prepared and presents a good reaction to intestinalbo＇＋ATprotein.
出处
《动物营养学报》
CAS
CSCD
北大核心
2013年第5期1025-1036,共12页
CHINESE JOURNAL OF ANIMAL NUTRITION
基金
国家973资金资助(2012CB124701)
