利用噬菌体肽库技术筛选猪生长激素抗原模拟表位

发布时间：2018-06-30 05:40

本文选题：猪生长激素 + 单克隆抗体　；参考：《延边大学》2011年硕士论文

【摘要】：猪生长激素是垂体分泌的多肽激素,与生长发育和繁殖功能有关,大量研究证明,猪生长激素能显著提高猪生长速度、瘦肉率和饲料报酬,对猪的生产、育种和饲料的调配产生重大的影响。本实验室对猪生长激素的研究是针对抗原表位的研究。本研究主要结果如下： 1.用实验室制备的杂交瘤细胞,注射到液体石蜡免疫一周后的经产BALB/c小鼠,腹水采用辛酸-饱和硫酸法进行粗提,再经层析系统纯化蛋白,最后用SDS-聚丙烯酞胺凝胶电泳检测其纯度,间接ELISA测定其OD490值,杂交瘤细胞培养上清效价为1：1600,制得的小鼠腹水效价为1：1.6×104 2.用猪生长激素单克隆抗体作为靶蛋白,对噬菌体随机12肽库进行了四轮淘洗,噬菌体克隆回收率从1.1×10-8增加到1.0×10-2,目标噬菌体克隆得到了有效富集。随机挑取47个蓝色噬菌斑扩增后,经间接ELISA检测,发现有9个噬菌斑克隆与单克隆抗体有较强的特异性结合,将该9个阳性克隆提取ssDNA进行序列测定及比对分析,结果中有7个阳性克隆的外源肽序列完全一致,与氨基酸序列进行比对,得到同源序列NxxxTSx。这些模拟表位对猪生长激素表位研究具有一定的参考价值。为进一步分析猪生长激素的结构与功能的研究奠定基础,
[Abstract]:Porcine growth hormone is a polypeptide hormone secreted by the pituitary gland, which is related to growth, development and reproductive function. A large number of studies have proved that pig growth hormone can significantly improve the growth rate, lean meat rate and feed reward of pigs. Breeding and forage blending have a significant impact. The study of porcine growth hormone in our laboratory is aimed at antigen epitopes. The main results of this study are as follows: 1. The hybridoma cells prepared in the laboratory were injected into liquid paraffin to immunize BALB / r / c mice one week later. Ascites were extracted by octanoic acid-saturated sulfuric acid method, and the protein was purified by chromatography system. The purity was detected by SDS-polyacrylamide gel electrophoresis, the OD490 value was determined by indirect Elisa, the titer of supernatant of hybridoma cell culture was 1: 1600, and the titer of mouse ascites was 1: 1.6 脳 104.2. Using porcine growth hormone monoclonal antibody as the target protein, the phage random 12 peptide library was washed for four rounds. The phage clone recovery increased from 1.1 脳 10 ~ (-8) to 1.0 脳 10 ~ (-2), and the target phage clone was effectively enriched. After random amplification of 47 blue plaque, the results of indirect Elisa showed that 9 clones of plaque had strong specific binding with monoclonal antibody, and the 9 positive clones were extracted from ssDNA for sequence analysis and comparative analysis. Results the sequence of exogenous peptide of 7 positive clones was identical with amino acid sequence, and the homologous sequence NxxxTSxwas obtained. These mimic epitopes have some reference value for the study of porcine growth hormone epitopes. It lays a foundation for further study on the structure and function of porcine growth hormone.
【学位授予单位】：延边大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R392

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