CSDC2蛋白免疫优势肽段分析及其多克隆抗体的制备  

Prediction of immunodominant peptide of CSDC2 protein and preparation of polyclonal antibody

作　　者：朱荷青 玛哈巴·肉孜 李莹莹 张洁[1] 许赫 李甜甜 韩吉龙 彭夏雨[1] 杨敏 ZHU Heqing;ROUZI Mahaba;LI Yingying;ZHANG Jie;XU He;LI Tiantian;HAN Jilong;PENG Xiayu;YANG Min(College of Animal Science and Technology,Shihezi University,Shihezi,Xinjiang 832000,China;Xinjiang Uygur Autonomous Regional Animal Husbandry Station,Urumqi,Xinjiang 830000,China)

机构地区：[1]石河子大学动物科技学院,石河子832000 [2]新疆维吾尔自治区畜牧总站,乌鲁木齐830000

出　　处：《石河子大学学报（自然科学版）》2023年第2期193-198,共6页Journal of Shihezi University(Natural Science)

基　　金：国家自然基金项目(31802045);石河子大学青年创新拔尖人才项目(CXBJ201903)。

摘　　要：目的为制备含有C2的冷休克结构域(cold shock domain containing C2,CSDC2)的特异性抗体,本试验预测了CSDC2蛋白的免疫优势肽段并制备多克隆抗体,可为进一步探究其功能奠定基础。方法本研究以绒山羊CSDC2蛋白全长氨基酸序列为基础,应用在线分析工具Expasy分析亲疏水性;SOPMA和Predictprotein共同分析二级结构;DNAstar分析抗原指数、表面可及性、柔韧性;TMHHM 2.0预测跨膜结构;SWISS-MODEL预测三级结构;SignalP 5.0预测信号肽;NetPhos 3.1预测磷酸化位点;SVMTriP在线分析软件预测抗原表位,综上分析获得CSDC2免疫优势肽段,制备多克隆抗体;ELISA检测多克隆抗体效价,Western blot验证抗体特异性。结果CSDC2为不稳定亲水蛋白,无跨膜结构和信号肽区域,具有22个磷酸化位点,二级结构主要由无规则卷曲、α-螺旋和延伸链构成。最终选取绒山羊CSDC2蛋白N-端15-33氨基酸序列(HSPKSPVWPTFPFHREGSR)为特异免疫肽段,诱导兔产生CSDC2蛋白特异性IgG抗体,Western blot结果表明该抗体可以与绒山羊CSDC2蛋白进行特异性结合。结论本研究得出绒山羊CSDC2的免疫优势肽段为15~33 aa(HSPKSPVWPTFPFHREGSR)并免疫试验兔制备了多克隆抗体,进一步实验证明该抗体具有较好生物学效价,为后续深入研究CSDC2在绒山羊绒毛周期性生长调控的作用奠定了理论基础。Objective To prepare specific antibodies of the cold shock domain containing C2(CSDC2),the study predicted the immunodominant peptide containing CSDC2 protein and prepared its polyclonal antibody,which can lay the foundation for further investigation of its function.Methods Based on the full-length amino acid sequence of CSDC2 protein,hydrophobicity and hydrophilia were analyzed by Expasy,and secondary structure of protein was predicted jointly by SOPMA and Predictprotein,and tertiary structure was analyzed by SWISS-MODEL.DNAstar was used to further analyzed antigenicity,surface probability,flexibility and polarity.Transmembrane structure was forecast by TMHHM 2.0,meanwhile,SignalP 5.0,NetPhos 3.1 and SVMTriP was used to predicted signal peptides,phosphorylation sites and epitope separately.In addition,ELISA method was used to measure the titer of the prepared polyclonal antibody,and its specificity and expression in skin was verified by Western blot.Results CSDC2 was an unstable hydrophilic protein,without transmembrane structure and signal peptide region,with 22 phosphorylation sites,and its secondary structure was mainly composed of irregular crimpα-Helix and extension chain.The amino acids 15~33(HSPKSPVWPTFPFHREGSR)at N-terminus was designed as the immunodominant peptide of CSDC2 protein,which induced CSDC2 protein-specific IgG antibody in rabbits.Western blot showed that the antibody can bind to CSDC2 protein specifically and expressed in goat skin.Conclusion The immune dominant peptide of cashmere goat CSDC2 was 15~33 aa(HSPKSPVWPTFPFHREGSR).The polyclonal antibody was prepared by immunizing rabbits.Further experiments proved that the antibody had effective biological titer,which laid a theoretical foundation for the follow-up in-depth study of the role of CSDC2 in the regulation of Cashmere hair follicle periodic growth.

关 键 词：绒山羊 CSDC2 生物信息学分析 毛囊周期性生长 多克隆抗体 

分 类 号：S827[农业科学—畜牧学]