鸭抗原相关转运体TAP1和TAP2原核表达及多克隆抗体的制备  

Prokaryotic expressions and polyclonal antibody preparations of duck transporter associated with antigen processing (TAP) 1 and TAP 2

作　　者：张晓娜[1] 吴少莲 文辉强[1] 廉传江[1] 陈洪岩[1] 韩凌霞[1] 

机构地区：[1]中国农业科学院哈尔滨兽医研究所兽医生物技术国家重点实验室/实验动物与比较医学研究团队,哈尔滨150001

出　　处：《黑龙江畜牧兽医》2015年第6期40-43,共4页Heilongjiang Animal Science And veterinary Medicine

基　　金：中央级公益性科研院所基本科研业务费项目(0302014021)

摘　　要：HBK鸭是国家禽类实验动物种子中心培育的我国首个无特定病原体(SPF)鸭,已封闭饲养9个世代。鸭抗原处理相关转运体(TAP)基因位于主要组织相容性复合体区域,TAP1和TAP2组成的二聚体具有将抗原肽从细胞质转运入内质网腔的重要作用。为了表达鸭抗原相关转运体蛋白,并制备特异性多克隆抗体,试验采用PCR方法扩增HBKB系鸭的TAP1完整基因和TAP2基因的预测肽结合区(PBD),分别命名为TAP1和TAP2PBD,克隆至原核表达载体pET-30a(+)中,获得重组质粒p ET-TAP1和p ET-TAP2PBD,转化大肠杆菌BL21感受态细胞,经IPTG诱导表达后进行SDSPAGE及Western-blot鉴定,表达产物经SDS-PAGE后分别切胶免疫Balb/c小鼠制备多克隆抗体。结果表明:两种目的蛋白均获得了正确表达。说明试验制备了高特异性鸭TAP1和TAP2多克隆抗体,可用于水禽TAP的结构和功能研究。HBK duck is China＇ s first specific pathogen free （SPF） duck bred by the national seed center for poultry laboratory animals, which has been closely reared for nine generations. The duck transporter associated with antigen processing （TAP） genes locate in the major histocom- patibility complex （MHC） region, and the dimer composed of TAP1 and TAP2, plays an important role on transport of antigenic peptldes from the cytosol to the lumen of the endoplasmic reticulum （ER）. To express the duck TAP proteins and prepare their specific polyclonal antibodies, the PCR assay was used to amplify the whole gene of TAP1 and the predicted peptide binding domain （PBD） of the TAP2 gene from the B line of HBK - SPF ducks. The PCR products were named as TAP1 and TAP2PBD, and then were cloned into the prokaryotic expression vector pET- 30a（ ＋ ） to obtain the recombinant plasmids pET- 30 -TAP1 and pET- 30 -TAP2PBD. The recombinant plasmids were transformed into E. coli BL- 21 （DE3）, and then the expressed recombinant proteins were identified by SDS -PAGE and Western blot after IPTG induced expression. The Balb/c mice were immunized with the recombinant proteins from the SDS - PAGE gel slice to prepare polyclonal antibodies. The results indicate that the two kinds of target proteins were correctly expressed. The result indicates that the duck TAP1 and TAP2 polyclonal antibodies with high specificity are prepared, which can be used to study the structure and function of waterfowl TAP.

关 键 词：鸭 抗原相关转运体 肽结合区 原核表达 多克隆抗体 

分 类 号：S834[农业科学—畜牧学]