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B19和B21单倍型鸡NK细胞受体B-NK19和B-NK21的多克隆抗体制备及鉴定 被引量：2

Preparation and characterization of polyclonal antibodies against haplotype chicken natural killer cell receptor B-NK19 and B-NK21
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摘要 目的利用大肠杆菌系统表达对多种禽病存在敏感性差异的B19和B21单倍型鸡NK细胞抑制性受体B-NK基因,并制备其多克隆抗体。方法分别提取B19和B21鸡外周血淋巴细胞总RNA,采用反转录PCR法扩增B-NK基因(分别命名为B-NK19和B-NK21),克隆至pET-30a原核表达载体中,经酶切和PCR鉴定构建重组质粒。转入到大肠杆菌BL21(DE3)中诱导表达,SDS-PAGE分析外源蛋白的表达。将含有与组氨酸(His)标签融合表达的外源蛋白胶条切下,多点皮下免疫新西兰兔,分别制备B-NK19和B-NK21特异性多克隆抗体。利用Western blot法鉴定其组织相容性复合体(MHC)单倍型特异性。结果成功构建了原核表达重组质粒pET-B-NK19和pET-B-NK21,利用IPTG在37℃诱导4 h后目的蛋白获得表达,免疫5次后收获兔多克隆抗体,Western blot表明B-NK19和B-NK21蛋白存在免疫学交叉反应和部分特异性。结论对多种禽病具有敏感性差异的MHC单倍体型B19和B21鸡的NK细胞抑制性受体B-NK蛋白存在抗原反应交叉性,其多克隆抗体具有部分特异性。 Objective To express natural killer（NK） cell inhibitory receptor gene B-NK of B19 and B21 haplotype chicken,which show different susceptible to various avian diseases,using prokaryotic expression system,and to prepare B-NK19 and B-NK21-specific rabbit polyclonal antibodies. Methods B-NK genes of B19（ named as B-NK19） and B21（ named as B-NK21） chicken were amplified by reverse transcription PCR from peripheral blood lymphocytes of one B19 or B21 chicken,respectively,and then inserted into an expression vector of pET-30 a. The recombinant plasmids of pET-B-NK19 and pETB-NK21 were transformed into E. coli（ DE3） and induced to express,respectively. Both His-tagged foreign proteins of B-NK19 and B-NK21 were seperated by SDS-PAGE and immunized to New Zealand rabbits to prepare polyclonal antibodies.The MHC-specificities of the sera were analyzed by Western blotting. Results The chicken NK cell inhibitory receptor gene B-NK from MHC haplotypes B19 and B21 were successfully amplified,cloned and expressed in E. coli after induction with1. 0 mmol/mL IPTG for 4 h at 37℃,respectively. Western blotting revealed that the prepared rabbit serum antibodies positively reacted to the expressed foreign proteins. Conclusion Chicken NK cell inhibitory receptor proteins B-NK of B19 and B21 haplotypes could immunologically cross-react and their polyclonal antibodies are partially specific to B complex.
作者 杨超 张晓娜 许峰铭 文辉强 廉传江 陈洪岩 韩凌霞
机构地区 中国农业科学院哈尔滨兽医研究所
出处 《细胞与分子免疫学杂志》 CAS CSCD 北大核心 2014年第12期1274-1277,共4页 Chinese Journal of Cellular and Molecular Immunology
基金 国家高技术研究发展技划(863)(2011AA100305) 国家自然科学基金(31101800) 国家科技支撑项目(2013BAK11B02)
关键词 鸡NK细胞受体 B-NK B19单倍型 B21单倍型 多克隆抗体 chicken natural killer cell receptor B-NK B21 haplotype B19 haplotype polyclonal antibody
分类号 R392.11 [医药卫生—免疫学] R392-33 [医药卫生—免疫学]
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参考文献19

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共引文献19

同被引文献26

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细胞与分子免疫学杂志
2014年 第12期

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