鸡TM9SF2蛋白胞外结构域的原核表达及其家兔抗血清的制备  

Prokaryotic expression of chicken TM9SF2 protein extracellular domain and preparation of rabbit antiserum

作　　者：张敏霞 杜寿文 李昌 廖明 瞿孝云 ZHANG Min-xia;DU Shou-wen;LI Chang;LIAO Ming;QU Xiao-yun(National and Regional Joint Engineering Laboratory for Medicament of Zoonosis Prevention and Control/College of Veterinary Medicine,South China Agricultural University,Guangzhou 510642,China;Key Laboratory for Prevention and Control of Avian Influenza and Other Major Poultry Disease,Ministry of Agriculture and Rural Affairs/Institute of Animal Health,Guangdong Academy of Agricultural Sciences,Guangzhou 510640,China;The Second Clinical Medical College,Jinan University,Shenzhen 518020,China;Changchun Veterinary Research Institute,Chinese Academy of Agricultural Sciences,Changchun 130122,China)

机构地区：[1]华南农业大学兽医学院,人兽共患病防控制剂国家地方联合工程实验室,广东广州510642 [2]广东省农业科学院动物卫生研究所,农业农村部禽流感等重大疾病防控重点实验室,广东广州510640 [3]暨南大学第二临床医学院,广东深圳518020 [4]中国农业科学院长春兽医研究所,吉林长春130122

出　　处：《中国兽医科学》2023年第7期878-883,共6页Chinese Veterinary Science

基　　金：国家自然科学基金项目(31972719);广东省科技计划项目(2021B1212030015);国家重点研发计划项目(2022-YFD1801000);“十四五”广东省农科科技创新十大主攻方向“揭榜挂帅”项目(2022SDZG02);国家肉鸡产业技术体系项目(CARS-41);农业农村部禽流感等重大疾病防控重点实验室开放课题项目(YDWS202204)。

摘　　要：为了探究鸡源九次跨膜超家族成员2(TM9SF2)的生物学功能,通过软件分析TM9SF2蛋白的氨基酸序列保守性,选择其胞外区,经密码子优化后进行基因合成,命名为chTM9ECD;将该基因亚克隆至原核表达载体pET-28a(+)中,成功构建重组表达质粒pET-chTM9ECD,并在大肠杆菌BL21(DE3)中诱导表达;用纯化的重组TM9SF2蛋白与佐剂混合后免疫新西兰大白兔制备多抗血清。SDS-PAGE和Western-blot均证实重组TM9SF2蛋白以包涵体形式表达,分子质量约为33.6 ku。经ELISA检测,抗体效价达1∶128 000,Western-blot证实家兔抗重组TM9SF2血清可特异性识别293细胞中表达的TM9SF2。结论,本试验应用大肠杆菌成功表达了重组TM9SF2胞外蛋白,具有免疫原性,并获得家兔源阳性血清,为TM9SF2生物学功能研究奠定基础。To investigate the biological function of chicken transmembrane 9 superfamily member 2(chTM9SF2),the amino acid sequence conservation of TM9SF2 protein was analyzed by software,and the coding sequences of chTM9SF2 extracellular domain(chTM9ECD)was codon-optimized and artificially synthesized based on the conservative analysis of chTM9SF2 in the amino acid levels,and the chTM9ECD genefragmentwassubclonedintoapET-28a(+)vector,and the recombinant expression plasmid pET-chTM9ECD was successfully constructed and induced to express in E.coli BL21(DE3).The chTM9ECD protein was induced to express and purified to immunize New Zealand white rabbits with the adjuvants.The chTM9ECD protein was expressed as an inclusion body and confirmed by SDS-PAGE and Western-blot,and its molecular mass was about 33.6 ku.The titer of anti-chTM9ECD rabbit serum reached 1∶128000 by ELISA after four times of immunization,and Western-blot confirmed that TM9SF2 rabbit antiserum can specifically recognize TM9SF2 in HEK293 cells.In conclusion,TM9SF2 ectodomain had been successfully expressed in E.coli and used as the immunogen to prepare rabbit antiserum,which provided a foundation for the study of chTM9SF2 physiological function.

关 键 词：鸡 TM9SF2 跨膜蛋白 原核表达 抗血清 

分 类 号：S852[农业科学—基础兽医学]