[1]高文昊,杜露平,侯立婷,等.猪CD205分子CysR-FN Ⅱ截短基因的克隆表达及应用[J].江苏农业学报,2022,38(05):1272-1277.[doi:doi:10.3969/j.issn.1000-4440.2022.05.014]
 GAO Wen-hao,DU Lu-ping,HOU Li-ting,et al.Cloning, expression and application of CysR-FN Ⅱ truncated gene of porcine CD205[J].,2022,38(05):1272-1277.[doi:doi:10.3969/j.issn.1000-4440.2022.05.014]
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猪CD205分子CysR-FN Ⅱ截短基因的克隆表达及应用()
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江苏农业学报[ISSN:1006-6977/CN:61-1281/TN]

卷:
38
期数:
2022年05期
页码:
1272-1277
栏目:
畜牧兽医·水产养殖
出版日期:
2022-10-31

文章信息/Info

Title:
Cloning, expression and application of CysR-FN Ⅱ truncated gene of porcine CD205
作者:
高文昊12杜露平13侯立婷13于晓明13陈瑾13冯秀丽2郑其升13程海卫13
(1.江苏省农业科学院动物免疫工程研究所/国家兽用生物制品工程技术研究中心/江苏省食品质量与安全重点实验室,江苏南京210014;2.南京农业大学动物医学院,江苏南京210095;3.江苏省动物重要疫病与人兽共患病防控协同创新中心,江苏扬州225009)
Author(s):
GAO Wen-hao12DU Lu-ping13HOU Li-ting13YU Xiao-ming13CHEN Jin13FENG Xiu-li2ZHENG Qi-sheng13CHENG Hai-wei13
(1. Institute of Veterinary Immunology & Engineering, Jiangsu Academy of Agricultural Sciences/National Research Center of Engineering and Technology for Veterinary Biologicals/Jiangsu Key Laboratory for Food Quality and Safety, Nanjing 210014, China;2.College of Veterinary Medicine, Nanjing Agricultural University, Nanjing 210095, China;3.Jiangsu Co-innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses, Yangzhou 225009, China)
关键词:
CD205CysR-FN Ⅱ截短基因原核表达生物学活性
Keywords:
pigCD205CysR-FN Ⅱ truncated geneprokaryotic expressionbiological activity
分类号:
S852.4
DOI:
doi:10.3969/j.issn.1000-4440.2022.05.014
文献标志码:
A
摘要:
为克隆表达编码猪CD205分子的CysR-FN Ⅱ截短基因片段,并验证其生物学活性,提取了猪淋巴组织总RNA,并合成cDNA,利用PCR方法扩增编码猪CD205分子的CysR-FN Ⅱ截短基因,构建pET-32a-CysR-FN Ⅱ原核重组表达质粒,转化至大肠杆菌BL21,分别进行诱导表达与蛋白质纯化,利用制备的目的蛋白免疫小鼠获得多克隆抗体,经间接免疫荧光试验和流式细胞分析鉴定。SDS-PAGE与Western Blot鉴定结果显示,目的蛋白以可溶性形式表达,大小约4.0×104,与理论值一致。一次免疫后14 d抗体效价达1∶3 200。间接免疫荧光试验和流式分析结果显示,获得的多克隆抗体可与猪骨髓来源树突状细胞发生特异性结合。说明该重组表达蛋白质具有相应的生物学活性,可用于猪CD205抗原靶向研究。
Abstract:
To clone and express the truncated CysR-FNⅡ gene fragment encoding porcine CD205 molecule and verify its biological activity, the total RNA of porcine lymphoid tissue was extracted, and cDNA was synthesized. The CysR-FNⅡ truncated gene encoding porcine CD205 molecule was amplified by PCR method, the recombinant expression plasmid pET-32a-CysR-FNⅡ was constructed and transformed into Escherichia coli BL21, and the induced expression and protein purification were performed, respectively. The polyclonal antibodies were obtained by immunizing mice with the prepared target protein, and identified by indirect immunofluorescence assay and flow cytometry. The results of SDS-PAGE and Western blot showed that the target protein was expressed in a soluble form, with a size of about 4.0×104, which was consistent with the theoretical value. The antibody titer reached 1∶3 200 after 14 days of primary immunization. The results of indirect immunofluorescence assay and flow cytometry analysis indicated that the polyclonal antibodies obtained in this study could specifically bind to porcine bone marrow-derived dendritic cells. The recombinant protein has the corresponding biological activity and can be used for the study of porcine CD205 antigen targeting.

参考文献/References:

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备注/Memo

备注/Memo:
收稿日期:2022-06-21基金项目:国家自然科学基金项目(32102690);江苏省农业科技自主创新项目[CX(20)3096]作者简介:高文昊(1999-),男,河南许昌人,学士,主要从事兽用免疫增强剂研究。(E-Mail)wenhao.gao@outlook.com通讯作者:程海卫,(E-Mail)chw5673@126.com
更新日期/Last Update: 2022-11-07