新城疫病毒7793株HN基因片段原核表达及功能鉴定

中国生物工程杂志

2013, Vol. 33

Issue (8): 32-37

研究报告

新城疫病毒7793株HN基因片段原核表达及功能鉴定

梁莹, 刘金颖, 樊晓辉, 宋德志, 肖庆, 殷君, 冯安林, 杨利, 周丹旎, 赖振屏

广西医科大学南宁 530022

The Prokaryotic Expression and Identification of HN Gene Fragment of Newcastle Disease Virus Strain 7793

LIANG Ying, LIU Jin-ying, FAN Xiao-hui, SONG De-zhi, XIAO Qing, YIN Jun, FENG An-lin, YANG Li, ZHOU Dan-ni, LAI Zhen-pin

Guangxi Medical University, Nanning 530022, China

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摘要： HN蛋白是新城疫病毒(Newcastle disease virus,NDV)的血凝素神经氨酸酶蛋白(hemagglutinin-neuraminidase,HN),是NDV识别宿主细胞的包膜蛋白。NDV-HN能与NK细胞的NKp46受体结合,活化NK细胞上调TNF-α和IFN-γ发挥抗瘤效应^[1]。然而HN蛋白是否能直接活化NK细胞上调TNF相关凋亡诱导配体(TNF-related apoptosis-inducing ligand ,TRAIL)尚待阐明。因此对新城疫病毒7793株(NDV 7793)HN基因主要抗原表位区域进行克隆和原核表达,并对重组HN(recombinant HN,rHN)蛋白是否能活化NK细胞上调TRAIL进行研究。流式细胞分析表明rHN蛋白的纯化产物能与小鼠胸腺的NK细胞膜分子NKp46结合,并上调NK细胞TRAIL的表达。HN抗体阻断试验能部分抑制HN-NKp46结合及NK细胞TRAIL 上调的效应。这一结果将为进一步研究NDV-HN直接活化NK细胞增强抗肿瘤效应的胞内信号传导提供依据。

关键词： 新城疫病毒; HN蛋白; 原核表达; 免疫机制

Abstract: The hemagglutinin-neuraminidase(HN)protein, which is an important spike on Newcastle disease virus (NDV) envelope. It not only has function binding the cell to initiate infection, but also involves in NDV oncolytic effects. HN of NDV serves as a ligand structure for NKp46.NDV-infected tumor cells stimulated NK calls to produce increased amounts of the effector IFN-γ and TNF-α. However, the mechanism about whether HN protein can bind to NKp46 receptor on NK cells and cause TRAIL up-regulation has not yet been clear. The purpose is to obtain HN recombinant protein for further signal transduction researches in NDV oncolytic mechanism. According to the sequence of NDV HN gene available in GenBank, a pair of primers is designed. The target sequence, coding for major antigenic determinants, was amplified by RT-PCR, and was ligated to prokaryotic expression vector pET30a to construct a recombinant expression plasmid pET-HNa. The pET-HNa was transformed to E.coli strain BL21(DE3), and the recombinant HN protein was induced and expressed by IPTG induction. The results of FCM showed that HN not only can bind to NKp46 receptor on resident mice NK cells but also can raise TRAIL production of NK cell. Blocking experiment shows that anti-HN partially inhibit this effect. The results provide basic material for further study on signal transduction in HN stimulated NK cells.

Key words: Newcastle disease virus HN protein Prokaryotic expression Immunity mechanism

收稿日期: 2012-09-19 出版日期: 2013-08-25

ZTFLH:

Q786

基金资助: 国家自然科学基金(30860328);广西自然科学基金(2011GXNSFA01822);广西教育厅科学基金(200810MS059);广西医学科学实验中心开放基金专项(KFJJ2011-21);广西研究生教育创新计划(2009105980710M191)资助项目

通讯作者: 樊晓辉,E-mail:fanxiaohui63@yahoo.com.cn E-mail: fanxiaohui63@yahoo.com.cn

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引用本文:

梁莹, 刘金颖, 樊晓辉, 宋德志, 肖庆, 殷君, 冯安林, 杨利, 周丹旎, 赖振屏. 新城疫病毒7793株HN基因片段原核表达及功能鉴定[J]. 中国生物工程杂志, 2013, 33(8): 32-37.

LIANG Ying, LIU Jin-ying, FAN Xiao-hui, SONG De-zhi, XIAO Qing, YIN Jun, FENG An-lin, YANG Li, ZHOU Dan-ni, LAI Zhen-pin. The Prokaryotic Expression and Identification of HN Gene Fragment of Newcastle Disease Virus Strain 7793. China Biotechnology, 2013, 33(8): 32-37.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/ 或 https://manu60.magtech.com.cn/biotech/CN/Y2013/V33/I8/32

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