Please wait a minute...

中国生物工程杂志

CHINA BIOTECHNOLOGY
中国生物工程杂志
中国生物工程杂志  2018, Vol. 38 Issue (7): 67-74    DOI: 10.13523/j.cb.20180709
技术与方法     
鸡OV启动子表达HA对禽流感病毒攻击提供完全保护 *
李亚芳1,2,赵颖慧2,刘赛宝1,2,王伟2,曾为俊1,2,王金泉1,**(),陈洪岩2,**(),孟庆文2,**()
1 新疆农业大学动物医学学院 乌鲁木齐 830000
2 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 黑龙江省实验动物与比较医学重点实验室 哈尔滨 150069
Chicken OV Promoter Expressed HA to Protect Chickens from Lethal Challenge of AIV
Ya-fang LI1,2,Ying-hui ZHAO2,Sai-bao LIU1,2,Wei WANG2,Wei-jun ZENG1,2,Jin-quan WANG1,**(),Hong-yan CHEN2,**(),Qing-wen MENG2,**()
1 College of Veterinary Medicine, Xinjiang Agricultural University, Urumqi 830000, China
2 State Key Laboratory of Veterinary Biotechnology,Heilongjiang Provincial Key Laboratory of Laboratory Animals and Comparative Medicine, Harbin Veterinary Research Institute of Chinese Academy of Agricultural Sciences,Harbin 150069, China
 全文: PDF(1029 KB)   HTML
摘要：

鸡卵清蛋白(ovalbumin,OV)基因5'调控序列是构建鸡输卵管生物反应器的首选调控元件。以EGFP为报告基因,构建OV启动子真核表达载体,转染原代输卵管上皮细胞和CHO细胞,筛选得到1.1kb的高效OV启动子。构建1.1kb OV启动子表达H5N1亚型禽流感病毒HA蛋白真核表达载体pOV1.1k-HA,转染CHO细胞。PCR、RT-PCR鉴定结果证明HA基因整合至CHO细胞基因组,并进行转录;SDS-PAGE、Western blot及HA试验结果证明HA蛋白在CHO细胞内的表达,并具有免疫反应性和血凝活性。以纯化的HA蛋白免疫4周龄SPF鸡,2周后加强免疫一次,加强免疫3周HI抗体水平为6.3log2;以106 EID50 H5N1(A/Goose/Guangdong/1/96)亚型禽流感病毒鼻腔接种SPF鸡,免疫组100%存活,无排毒现象,对照组100%死亡。结果表明,筛选的1.1kb OV启动子可有效驱动HA蛋白表达,表达的HA蛋白免疫SPF鸡对禽流感病毒攻击提供完全保护;为鸡输卵管生物反应器表达保护性抗原和珍贵药物蛋白奠定了基础。

关键词： 鸡卵清蛋白启动子;  HA;  禽流感病毒;  免疫保护;  输卵管生物反应器    
Abstract:

The 5'regulation sequence of the chicken ovalbumin (ovalbumin, OV) gene is the preferred regulatory element for the development of poultry oviduct bioreactor. Using EGFP as reporter gene,the eukaryotic expression vector containing the OV promoter is constructed and effective promoter is screened by analyzing the expression of GFP in the primary cell cultures of chicken oviduct and CHO cell, OV promoter of 1.1kb is identified which is used for further experiments. A recombinant vector named pOV1.1k-HA to express HA protein of H5N1 subtype influenza virus is constructed. It is subsequently transfected into CHO cells, PCR and RT-PCR analysis of HA gene suggested that the vector could be delivered into CHO cells and then get them transcribed. The immunoreactivity and hemagglutination activity of HA protein are determined by Western blot and HA test. The 4-week-old SPF chickens are vaccinated with purified HA protein and boosts were conducted with the same dosage after two weeks. The HI antibody level is 6.3log2 of three weeks after the boost. All chickens are challenged with 106 EID50 of H5N1 virus ((A/Goose/Guangdong/1/96). The survival rate of all vaccinated chickens is 100% and that of control group is 0, also no detoxification in the vaccine group. It is indicated that complete protection is provided. Results show that the screened 1.1kb OV promoter could effectively drive the expression of HA protein and the expression of HA protein immunizing SPF chicken provided complete protection against avian influenza virus attack. The basis for the expression of protective antigens and precious drug proteins in the chicken oviduct bioreactor will be founded.

Key words: Chicken ovalbumin promoter    HA    Avian influenza virus    Immune protection    Oviduct bioreactor
收稿日期: 2018-03-11 出版日期: 2018-08-13
ZTFLH:  Q819  
基金资助: 兽医生物技术国家重点实验室自主课题(SKLVBP2017010);国家自然基金项目(30771615);“十二五”农村领域国家科技计划课题(2011AA100305)
通讯作者: 王金泉,陈洪岩,孟庆文     E-mail: wangjinquan163@163.com;chenhongyan@caas.cn;mqw@hvri.ac.cn
服务　　
把本文推荐给朋友
加入引用管理器
E-mail Alert
RSS
作者相关文章　　
李亚芳
赵颖慧
刘赛宝
王伟
曾为俊
王金泉
陈洪岩
孟庆文

引用本文:

李亚芳,赵颖慧,刘赛宝,王伟,曾为俊,王金泉,陈洪岩,孟庆文. 鸡OV启动子表达HA对禽流感病毒攻击提供完全保护 *[J]. 中国生物工程杂志, 2018, 38(7): 67-74.

Ya-fang LI,Ying-hui ZHAO,Sai-bao LIU,Wei WANG,Wei-jun ZENG,Jin-quan WANG,Hong-yan CHEN,Qing-wen MENG. Chicken OV Promoter Expressed HA to Protect Chickens from Lethal Challenge of AIV. China Biotechnology, 2018, 38(7): 67-74.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/10.13523/j.cb.20180709    或    https://manu60.magtech.com.cn/biotech/CN/Y2018/V38/I7/67

图1  pOV1.1k-GFP、pOV2.7k-GFP、pOV3.0k-GFP、pcDNA6.2-GFP在原代输卵管上皮细胞中的表达(10×)
图2  转染原代输卵管上皮48h后各转染组阳性率(a)和平均荧光强度(b)
图3  pOV1.1k-GFP、pOV2.7k-GFP、pOV3.0k-GFP、pcDNA6.2-GFP质粒在CHO细胞内表达(10×)
图4  转染CHO细胞48h后各转染组阳性率(a)和平均荧光强度(b)
图5  重组质粒酶切鉴定
图6  转染CHO细胞HA基因PCR(a)RT-PCR(b)结果
图7  SDS-PAGE(a)及Western blot(b)分析HA蛋白表达
组别 HI抗体(log2) 攻毒后排毒情况(lgEID50) 存活/总数
DPI=3 DPI=6
喉拭子 泄殖腔拭子 喉拭子 泄殖腔拭子
HA蛋白组 6.3±0.96 0/4 0/4 0/4 0/4 4/4
PBS组 - 4/4 4/4 / / 0/4
表1  加强免疫3周后HI抗体水平及攻毒保护分析
[1] Lillico S G , McGrew M J, Sherman A, et al. Transgenic chickens as bioreactors for protein-based drugs. Drug Discov Today, 2005,10(3):191-196.
doi: 10.1016/S1359-6446(04)03317-3 pmid: 15708533
[2] Sheridan C . FDA approves ‘farmaceutical’ drug from transgenic chickens. Nature Biotechnology, 2016,34(2):117-119.
doi: 10.1038/nbt0216-117 pmid: 26849497
[3] Sanders M M, Mcknight G S . Positive and negative regulatory elements control the steroid-responsive ovalbumin promoter. Biochemistry, 1988,27(17):6550-6557.
doi: 10.1021/bi00417a053 pmid: 3064812
[4] Haecker S A, Muramatsu T, Sensenbaugh K R , et al. Repression of the ovalbumin gene involves multiple negative elements including a ubiquitous transcriptional silencer. Molecular Endocrinology, 1995,9(9):1113-1126.
[5] 杨鹏翔, 王曦晨, 王宇祥 , 等. 转基因鸡生物反应器载体的构建及其表达特性分析. 生物工程学报, 2011,27(8):1215-1224.
Yang P X, Wang X C, Wang Y X , et al. Construction and expression characterization of transgenic chicken bioreactor vector. Chinese Journal of Biotechnology, 2011,27(8):1215-1224.
[6] Oishi I, Kim S, Yoshii K , et al. Cre-LoxP-regulated expression of monoclonal antibodies driven by an ovalbumin promoter in primary oviduct cells. BMC Biotechnology, 2011,11(1):1-8.
doi: 10.1186/1472-6750-11-1
[7] Kodama D, Nishimiya D, Nishijima K , et al. Chicken oviduct-specific expression of transgene by a hybrid ovalbumin enhancer and the Tet expression system. Journal of Bioscience & Bioengineering, 2012,113(2):146-153.
[8] Liu G L, Zhang F F, Shi J Z , et al. A subunit vaccine candidate derived from a classic H5N1 avian influenza virus in China protects fowls and BALB/c mice from lethal challenge. Vaccine, 2013,31(46):5398-5404.
doi: 10.1016/j.vaccine.2013.09.009
[9] Meng Q W, Liu G L, Liu Y G , et al. A broad protection provided by matrix protein 2 (M2) of avian influenza virus. Vaccine, 2015,33(31):3758-3765.
doi: 10.1016/j.vaccine.2015.05.045 pmid: 26036948
[10] Stadnicka K, Bodnar M, Marszałek A , et al. Efficient source of cells in proximal oviduct for testing non-viral expression constructs in the chicken bioreactor model and for other in vitro studies. Folia Biologica, 2016,64(1):37-46.
doi: 10.3409/fb64_1.37
[11] Park H M, Okumura J, Muramatsu T , et al. Modulation of transcriptional activity of the chicken of albumin gene promoter in primary cultures of chicken oviduct cells: effects of putative regulatory elements in the 5'-flanking region. Biochem Mol Biol Int, 1995,36(4):811-816.
[12] Palmiter R D, Sandgren E P, Avarbock M R , et al. Heterologous introns can enhance expression of transgenes in mice. Proc Natl Acad Sci USA, 1991,88(2):478-482.
doi: 10.1073/pnas.88.2.478
[13] 房浩霞, 王安平, 高波 , 等. 鸡卵清蛋白基因第一内含子和3'-调控区对外源基因表达的调控作用. 生物工程学报, 2008,24(2):333-338.
doi: 10.3321/j.issn:1000-3061.2008.02.028
Fang H, Wang A, Gao B , et al. The regulatory effect of the first intron and 3'-regulatory region of ovalbumin gene on transgene expression. Chinese Journal of Biotechnology, 2008,24(2):333-338.
doi: 10.3321/j.issn:1000-3061.2008.02.028
[14] 黄菁, 朱志伟, 陈晓宇 , 等. 鸡卵清蛋白基因调控序列的克隆与载体构建. 浙江农业学报, 2016,28(3):412-419.
doi: 10.3969/j.issn.1004-1524.2016.03.09
Huang J, Zhu Z W, Chen X Y , et al. Cloning and vector construction of chicken ovalbumin gene regulatory sequences. Acta Agriculturae Zhejiangensis, 2016,28(3):412-419.
doi: 10.3969/j.issn.1004-1524.2016.03.09
[15] 赵颖慧, 王伟, 李越 , 等. 1.1kb、2.7kb、3.0kb鸡输卵管组织特异性启动子的克隆及细胞评价. 中国家禽, 2014,36(9):6-11.
doi: 10.3969/j.issn.1004-6364.2014.09.003
Zhao Y H, Wang W, Li Y , et al. Cloning and evaluation of 1.1kb,2.7kb and 3.0kb of oviduct-specific promoter of chicken. China Poultry, 2014,36(9):6-11.
doi: 10.3969/j.issn.1004-6364.2014.09.003
[16] Ochiai H, Park H M, Nakamura A , et al. Synthesis of human erythropoietin in vivo in the oviduct of laying hens by localized in vivo gene transfer using electroporation. Poultry Science, 1998,77(2):299-302.
doi: 10.1093/ps/77.2.299
[17] Jayapal K P, Wlaschin K F, Hu W S , et al. Recombinant protein therapeutics from CHO cells - 20 years and counting. Chemical Engineering Progress, 2007,103(10):40-47.
doi: 10.1021/cen-v085n040.p035
[18] 逄越, 李庆伟 . 鸡卵清蛋白基因启动子调控GFP基因在鸡原代输卵管上皮细胞和中国仓鼠卵巢细胞的表达. 生物工程学报, 2005,21(1):154-158.
doi: 10.3321/j.issn:1000-3061.2005.01.028
Pang Y, Li Q W . GFP reporter gene under the direction of chicken ovalbumin gene promoter expressed in the CHO cell and in the primary cell cultures of chicken oviduct. Chinese Journal of Biotechnology, 2005,21(1):154-158.
doi: 10.3321/j.issn:1000-3061.2005.01.028
[1] 张文玉,魏东升,钱江潮. 共表达PDI1、MDH1和HAC1基因对重组毕赤酵母分泌表达葡糖氧化酶的影响 *[J]. 中国生物工程杂志, 2019, 39(10): 24-33.
[2] 刘赛宝,李亚芳,王辉,王伟,冉多良,陈洪岩,孟庆文. 利用CRISPR/Cas9技术构建流感病毒高产细胞系MDCK-Tpl2 -/-*[J]. 中国生物工程杂志, 2019, 39(1): 46-54.
[3] 马淑霞,张玲,闫晋飞,游松. 裂壶藻脂肪酸合酶途径合成多不饱和脂肪酸的研究 *[J]. 中国生物工程杂志, 2018, 38(9): 27-34.
[4] 秦梦菲, 孙鸿, 宋浩. 分枝杆菌细胞裂解液催化甾体激素C1,2位脱氢反应的研究[J]. 中国生物工程杂志, 2017, 37(8): 23-30.
[5] 王小莉, 余庆, 袁雅红, 腾智平, 李东升, 曾毅. 打靶恒河猴CD4+ T细胞的TRIM5α基因影响其感染HIV的能力[J]. 中国生物工程杂志, 2017, 37(2): 15-19.
[6] 罗枫雪, 李佛生, 姚民, 徐莺. 水稻OsHAK26启动子的克隆及瞬时表达分析[J]. 中国生物工程杂志, 2017, 37(2): 33-39.
[7] 何石宝, 杨成飞, 尚杉, 王凌燕, 唐文超, 朱勇. 家蚕保幼激素结合蛋白Bmtol基因的克隆及表达分析[J]. 中国生物工程杂志, 2017, 37(10): 16-25.
[8] 卓丽霞, 王莹, 张春萍, 段静, 张亚妮. α-溶血素的表达及其纳米孔的制备[J]. 中国生物工程杂志, 2017, 37(1): 53-57.
[9] 张宇萌, 童梅, 陆小冬, 米月, 莫婷, 刘金毅, 姚文兵. 大肠杆菌可溶性表达抗TNF-α Fab的工艺优化[J]. 中国生物工程杂志, 2016, 36(9): 31-37.
[10] 李成媛, 张晶晶, 钱凯, 缪亚娜, 蔡燕飞, 杨剑峰, 何杨, 金坚. 人血清白蛋白-干扰素α2b融合蛋白在CHO细胞中的表达[J]. 中国生物工程杂志, 2016, 36(7): 7-14.
[11] 庞倩, 马榆, 李诚, 刘韫滔, 刘书亮, 王小红, 刘爱平. 基于CDR2和CDR3区随机突变筛选抗黄曲霉毒素B1单域重链抗体的研究[J]. 中国生物工程杂志, 2016, 36(7): 21-26.
[12] 堵晶晶, 李强, 程霄, 沈林園, 李学伟, 张顺华, 朱砺. CRISPR/Cas系统的研究进展及其在畜禽遗传改良中的应用前景[J]. 中国生物工程杂志, 2016, 36(7): 92-103.
[13] 高翠娟, 连思琪, 祁庆生. 代谢工程构建重组耶氏解脂酵母生产中长链聚羟基脂肪酸酯[J]. 中国生物工程杂志, 2016, 36(5): 53-58.
[14] 朱志坚, 连凯琪, 杨帆, 张伟, 郑海学, 杨孝朴. 稳定表达鼠源整联蛋白αvβ6的CHO-677细胞系的构建[J]. 中国生物工程杂志, 2015, 35(8): 23-29.
[15] 王兰, 徐刚领, 高凯, 王军志. 抗体类生物治疗药物活性测定方法[J]. 中国生物工程杂志, 2015, 35(6): 101-108.
主管：中国科学院 　主办：中国科学院文献情报中心 　中国生物技术发展中心 　中国生物工程学会