|
|
|
| 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 |
|
|
|
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.
|
|
Received: 11 March 2018
Published: 13 August 2018
|
|
|
|
Corresponding Authors:
Jin-quan WANG,Hong-yan CHEN,Qing-wen MENG
E-mail: wangjinquan163@163.com;chenhongyan@caas.cn;mqw@hvri.ac.cn
|
|
|
| [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.
|
|
|
| [5] |
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
|
|
|
| [13] |
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
|
|
|
| [14] |
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
|
|
|
| [15] |
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
|
|
|
| [18] |
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
|
|
|
|
Viewed |
|
|
|
Full text
|
|
|
|
|
Abstract
|
|
|
|
|
Cited |
|
|
|
|
| |
Shared |
|
|
|
|
| |
Discussed |
|
|
|
|
