家蚕丝腺生物反应器表达狂犬病病毒核蛋白

doi:10.3864/j.issn.0578-1752.2013.18.023

中国农业科学 ›› 2013, Vol. 46 ›› Issue (18): 3922-3929.doi: 10.3864/j.issn.0578-1752.2013.18.023

• 畜牧.兽医.资源昆虫 • 上一篇下一篇

家蚕丝腺生物反应器表达狂犬病病毒核蛋白

尤征英1, 危浩13, 阮系真2, 叶露鹏1, 王少华1, 周继勇2, 钟伯雄1

1.浙江大学动物科学学院特种经济动物科学系，杭州 310029
2.浙江大学农业部动物病毒学重点实验室，杭州310029
3.湖北省畜禽育种中心，武汉430070

收稿日期:2013-03-01 出版日期:2013-09-15 发布日期:2013-04-26
通讯作者: 通信作者钟伯雄，Tel/Fax：0571-86971302；E-mail：bxzhong@zju.edu.cn
作者简介:尤征英，Tel：13989832857；E-mail：youzhengying85@163.com
基金资助:
国家重点基础研究发展“973”计划（2012CB114601）、国家高技术研究发展 “863”计划（2012AA101303）、农业公益性行业技术专项（201103032）

Expression of RVNP in the Silk Gland of Transgenic Silkworm (Bombyx mori)

YOU Zheng-Ying-1, WEI Hao-13, RUAN Xi-Zhen-2, YE Lu-Peng-1, WANG Shao-Hua-1, ZHOU Ji-Yong-2, ZHONG Bo-Xiong-1

1.Department of Special Economic Animal Science of College of Animal Science, Zhejiang University, Hangzhou 310029
2.Key Laboratory of Animal Virology of Ministry of Agriculture, Zhejiang University, Hangzhou 310029
3.Animal & Domestic Breeding Center of Hubei Province, Wuhan 430070

Received:2013-03-01 Online:2013-09-15 Published:2013-04-26

摘要/Abstract

摘要： 【目的】采用家蚕丝腺生物反应器这一蛋白高效表达系统，尝试建立一种高效、低廉、稳定生产狂犬病病毒核蛋白（RVNP）的技术体系，为生产基因工程疫苗奠定基础。【方法】通过RT-PCR方法克隆获得RVNP序列，采用家蚕丝胶蛋白基因（Ser1）启动子为特异性启动子，将RVNP构建到含有增强型绿色荧光蛋白（EGFP）筛选标记基因的piggyBac转座子表达载体（pBA3EGFP）中，并采用显微注射转基因家蚕技术构建家蚕丝腺生物反应器。【结果】G1代通过EGFP标记的筛选获得转基因家蚕阳性蛾区26个，蛾圈阳性率为59.09%。PCR实验证实RVNP已经整合到家蚕基因组中，Western blot分析表明RVNP蛋白可能附着于丝胶蛋白并随家蚕吐丝行为分泌到蚕茧中。【结论】获得了能够表达外源的RVNP蛋白的转基因家蚕品系，该系统有望成为高效、低廉、稳定地生产狂犬病基因工程疫苗的技术体系之一。

关键词: 狂犬病 , 丝腺生物反应器 , piggyBac , 家蚕

Abstract: 【Objective】In order to obtain a potent technology which can produce high value RVNP protein and then lay a foundation for the production of vaccines, the silk gland bioreactor was employed as an expression system in this study.【Method】The nucleoprotein gene of rabies virus ERA strain (RVNP) was cloned by RT-PCR, and the RVNP was firstly connected to the downstream of middle silk gland-specific Ser1 promoter, and then inserted into pBA3EGFP, an expression vector which contained an EGFP reporter gene. By using the transgenic method, the silk gland bioreactor of piggyback-mediated transgenic silkworm was obtained. 【Result】Twenty-six broods of transgenic silkworms (G1) were obtained and the GFP-positive percentage of broods was 59.09%. Further PCR identification indicated that RVNP had been integrated into silkworm genome, and Western blot analysis showed that the protein might be attached to the sericin which was secreted into the cocoon with the spinning of transgenic silkworms. 【Conclusion】Transgenic silkworm strain able to express exogenous RVNP protein was obtained. The present system is expected to become a high efficiency, low-cost and stable technical system to produce genetic engineering rabies vaccine.

Key words: rabies , silk gland bioreactor , piggyBac , silkworm (Bombyx mori)

尤征英1, 危浩13, 阮系真2, 叶露鹏1, 王少华1, 周继勇2, 钟伯雄1. 家蚕丝腺生物反应器表达狂犬病病毒核蛋白[J]. 中国农业科学, 2013, 46(18): 3922-3929.

YOU Zheng-Ying-1, WEI Hao-13, RUAN Xi-Zhen-2, YE Lu-Peng-1, WANG Shao-Hua-1, ZHOU Ji-Yong-2, ZHONG Bo-Xiong-1. Expression of RVNP in the Silk Gland of Transgenic Silkworm (Bombyx mori)[J]. Scientia Agricultura Sinica, 2013, 46(18): 3922-3929.

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参考文献

[1]Abe C. Anti-tuberculosis drug resistance in Japan and in the world. Kekkaku, 2001, 76(11): 699-706.

[2]殷震, 刘景华. 动物病毒学. 2版. 北京: 科学出版社, 1997.

Yin Z, Liu J H. Animal Virology. 2nd ed. Beijing: Science Press, 1997. (in Chinese)

[3]Liu P H, Yang J, Wu X F, Fu Z F. Interactions amongst rabies virus nucleoprotein, phosphoprotein and genomic RNA in virus-infected and transfected cells. Journal of General Virology, 2004, 85(12): 3725-3734.

[4]Mannen K, Hiramatsu K, Mifune K, Sakamoto S. Conserved nucleotide sequence of rabies virus cDNA encoding the nucleoprotein. Virus Genes, 1991, 5(1): 69-73.

[5]蔡月琴, 马益萍, 申会刚, 郭军庆, 周继勇. 狂犬病病毒核蛋白基因在大肠杆菌中的高效表达. 中国兽医科学, 2007, 37(2): 145-149.

Cai Y Q, Ma Y P, Shen H G, Guo J Q, Zhou J Y. High-level expression of nucleoprotein gene of rabies virus in Escherichia coli. Veterinary Science in China, 2007, 37(2): 145-149. (in Chinese)

[6]尹伟, 徐洁萍, 张金阳, 颜焰, 周继勇. 狂犬病病毒核蛋白在 Bac-To-Bac/AcMNPV 杆状病毒系统的表达. 中国预防兽医学报, 2010, 32(2): 86-89.

Yin W, Xu J P, Zhang J Y, Yan Y, Zhou J Y. Expression of nucleoprotein of Rabies virus in Bac-To-Bac/AcMNPV Baculovirus expression system. Chinese Journal of Preventive Veterinary Medicine, 2010, 32(2): 86-89. (in Chinese)

[7]Arango I P, Rubio E L, Anaya E R, Flores T O, de la Vara L G, Lim M A G. Expression of the rabies virus nucleoprotein in plants at high-levels and evaluation of immune responses in mice. Plant Cell Reports, 2008, 27(4): 677-685.

[8]杨卉娟, 陈俊英, 孙明波, 张新文, 钱源, 段骞, 王东宝, 姜述德, 廖国阳, 李卫东. 狂犬病毒aG株核蛋白和糖蛋白双表达质粒的构建及其在真核细胞中的表达. 中国生物制品学杂志, 2006, 19(6): 564-567.

Yang H J, Chen J Y, Sun M B, Zhang X W, Qian Y, Duan Q, Wang D B, Jiang S D, Liao G Y, Li W D. Construction of recombinant plasmid for co-expression of nucleoprotein and glycoprotein of Rabies virus aG strain in eukaryotic cells. Chinese Journal of Biologicals, 2006, 19(6): 564-567. (in Chinese)

[9]钟伯雄, 危浩, 庄兰芳. piggyBac转座子介导的转基因家蚕丝腺生物反应器研究进展. 中国农业科学, 2011, 44(21): 4488-4498.

Zhong B X, Wei H, Zhuang L F. Advances in research of silk gland bioreactor of piggyBac-mediated transgenic silkworm. Scientia Agricultural Sinica, 2011, 44(21): 4488-4498. (in Chinese)

[10]Toshiki T, Chantal T, Corinne R, Toshio K, Eappen A, Mari K, Natuo K, Jean-Luc T, Bernard M, Gerard C, Paul S, Malcolm F, Jean-Claude P, Piette C. Germline transformation of the silkworm Bombyx mori L. using a piggyBac transposon-derived vector. Nature Biotechnology, 2000, 18(1): 81-84.

[11]Tatematsu K, Kobayashi I, Uchino K, Sezutsu H, Iizuka T, Yonemura N, Tamura T. Construction of a binary transgenic gene expression system for recombinant protein production in the middle silk gland of the silkworm Bombyx mori. Transgenic Research, 2010, 19(3): 473-487.

[12]Xue R Y, Wang Y, Cao G L, Pan Z H, Zheng X J, Zhou W L, Gong C L. Lowering the blood glucose of diabetes mellitus mice by oral administration with transgenic human insulin-like growth factor I silkworms. Journal of Agricultural and Food Chemistry, 2012, 60(26): 6559-6564.

[13]Sato M, Kojima K, Sakuma C, Murakami M, Aratani E, Takenouchi T, Tamada Y, Kitani H. Production of scFv-conjugated affinity silk powder by transgenic silkworm technology. PLoS One, 2012, 7(4): e34632.

[14]Tatemastu K I, Sezutsu H, Tamura T. Utilization of transgenic silkworms for recombinant protein production. Journal of Biotechnology & Biomaterials, 2012, S9: 1-8.

[15]Adachi T, Wang X, Murata T, Obara M, Akutsu H, Machida M, Umezawa A, Tomita M. Production of a non-triple helical collagen α chain in transgenic silkworms and its evaluation as a gelatin substitute for cell culture. Biotechnology and Bioengineering, 2010, 106(6): 860-870.

[16]Xue R Y, Chen H M, Cui L L, Cao G L, Zhou W L, Zheng X J, Gong C L. Expression of hGM-CSF in silk glands of transgenic silkworms using gene targeting vector. Transgenic Research, 2012, 21(1): 101-111.

[17]Zhao A C, Zhao T F, Zhang Y S, Xia Q Y, Lu C, Zhou Z Y, Xiang Z H, Nakagaki M. New and highly efficient expression systems for expressing selectively foreign protein in the silk glands of transgenic silkworm. Transgenic Research, 2010, 19(1): 29-44.

[18]Tomita M, Hino R, Ogawa S, Iizuka M, Adachi T, Shimizu K, Sotoshiro H, Yoshizato K. A germline transgenic silkworm that secretes recombinant proteins in the sericin layer of cocoon. Transgenic Research, 2007, 16(4): 449-465.

[19]Wen H X, Lan X Q, Zhang Y S, Zhao T F, Wang Y J, Kajiura Z, Nakagaki M. Transgenic silkworms (Bombyx mori) produce recombinant spider dragline silk in cocoons. Molecular Biology Reports, 2010, 37(4): 1815-1821.

[20]Tomita M. Transgenic silkworms that weave recombinant proteins into silk cocoons. Biotechnology Letters, 2011, 33(4): 645-654.

[21]Zhong B X, Li J Y, Chen J E, Ye J, Yu S D. Comparison of transformation efficiency of piggyBac transposon among three different silkworm Bombyx mori strains. Acta Biochimica et Biophysica Sinica, 2007, 39(2): 117-122.

[22]Inoue S, Kanda T, Imamura M, Quan G X, Kojima K, Tanaka H, Tomita M, Hino R, Yoshizato K, Mizuno S, Tamura T. A fibroin secretion-deficient silkworm mutant, Nd-sD, provides an efficient system for producing recombinant proteins. Insect Biochemistry and Molecular Biology, 2005, 35(1): 51-59.

[23]Wang F, Xu H F, Yuan L, Ma S Y, Wang Y C, Duan X L, Duan J P, Xiang Z H, Xia Q Y. An optimized sericin-1 expression system for mass-producing recombinant proteins in the middle silk glands of transgenic silkworms. Transgenic Research, 2013: DOI 10.1007/s11248-013-9695-6.

[24]Liu Y, Yu L, Guo X Y, Guo T Q, Wang S P, Lu C D. Analysis of tissue-specific region in sericin 1 gene promoter of Bombyx mori. Biochemical and Biophysical Research Communications, 2006, 342(1): 273-279.

[25]Cong L, Cao G L, Xue R Y, Pan Z H, Zheng X J, Zhou W L, Gong C L. Reducing blood glucose level in TIDM mice by orally administering the silk glands of transgenic hIGF-I silkworms. Biochemical and Biophysical Research Communications, 2011, 410(4): 721-725.

家蚕丝腺生物反应器表达狂犬病病毒核蛋白

Expression of RVNP in the Silk Gland of Transgenic Silkworm (Bombyx mori)

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