拯救细胞系恢复重组家蚕核型多角体病毒包涵体实现外源蛋白在家蚕中产业化生产

doi:Q789

中国生物工程杂志

2010, Vol. 30

Issue (05): 96-102 DOI: Q789

技术与方法

拯救细胞系恢复重组家蚕核型多角体病毒包涵体实现外源蛋白在家蚕中产业化生产

吴坤^1,2,冯娟^1,2,姚伦广^1**,楚素霞^1,3,阚云超¹

1.中英南阳洛桑昆虫生物学联合实验室河南省伏牛山昆虫生物学重点实验室南阳师范学院生物科学与技术学院南阳473061
2.河南农业大学生命科学学院郑州 450002
3 河南师范大学生命科学学院新乡 453007

Potential Industrial Production of Foreign Protein in Silkworm Using the Collusion Bodies of Recombinant Bombyx mori(silkworm) Multiple Nucleopolyhedrovirus Rescuesd by Polyhedrin Expressing Stable Cell Line

WU Kun^1,2,FENG Juan^1,2,YAO Lun-guang¹,CHU Su-xia^1,3,KAN Yun-chao¹

1.ChinaUK, Nan Yang Normal UniversityRothamsted Research Joint Laboratory of Insect Biology, Henan Provincial Key Laboratory of Funiu Mountain Insect Biology, Nanyang Normal University, Nanyang 473061,China
2.Life Science College of Henan agriculture University, Zhengzhou 450002, China
3.Life Science College of Henan Normal University, Xinxiang 453007,China

全文: PDF(824 KB) HTML

摘要：

构建家蚕Bombyx mori肌动蛋白（BmA3）启动子驱动的家蚕核型多角体病毒（BmNPV）多角体基因（ph）和OpNPV极早期启动子（IE1）驱动的zeocin抗性筛选基因转座供体载体，与鳞翅目辅助转座质粒pie2piggyBac共转染家蚕卵巢细胞BmN，经200μg/ml zeocin抗生素筛选一个月，成功获得持续表达BmNPV多角体蛋白的稳定细胞系BmN-A3ph。多角体缺陷型重组病毒BmBac-GF P感染拯救细胞系BmN- A3ph, 细胞成功装配出病毒包涵体颗粒，其包装效率约为野生型病毒感染正常BmN细胞的8%。用拯救型包涵体病毒颗粒喂食家蚕幼虫进行复感染，结果表明稳定细胞系所包装的包涵体病毒与野生型病毒一样能够通过口服途径感染宿主，却并不在宿主体内形成包涵体，从而保证外源基因高效表达。拯救型包涵体病毒可望解决传统注射感染效率较低问题，通过喂食感染可促进杆状病毒介导的家蚕生物反应器产业化进程。

关键词： 拯救细胞系; piggyBac转座; 包涵体; 家蚕生物反应器

Abstract:

The transposon donor vector harboring both polyhedrin gene droved by BmA3 promoter and zeocin resistant gene controlled by IE1 promoter from OpNPV co-transefeced with a lepidoptera transposition helper plasmid pie2piggBac into BmN cells. A polyhedrin expressing stable cell line was constructed by screening with the 200μg/ml zeocin selection medium for one month. Occlusion bodies (OB) were rescued successfully in the polyhedrin expressing BmN cells infected with recombinant BmBac-gfp budded virus without polyhedrin gene. The amount of rescued OB produced in polyhedrin expressing cells is only 8% of wild type BmNPV OB produced in normal cells. The silkworm larvae were infected efficiently when they were feed by the rescued OB sprayed mulberry leaves. Foreign GFP was expressed efficiently and no OB was observed in the hemolymph of silkworm larvae. This rescued OB oral infection method is benefit to promote the industrial progress of silkworm bioreactor by eliminating the tedious injection budded virus solution by hand.

Key words: Rescuing cell line Piggybac transposon Occlusion body Silkworm bioreactor

收稿日期: 2009-12-31 出版日期: 2010-05-25

基金资助:

国家自然科学基金（30700750）、河南省省教育厅科技攻关项目(2008A180020)资助项目

通讯作者: 姚伦广 E-mail: lunguangyao@163.com

	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
	吴坤
	冯娟
	姚伦广
	楚素霞
	阚云超

引用本文:

吴坤冯娟姚伦广楚素霞阚云超. 拯救细胞系恢复重组家蚕核型多角体病毒包涵体实现外源蛋白在家蚕中产业化生产[J]. 中国生物工程杂志, 2010, 30(05): 96-102.

TUN Kun, FENG Juan, TAO Lun-An, CHU Su-Xia, HAN Yun-Chao. Potential Industrial Production of Foreign Protein in Silkworm Using the Collusion Bodies of Recombinant Bombyx mori(silkworm) Multiple Nucleopolyhedrovirus Rescuesd by Polyhedrin Expressing Stable Cell Line. China Biotechnology, 2010, 30(05): 96-102.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/Q789 或 https://manu60.magtech.com.cn/biotech/CN/Y2010/V30/I05/96

［1］姚宁，姚伦广，阚云超，等.杆状病毒表达系统的改进及IL6在昆虫细胞内的表达和纯化.生物工程学报，2006, 22（4）: 572580. Yao N,Yao L G,Kan Y C,Chin J Biotech, 2006, 22（4）: 572580.
［2］ Kost T A, Condreay J P,Jarvis D L. Baculovirus as versatile vectors for protein expression in insect and mammalian cells. Nat. Biotechnol, 2005, 23: 567575.
［3］ Smith G E, Summers M D, Fraser M J. Production of human beta interferon in insect cells infected with a baculovirus expression vector. Mol. Cell. Biol, 1983, 3 (12):21562165.
［4］ Maeda S, Kawai T, Obinata M, et al. Production of human alphainterferon in silkworm using a baculovirus vector. Nature, 1985, 315: 592594.
［5］张颖，吴祥甫.重组家蚕病毒表达系统转移载体的构建和β半乳糖苷酶的表达. 病毒学报,1994, 3: 251256. Zhang Y,Wu X F.Chinese Journal of Virology,1994, 3: 251256.
［6］ Motohashi T, Shimojima T, Fukagawa T,et al. Efficient largescale protein production of larvae and pupae of silkworm by Bombyx mori nuclear polyhedrosis virus bacmid system.Biochem Biophys Res Commun, 2005, 326: 564569.
［7］ Yao L G, Liu Z C, Zhang X M, et al. A highly efficient method for the generation of a recombinant Bombyx mori nuclearpolyhedrosisvirus Bacmid and largescale expression of foreign proteins in silkworm (B. mori) larvae. Biotechnol Appl Biochem, 2007, 48: 4553.
［8］姚伦广, 张红玲,冯娟，等.零背景转座技术高效构建重组家蚕杆状病毒表达系统.微生物学报，2009, 6: 831837. Yao L G, Zhang H L,Feng J,et al.Acta Microbiologica Sinica, 2009, 6: 831837.
［9］ Lun G Y, Jing C S, Hua X, A novel economic method for high throughput production of recombinant baculovirus by infecting insect cells with bacmidcontaining diminopimelateauxotrophic escherichia coli. Journal of Biotechnolgy,2010 (1):2329.
［10］彭建新.杆状病毒分子生物学.武汉：华中师范大学出版社，2000. Peng X J. Baculovirus Molecular Biology.Wuhan:Central China Normal University Press, 2000.
［11］ Berger I, Fitzgerald D J, Richmond T J. Baculovirus expression system for heterologous multiprotein complexes. Nat Biotechnol, 2004, 22(12):15831587.
［12］ Horn C, Wimmer E A. A versatile vector set for animal transgenesis. Dev. Genes Evol, 2000, 210: 630–637.
［13］ Sambrook J, Russell D W. Molecular Cloning:A Laboratory Manual.NY:Cold Spring Harbor Laboratory Press, 2001.
［14］ F M奥斯伯,R E金斯顿,J G赛德曼,等.精编分子生物学实验指南.第4版,北京：科学出版,2005. Ausubel F M,Kingston R E,et al.Short Protocols in Molecalar Biology:4th ed.Beijing:Science Press,2005.
［15］吕鸿声.昆虫病毒分子生物学.北京：中国农业科技出版社,1998. Lv H S. Molecular Biology of Insect Virus.Beijing: Chinese Agricultural Science and Technology Press, 1998.
［16］ Jarvis D L, Weinkauf C, Guarino L A. Immediateearly baculovirus vectors for foreign gene expression in transformed or infected insect cells. Protein Expression and Purification 8, 1996, 8:191203.

[1]	刘亚龙, 闫东明, 翁樑, 邹雪, 刘丹, 彭超, 苏亚南, 闫锦锦, 张静, 郭志燕. 重组大肠杆菌不耐热肠毒素B亚单位的中试发酵及纯化工艺[J]. 中国生物工程杂志, 2015, 35(2): 78-83.
[2]	罗莉, 秦娇荣, 王明蓉. 重组sTNFR1蛋白包涵体洗涤方法的研究[J]. 中国生物工程杂志, 2013, 33(9): 45-52.
[3]	苏燕南, 薛正莲, 陈涛, 马琦亚. 粘质沙雷氏菌PL-06磷脂酶A1基因大肠杆菌优化表达[J]. 中国生物工程杂志, 2013, 33(7): 36-42.
[4]	黄鹏煌, 王泽, 田海山, 赵海洋, 李海燕, 李校堃. 重组人成纤维细胞生长因子8b原核表达载体的构建和纯化研究[J]. 中国生物工程杂志, 2013, 33(1): 14-19.
[5]	罗莉, 何勇智, 张勇侠, 王明蓉. 功能性包涵体的研究进展[J]. 中国生物工程杂志, 2013, 33(1): 114-121.
[6]	李倩倩, 李中媛, 冯舵, 黄火清, 韩翠晓, 杨培龙, 姚斌, 高伟. 芽孢杆菌β-折叠桶植酸酶的原核可溶性表达优化及包涵体复性研究[J]. 中国生物工程杂志, 2012, 32(08): 49-55.
[7]	王振东, 王林林, 杨宇, 杨永莉, 王静. 委内瑞拉马脑炎病毒重组抗原的制备纯化及其胶体金免疫层析检测方法的建立[J]. 中国生物工程杂志, 2011, 31(7): 104-108.
[8]	李伟. 重组PLZF蛋白锌指结构域的表达、提纯和活性分析[J]. 中国生物工程杂志, 2011, 31(11): 1-5.
[9]	刘晓飞, 裴剑竹, 杜国俊, 杨章民. 蛇毒蛋白原核表达包涵体复性研究进展[J]. 中国生物工程杂志, 2011, 31(03): 113-119.
[10]	舒梅, 许杨, 徐熙, 涂追. 两种水生动物抗菌肽的原核表达及活性分析[J]. 中国生物工程杂志, 2011, 31(02): 56-61.
[11]	王增1,马会勤1,张文1,陈尚武2. 包涵体蛋白的分离和色谱法体外复性纯化研究进展[J]. 中国生物工程杂志, 2009, 29(07): 102-107.
[12]	甘慧,周勇,王全立,詹林盛. 补体C3与BPI活性区融合蛋白（CB）的构建与表达[J]. 中国生物工程杂志, 2007, 27(6): 31-37.
[13]	窦佳,蔡河,姬芳玲,崔文举,王静云,包永明,安利佳. Gln49-磷脂酶A2基因工程定点突变及酶活性分析[J]. 中国生物工程杂志, 2007, 27(5): 21-27.
[14]	贾仟涛,徐丽慧,迟晓云,查庆兵,李丰耀,何贤辉. *可溶性HLA-A0203-BSP原核表达载体的构建及其在大肠杆菌中的高效表达**[J]. 中国生物工程杂志, 2006, 26(09): 5-10.
[15]	陶永辉, 赵砚婷, 张莲芬, 蔡刚明, 金坚. 从包涵体中制备重组人血管抑素[J]. 中国生物工程杂志, 2005, 25(S1): 249-252.

Viewed

Full text

Abstract

Cited

Shared

Discussed