重组腺病毒介导的人尿激酶原突变体在山羊乳汁中的表达

中国生物工程杂志

2009, Vol. 29

Issue (02): 29-33

研究报告

重组腺病毒介导的人尿激酶原突变体在山羊乳汁中的表达

于永生^1,2,吴晓洁¹,罗晓彤²,周颜荣¹,陈红星¹,邓继先¹

1. 军事医学科学院生物工程研究所
2. 吉林省农业科学院畜牧分院

Expression of Human Prourokinase Mutant in the Milk of Goat Mediated by the Recombinant Adenovirus

全文: PDF(583 KB) HTML

摘要： 构建携带人尿激酶原突变体cDNA的重组腺病毒，通过该腺病毒介导实现外源基因在山羊乳腺中表达。通过大肠杆菌内同源重组将人尿激酶原突变体cDNA插入到腺病毒载体中，经过293细胞包装获得重组腺病毒，直接注射到泌乳山羊乳腺，收集感染后1~4天的乳汁，利用纤维蛋白溶圈试验、Western blot、ELISA检测乳清中尿激酶原突变体的表达。结果显示病毒注射后1~4天乳清中均可检测到尿激酶原的表达，其表达量可达0.41mg/ml。该方法可以实现重组蛋白在山羊乳腺的短期表达，可能是大规模生产临床医疗蛋白的一条经济有效的途径。

关键词： 人尿激酶原突变体;重组腺病毒;乳腺;乳汁;山羊

Abstract:

In order to express the recombinant proteins in the milk of goat, a recombinant adenovirus was constructed in which the Pro-urokinase mutant cDNA was cloned into the adenovirus vector, and then was transfected to 293 for packaging. The recombinant adenovirus were directly injected into the goat mammary glands, the milk serum was collected 1-4d post-infection and then subjected to fibrin plate assay，Western blotting and ELISA assay. The results showed that the human pro-urokinase was expressed efficiently in the milk of goat, even attained to 0.41mg/mL in the 2rd day post-infection. Taken together, the method is quite useful for the transient expression of recombinant proteins, and the approach established here is probably one of the most economical and efficient ways for large-scale production of recombinant proteins of biopharmaceutical interest.

收稿日期: 2008-10-23 出版日期: 2009-03-31

ZTFLH:

S814.8

基金资助:

国家“863”计划资助项目

通讯作者: 陈红星邓继先

	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
	于永生吴晓洁罗晓彤周颜荣陈红星邓继先

引用本文:

于永生,吴晓洁,罗晓彤,周颜荣,陈红星,邓继先. 重组腺病毒介导的人尿激酶原突变体在山羊乳汁中的表达[J]. 中国生物工程杂志, 2009, 29(02): 29-33.

XU Yong-Sheng- Tun-Xiao-Ji- Luo-Xiao-Tong- Zhou-Ya-Rong- Chen-Gong-Xing- Deng-Ji-Xian. Expression of Human Prourokinase Mutant in the Milk of Goat Mediated by the Recombinant Adenovirus. China Biotechnology, 2009, 29(02): 29-33.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/ 或 https://manu60.magtech.com.cn/biotech/CN/Y2009/V29/I02/29

［1］殷震,刘景华.动物病毒学.北京：科学出版社，1997.1105 Yin Z,Liu J H.Animal Virology.Beijing:Science Press,1997.1105 ［2］ Echavarría M. Adenoviruses in immunocompromised hosts. Clin Microbiol Rev,2008，21(4):704~715 ［3］尚智,何金生,张梅,等.人呼吸道合胞病毒M蛋白非复制型重组腺病毒的构建和鉴定.中国生物工程杂志, 2008, 28 (5) : 6～11 Shan Z,He J S,Zhang M,et al.China Biotechnology, 2008, 28 (5) : 6~11 ［4］杨松海,林天歆,刘珊英,等. 重组Osx腺病毒载体的构建及其调控成骨细胞增殖与分化的作用. 中国生物工程杂志, 2008, 28 (3) : 84～88 Yang S H, Lin T X, Liu S Y,et al. China Biotechnology, 2008, 28 (3) : 84～88 ［5］ Kost T A. Expression vectors and delivery systems tools for determining gene function and gene therapy. Curr Opin Biotech,1999,10(5):409～410 ［6］于永生，周艳荣，卢建申，等.人尿激酶原突变体的构建及其特性的研究.生物工程学报，2005，21（4）：6974 Yu Y S, Zhou Y R, Lu J S,et al. Chinese Journal of Biotechnology,2005，21（4）：69~74 ［7］ Inoue H，Nojima H,Okayama H.High efficiency transformation Escherichia coli with plasmids.Gene,1990,96:23～28 ［8］ Miller E M,Nickoloff J A. Escherichia coli electrotransformation.Methods Mol Biol,1995,47:105～113 ［9］辛艳丽.利用BAC技术制备转基因小鼠.北京：军事医学科学院,2005 Xin Y L.Preparing Transgene Mouse with BAC Technigue.Beijing:Academy of Military Medical Sciences,2005 ［10］ Chuang S T, Ji W T, Chen Y T, et al. Suppression of bovine ephemeral fever virus by RNA interference. J Virol Methods,2007,145(1):84~87 ［11］ Archer J S, Kennan W S, Gould M N, et al. Human growth hormone (hGH) secretion in milk of goats after direct transfer of the hGH gene into the mammary gland by using replicationdefective retrovirus vectors. Proc Natl Acad Sci USA, 1994,91: 6840~6844 ［12］ Han Z S, Li Q W, Zhang Z Y, et al. Highlevel expression of human lactoferrin in the milk of goats by using replicationdefective adenoviral vectors. Protein Expr Purif,2007 ,53(1):225~231 ［13］ van Berkel P H, Welling M M, Geerts M, et al. Largescale production of recombinant human lactoferrin in the milk of transgenic cows. Nat Biotechnol, 2002,20: 484~487 ［14］ Sanchez O, Toledo J R, Rodriguez M P,et al. Adenoviral vector mediates high expression levels of human growth hormone in the milk of mice and goats. J Biotechnol, 2004,114: 89~97 ［15］ Han Z S, Li Q W, Zhang Z Y,et al. Highlevel expression of human lactoferrin in the milk of goats by using replicationdefective adenoviral vectors. Protein Expr Purif,2007,53(1):225~231

No related articles found!

Viewed

Full text

Abstract

Cited

Shared

Discussed