乳腺生物反应器特异高效表达载体的构建

doi:10.13523/j.cb.20140708

中国生物工程杂志

2014, Vol. 34

Issue (7): 49-55 DOI: 10.13523/j.cb.20140708

技术与方法

乳腺生物反应器特异高效表达载体的构建

张斯敏¹, 高越¹, 方彧聃^1,2, 张金脉¹, 张敬之^1,2

1. 上海交通大学附属儿童医院上海市儿童医院上海交通大学医学遗传研究所上海 200040;
2. 卫生部医学胚胎分子生物学重点实验室及上海市胚胎与生殖工程重点实验室上海 200040

Construction of Mammary Gland-specific and Effective Expression Vector for Mammary Gland Bioreactor

ZHANG Si-min¹, GAO Yue¹, FANG Yu-dan^1,2, ZHANG Jin-mai¹, ZHANG Jin-zhi^1,2

1. Shanghai Children's Hospital, Shanghai Jiao Tong University, Shanghai Institute of Medical Genetics, Shanghai Jiao Tong University, Shanghai 200040, China;
2. Key Laboratory of Embryo Molecular Biology, Ministry of Health & Shanghai Key Laboratory of Embryo and Reproduction Engineering, Shanghai 200040, China

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摘要：

乳腺生物反应器具有广阔的开发前景，而提高目的基因的表达量是该领域一个重要的研究课题。因此，选用强的非特异性启动子pCAG，而不是乳腺特异性启动子来实现高效表达；通过Cre/loxP系统和山羊β-乳球蛋白启动区（pBLG）表达Cre重组酶来实现载体的自删除以达到乳腺特异表达的目的。方法：构建含PolyA终止信号的Cre乳腺特异表达元件PolyA-pBLG-cre，插至强启动子pCAG驱动的报告基因lacZ表达载体中，构建成乳腺特异表达载体pCBCZ（pCAG-loxP-PolyA-pBLG-cre-loxP-lacZ）。转染细胞实验结果：PCR鉴定确认pCBCZ载体在小鼠乳腺上皮细胞（HC11）中发生Cre-loxP同源重组。X-Gal染色表明载体能驱动lacZ在HC11细胞中高效表达β-半乳糖苷酶，而在NIH 3T3细胞中仅少量表达。结论：构建的pCBCZ载体能高效驱动外源基因在乳腺细胞中表达，且具有较好的乳腺特异性，为研发乳腺生物反应器表达载体提供新的方法。

关键词： 乳腺生物反应器; Cre/loxP系统; 特异表达; 报告基因lacZ

Abstract:

Mammary gland bioreactor is considered as a promising approach for generating pharmaceutical proteins. Yet, the low-level expression of the transgene is one of the bottlenecks in this technique. Here, a strong and constitutively expressing promoter instead of a mammary gland specific but weak promoter was used to improve transgene expression; meanwhile, a Cre/loxP system driven by a β-lactoglobulin (BLG) gene promoter was used to restrict the strong expression in mammary gland. Methods: Construct the mammary gland-specific expression unit (Polyadenylation signal-pBLG-cre), this unit was then flanked by a pCAG strong promoter and a lacZ reporter. Thus, a mammary gland-restricted expression vector named pCBCZ (pCAG-loxP-PolyA-pBLG-cre-loxP-lacZ) was constructed. The in vitro experiment results: Cre-loxP recombination was detected in the mouse epithelial (HC11) cells transfected with pCBCZ vector. X-Gal staining experiment showed that the vector can strongly drive β-galactosidase expression in HC11 cells, but weak in NIH 3T3 cells. These data indicate that the pCBCZ vector can effectively drive gene expression at a mammary gland-restricted manner, which provide a new strategy for the generation of an effective mammary gland bioreactor.

Key words: Mammary gland bioreactor Cre/loxP system Tissue specific expression lacZ reporter

收稿日期: 2014-02-21 出版日期: 2014-07-25

ZTFLH:

Q78

基金资助:

国家“973”计划（2010CB945202），国家自然科学基金（81271690）资助项目

通讯作者: 张敬之 E-mail: jzhang38@hotmail.com

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引用本文:

张斯敏, 高越, 方彧聃, 张金脉, 张敬之. 乳腺生物反应器特异高效表达载体的构建[J]. 中国生物工程杂志, 2014, 34(7): 49-55.

ZHANG Si-min, GAO Yue, FANG Yu-dan, ZHANG Jin-mai, ZHANG Jin-zhi. Construction of Mammary Gland-specific and Effective Expression Vector for Mammary Gland Bioreactor. China Biotechnology, 2014, 34(7): 49-55.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/10.13523/j.cb.20140708 或 https://manu60.magtech.com.cn/biotech/CN/Y2014/V34/I7/49

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