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携带人I型胶原蛋白基因片段的牛核移植胚胎的研究
彭涛
Subtype硕士
Thesis Advisor王亮
2009-06-04
Degree Grantor中国科学院研究生院
Place of Conferral北京
Degree Discipline生物有机化学
KeywordI型胶原蛋白 核移植 克隆 成纤维细胞 转染
Other Abstract
目前,胶原蛋白已经广泛应用于许多领域,国内外市场对胶原蛋白的需求量不断增长,传统的生产方法已经不能够满足市场需求,随着转基因技术的发展,利用转基因生物反应器生产胶原蛋白解决此问题成为研究的热点。 转基因乳腺生物反应器生产胶原蛋白这样结构复杂的蛋白时,相对于其它的转基因生物反应器具有更大的优势,因为其翻译加工后生产的重组蛋白更接近天然的结构。 本实验将含有人I型胶原蛋白基因(COL1A1)cDNA片段的乳腺特异表达的载体pGC1转化犊牛的耳部成纤维细胞中。在转染的22个细胞系中设计特异引物利用PCR检测出16个成功转染目的基因的细胞系。以此细胞作为核供体细胞通过核移植技术尘垢构建转基因牛胚胎,并将胚胎培养到囊胚阶段,囊胚率达到6.25%,对囊胚中内细胞团进行PCR检测,证明所有的囊胚中均转染了目的基因。 此次实验这对于正确评估乳腺特异表达载体的表达效率有很大意义,建立的转基因牛胚胎为制作高效表达重组蛋白的牛乳腺生物反应器奠定了基础。
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In these years, with the wide application of collagen in many fields as biomaterial, the market for collagen is increasing. Traditional production methods of collagen can not satisfy the increasing demands. As the transgenic technique developes, using transgenic bioreactor to produce collagen becomes the key to solve this problem,this research is hot spot. Transgenic mammary gland reactor possesses more advantages in producing highly-complex protein than other bioreactors. Proteins expression in mammary gland after translation and elaboration is very similar to their native counterpart in structure and function. In this test, the human COL1A1 (typeI collagen, α1-chain) gene fragment was successfully subcloned it into mammary gland specific vector. Finally, the Holstein cow fetal fibroblast cells were successfully transfected with this recombinant construct. 22 Holstein cow fetal fibroblast cell lines was established. The cells were transfected by the recombinant mammry specific construct. We obtained 16 transgenic cell line frome 22 cell line by PCR with verification primer. This test could be used to correctly evaluated expression efficiency of mammary specific construct. Our test underlied the high expression of recombinant proteins in mammary gland.
Document Type学位论文
Identifierhttp://ir.xjipc.cas.cn/handle/365002/3563
Collection资源化学研究室
Affiliation中国科学院新疆理化技术研究所
Recommended Citation
GB/T 7714
彭涛. 携带人I型胶原蛋白基因片段的牛核移植胚胎的研究[D]. 北京. 中国科学院研究生院,2009.
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