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人 I 型胶原蛋白基因片段的克隆及其转化乳腺上皮细胞的研究
Thesis Advisor王亮
Degree Grantor中国科学院研究生院
Place of Conferral北京
Degree Discipline生物有机化学
Keyword人i 型胶原蛋白 基因克隆 乳腺上皮细胞 转染

近年来,随着胶原蛋白广泛应用于很多领域,国内外市场对胶原蛋白的需求量越来越大,传统的生产方法不能够满足不断增长的市场需求量,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成为目前研究的焦点。转基因乳腺生物反应器生产诸如胶原蛋白这样的结构复杂蛋白时较目前其它的转基因生物反应器具有更大的优势,因为其生产的重组蛋白更接近其天然的结构。本实验从人胎盘基因组DNA 成功地扩增出人I 型胶原蛋白基因(COL1A1)片段,并构建其乳腺特异表达的载体,将其成功的转化了奶牛乳腺上皮细胞。首先,本实验在国内首次克隆出人I 型胶原蛋白基因片段,测序结果表明,其外显子部分与GenBank 中所公布的序列(Access NO.:AF017178)完全一致,但是在内含子部分,尤其是第一个内含子与第二个内含子与GenBank 中公布的序列有很大差异。然后,将连接上组氨酸标签的目的基因构建至乳腺特异表达载体上,与此同时将绿色荧光蛋白与新霉素抗性基因表达框构建到带有组氨酸标签的人I 型胶原乳腺特异表达载体上。最后,建立了奶牛泌乳期乳腺上皮原代细胞系,并将构建好的乳腺特异表达载体成功转染其中,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,并为制备高效表达重组胶原蛋白的乳腺生物反应器奠定了基础。

Other Abstract

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 transgene technique develops, using transgenic bioreactor to produce collagen becomes research spotlight today. Transgenic mammary gland bioreactor possesses more advantages in producing highly-complex protein, e.g. collagen, than other bioreactors, because proteins expressed in mammary gland are very similar to their natural counterparts in structure and function. In this study, the human COL1A1 (typeI collagen, α1-chain) gene fragment was successfully cloned, and then subcloned into mammary gland specific vector. Finally, the bovine mammary epithelial cells were successfully transfected with this recombinant construct. Firstly, human COL1A1 gene fragment was successfully amplified via PCR from human placenta genomic DNA for the first time in China. Sequencing result showed that Chinese COL1A1 fragment was 99.5% identical to the counterpart reported in GenBank (Access NO. AF017178). Their exons were completely identical; but for introns, there were some differences between Chinese COL1A1 fragment and its counterpart in GenBank. The inconsistency was especially obvious in intron1 and intron2. Secondly, COL1A1 gene fragment with His tag was subcloned into mammary specific vector, pBC1, into which Selective Marker gene (GFP and Neomycin expression cassette) were ligated. Finally, primary bovine lactating mammary epithelial cell line was established and then transfected with the recombinant mammary specific construct. This system could be used to correctly evaluated the expression efficiency of mammary specific vector. Above results made good preparations for high expression of recombinant proteins in mammary gland.

Document Type学位论文
Recommended Citation
GB/T 7714
张达江. 人 I 型胶原蛋白基因片段的克隆及其转化乳腺上皮细胞的研究[D]. 北京. 中国科学院研究生院,2007.
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