【作者】 王云龙；

【导师】 欧阳红生；

【作者基本信息】 吉林大学 ， 生物化学与分子生物学， 2005， 硕士

【摘要】 在本实验中我们要构建出可行的乳腺特异表达载体。根据GenBank上提供的人血清白蛋白基因序列,采用Oligo 6.0 软件设计一对引物,经PCR 扩增得到1833bp 的HSA 基因。以pCDNA3.1 为模板,设计特异性的引物,扩增出筛选标记NEO 基因,然后分别将两段基因HSA、NEO 插入pBC1 载体中,经过酶切、PCR 鉴定以及验证性核苷酸序列分析最终得到乳腺特异表达载体pBC-NEO-HSA。我们将构建好的载体导入到动物乳腺中检测相应的乳汁,目的基因在载体上的调控序列指导下,能够实现表达,这不仅说明载体构建的可行性,而且还表明我们所建立的检测载体系统的合理性与有效性。从而为转基因供体细胞的构建,甚至转基因牛的产生奠定坚实的基础。更多还原

【Abstract】 Human serum albumin is the most richest protein of human blood plasma,and it is about 60% of total protein in blood plasma.HSA plays an important role on maintain blood infiltration pressure,transport nutrition and others.Because HSA acts as the most important means of delivery and plasma protein component and exerts important physiological function.thus have quite a few uses in clinic medical practice,it can be used to treat all kinds of patients whose blood capacity reduced by shock, burn, wound, operation, accident and so on,it also can be used for patients of chronic nephritis,hepatitis,liver cirrhosis,terminal malignant tumour,hydropsy of malnutrition,blood disease of low albumin,blood disease of no albumin and so on.because HSA has great requirement(the dosage of HSA for each patient can reach a few gram),so HSA possesses of great market demand.According to recent report,the distribution of HSA can reach 600t/year throughout the world at present,according to 50dollar/10g,the sale exceed 300 hundred million each year.The distribution of Chinese market is about 70t recent years,the sale is about 300 hundred million RMB one year.The requirement of HSA shows ascend trend throughout the world.But HSA saled at town is mostly distilled from blood plasma of donor or human placenta.But more and more people have infect contagious diseases such as AIDS,hepatitis and so on because input HSA blood production.Because blood has been contaminated or other causation,and country strict control import blood product in addition,makes the supply of HSA quite intensity,people quite expect to explore new sources.Gordon et al obtain the tPA from latex of transgenic mouse in 1987 for the first time,thus makes it possible for transgenic animal to produce protein for medicine . The final goal of this study is to cultivate the donor’s cells which have HSA gene on it to HSA transgenic cow through nuclear transplant technique. The construct of HSA galactophore differential expression carrier is it’s most important foundation and crutial step to cultivate HSA transgenic cattle. According to the gene sequence of HSA protein provides by GenBank, design a pair of primer by Oligo 6.0 software, and add Xhol site on both sides,cDNA library of human liver conserved in our laboratory was used as template,we obtained HSA gene of 1833bp by PCR,pCDNA3.1 was also used as template to design specific primer ,and added Not1 to upstream primer,Apa1 and Not1 to downstream primer we obtained neo filter marker by PCR,then cloned HSA and NEO gene into pGEM-T carrier separatedly,the segment we obtained was approved correct by PCR, restriction analysis,and DNA nucleotide sequence analysis ,then we used pBC1 as basic framework,insert HSA and NEO at Not1 and Xho1 position separately,because these two genes that inserted are both single enzyme digestion,so direction identity was essential after constructed,thus we design a primer at upstream 100bp of the inserted position to match the downstream of the gene,if we obtained the gene that we wanted,thus indicated that the insert direction was the positive direction,we can not obtained the target gene by PCR if we inserted at reverse direction,we obtained specific expression carrier pBC-NEO-HSA finally by restriction analysis,PCR identity and validating nucleotide sequence analysis. Because the manufacture period ofgalactophore biological reactor was long, sheep and cattle only become pregnant in oestrum,so carry out the更多还原