【作者】 赵大鹏；

【导师】 付学奇； 盛军；

【作者基本信息】 吉林大学 ， 生物工程， 2007， 硕士

【摘要】 本研究用改进的RT-PCR法合成人肝脏cDNA,构建出cDNA文库,并从中分离出人胰岛素样生长因子1(human insulin like growth factor 1,hIGF-1)基因。将hIGF-1基因酶切后插入到pGEX-1λT载体中,连接转化大肠杆菌NM522,筛选克隆,酶切鉴定后测序,并进行了表达。将IPTG诱导表达的菌体离心收集,取少量菌体进行SDS-PAGE分析,在分子量34KD处可见明显高表达带(对照菌没有表达),Western印迹表明重组蛋白具有hIGF-1的抗原性。在完成以上研究的基础上,鉴于原核分泌型表达技术的优势,我们根据原核细胞分泌表达机制及需要,重新设计引物,利用PCR法分别分离出碱磷酸酶启动子(phoA),STII信号肽及hIGF-1编码基因,将它们连接到pUC载体,测序后,通过点突变技术对全基因序列进行了校正。分别构建出两套分泌型表达载体,并将其命名为pCSA和pCST,随后将上述基因装入到pCSA和pCST中,转化大肠杆菌W3110,得到工程菌pCSA/IGF-1/W3110,pCST/IGF-1/W3110,并进行了表达。经SDS-PAGE检测,在分子量7.6KD处有明显高表达带,Western印迹表明重组蛋白具有hIGF-1的抗原性。根据中国生物制品检定规程的有关要求,我们进行了基因工程大肠杆菌工程菌的筛选和鉴定,得到工程菌株pCSA/IGF-1/W3110 ,pCST/IGF-1/W3110,经传代培养,提质粒酶切鉴定及蛋白表达测定表明,经100次传代后质粒不丢失,酶切鉴定表明为阳性克隆,蛋白表达稳定。根据中试工艺的要求,我们初步建立了分泌型hIGF-1的生产工艺,即为;种子液制备→发酵培养→离心收集菌体深度冷冻→rhIGF-1低渗震荡溶出→层析前处理→疏水层析→分子筛→离子交换→半成品。参照有关文献,以NIH3T3作为靶细胞,用XTT掺入法,初步建立了hIGF-1生物活性检定方法。更多还原

【Abstract】 Human insulin-like growth factor I (hIGF-1) is a kind of multipotential regulatory factor secreted by cell, and has wide biological effects. It can promote proliferation and differentiation of many kind of cell. As a result human IGF-1 (hIGF-1) has been put in use to treat many diseases such as diabetes, insulin-resistant syndrome, dwarfism, some nervous system deseases and other obstinate diseases. The clinic outcomes were rather encounging.Unfortunately the quantity of hIGF-1 in blood is very limited, moreover it mainl exists in the form of inactive complex. In order to get sufficient quantities of pure hIGF-1 with bioactivity to meet the demand of basic research and clinical uses, We first builded the human liver tissue cDNA library and from it gene fragment of hIGF-1 was isolated. After DNA sequencing result proved to be the right sequence, It was cloned into vector pGEX-1λT so that the fusion expressing vector pGEX/hIGF-1 was constructed. When it was transformed into E.coli NM522 the fusion protein was expressed. The protein was indicated hIGF-1 antigenicity by analysis of SDS-PAGE and Western blot.At the same time, we construct two prokaryotic secretory expressing vector pCSA and pCST according to the principle of prokaryotic secretory expression. First, promotor alkaline phosphatase (phoA), signal peptide STII and hIGF-1 gene were obtained by PCR, and then they were inserted into vector pUC. After the whole gene sequence was corrected by technology of point mutation, it was inserted into vector pCSA and pCST, which came to the two secretory expressing prokaryotic vector of pCSA and pCST. After it was transformed to E.coli W3110, two engineering stains of pCSA/IGF-1/W3110 and pCST/IGF-1/W3110 were obtained to express corresponding protein. The protein expressed detected by SDS-PAGE showed a high expressed protein band with a relative molecular weight of 7.6KD, western blot showed the hIGF-1 antigenicity.Then Stability of the engineering strain and the method for its screening and preservation were studied, as well as the semi-works technology of secretory expressed hIGF-1 and detective method of its bioactivity.In the end, the fermentation, purification technology on secretory expression of hIGF-1 was primarily set up, as well as the detective method of the bioactivity of hIGF-1. The construction of semi-works technology of secretory-expressed hIGF-1 and the establishment of detective method of hIGF-1 bioactivity will play an elementary role of further study on the function and characteristics of hIGF-1, as well as the industrialization of hIGF-1.更多还原