人c-Src全长基因的克隆及其在大肠杆菌中表达的研究

发布时间：2018-03-12 08:19

  本文选题：c-Src　切入点：原核表达　出处：《浙江大学》2005年硕士论文　论文类型：学位论文

【摘要】：非受体酪氨酸蛋白激酶Src是最早发现的原癌基因表达蛋白之一。其以两种形式存在:病毒原癌基因表达蛋白v-Src和细胞原癌基因表达蛋白c-Src,两者在进化上具有高度同源性。真核细胞中c-src基因普遍存在,调节细胞的正常生长、增殖、分化、运动和凋亡等生理功能,c-Src蛋白在细胞信号转导过程中扮演重要角色。同时,c-Src蛋白的过量表达或激酶活性异常,与许多肿瘤的生成、入侵及转移密切相关,是一种公认的癌蛋白。 目前,国内外的研究主要侧重于c-Src与疾病相关的定位定性及调控细胞功能的分子机制等方面;而在应用上,人们已经预测c-Src蛋白激酶将成为研究疾病发生机理和筛选治疗药物的重要靶蛋白。因此,运用基因工程技术快速简便地克隆表达大量高活性的c-Src蛋白,用于激酶固定化筛选肿瘤抑制剂,将具有广阔的应用前景。 本文通过构建特异性c-src的原核表达重组质粒,转化大肠杆菌克隆表达得到完整的人源c-Src蛋白。首先,运用PCR技术,分别从人肺腺癌细胞总mRNA以及获赠的pcDNA3.1/Hygro质粒中,扩增出全长的人c-src基因。经测序鉴定该序列完全正确。然后,以克隆质粒pUCm-T为中介酶切连接,将c-src基因重组到pET-28a(+)、pET-22b(+)和pP_(RO)EX HTc等备选的表达载体,转化大肠杆菌中筛选阳性重组子,并用IPTG诱导表达、SDS-PAGE电泳检测,发现pP_(RO)EXHTc/c-src为高效的表达重组质粒,而在pET系列的重组质粒中c-src基因得不到明显表达。工程菌通过正交实验优化的表达条件是:30℃、0.1mM IPTG、5h,然后大量表达得到(His)_6/c-Src融合蛋白,在全菌蛋白表达含量中可以占20%以上。最后用(His)_6-Tag特异性亲和的Ni~(2+)-NTA层析柱分离纯化,得到的高纯度c-Src融合蛋白,与经变性透析处理的包涵体蛋白分别测定酪氨酸蛋白磷酸化活性。结果证明,表达的c-Src融合蚩白具有一定的激酶活性,短小的(His)_6-Tag并不影响其结构和功能。这是国内首次在大肠杆菌原核表达体系中克隆表达得到完整的人c-Src蛋白激酶,为后期的进一步研究以及抗癌药物筛选奠定了基础。
[Abstract]:Non-receptor tyrosine protein kinase (Src) is one of the earliest proto-oncogene expression proteins. It exists in two forms: virogen oncogene expression protein v-Src and cell oncogene expression protein c-Src. both have a high degree of evolution. Homology. The c-src gene is ubiquitous in eukaryotic cells. Regulation of the normal growth, proliferation, differentiation, movement and apoptosis of cells plays an important role in the signal transduction of cells. At the same time, the overexpression of c-Src protein or abnormal kinase activity are associated with the formation of many tumors. Invasion and metastasis are closely related, is a recognized oncoprotein. At present, domestic and foreign studies mainly focus on the localization and characterization of c-Src related to disease and the molecular mechanism of regulating cell function. It has been predicted that c-Src protein kinase will become an important target protein for studying the pathogenesis of disease and screening therapeutic drugs. Therefore, a large number of highly active c-Src proteins can be cloned and expressed quickly and easily by genetic engineering. It can be used to immobilize kinase to screen tumor inhibitors, which will have a wide application prospect. In this paper, the prokaryotic expression plasmid of specific c-src was constructed and transformed into E. coli to obtain the complete human c-Src protein. Firstly, the total mRNA of human lung adenocarcinoma cells and the donated pcDNA3.1/Hygro plasmid were obtained by PCR technique, respectively. The full-length human c-src gene was amplified. The sequence was confirmed to be correct by sequencing. Then, the c-src gene was ligated into alternative expression vectors pET-28a (pET-22b ()) and pP_(RO)EX HTc, using the cloned plasmid pUCm-T as the intermediate enzyme to ligate the c-src gene. The positive recombinant plasmid was screened in E. coli and detected by SDS-PAGE electrophoresis of IPTG induced expression. It was found that pP_(RO)EXHTc/c-src was an efficient recombinant plasmid. The expression conditions of c-src gene in recombinant plasmids of pET series were optimized by orthogonal experiment. The optimal expression conditions were as follows: 1. 30 鈩，

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