本文選題：c-Src　切入點(diǎn)：原核表達(dá)　出處：《浙江大學(xué)》2005年碩士論文　論文類型：學(xué)位論文

【摘要】：非受體酪氨酸蛋白激酶Src是最早發(fā)現(xiàn)的原癌基因表達(dá)蛋白之一。其以兩種形式存在:病毒原癌基因表達(dá)蛋白v-Src和細(xì)胞原癌基因表達(dá)蛋白c-Src,兩者在進(jìn)化上具有高度同源性。真核細(xì)胞中c-src基因普遍存在,調(diào)節(jié)細(xì)胞的正常生長、增殖、分化、運(yùn)動(dòng)和凋亡等生理功能,c-Src蛋白在細(xì)胞信號(hào)轉(zhuǎn)導(dǎo)過程中扮演重要角色。同時(shí),c-Src蛋白的過量表達(dá)或激酶活性異常,與許多腫瘤的生成、入侵及轉(zhuǎn)移密切相關(guān),是一種公認(rèn)的癌蛋白。 目前,國內(nèi)外的研究主要側(cè)重于c-Src與疾病相關(guān)的定位定性及調(diào)控細(xì)胞功能的分子機(jī)制等方面;而在應(yīng)用上,人們已經(jīng)預(yù)測(cè)c-Src蛋白激酶將成為研究疾病發(fā)生機(jī)理和篩選治療藥物的重要靶蛋白。因此,運(yùn)用基因工程技術(shù)快速簡便地克隆表達(dá)大量高活性的c-Src蛋白,用于激酶固定化篩選腫瘤抑制劑,將具有廣闊的應(yīng)用前景。 本文通過構(gòu)建特異性c-src的原核表達(dá)重組質(zhì)粒,轉(zhuǎn)化大腸桿菌克隆表達(dá)得到完整的人源c-Src蛋白。首先,運(yùn)用PCR技術(shù),分別從人肺腺癌細(xì)胞總mRNA以及獲贈(zèng)的pcDNA3.1/Hygro質(zhì)粒中,擴(kuò)增出全長的人c-src基因。經(jīng)測(cè)序鑒定該序列完全正確。然后,以克隆質(zhì)粒pUCm-T為中介酶切連接,將c-src基因重組到pET-28a(+)、pET-22b(+)和pP_(RO)EX HTc等備選的表達(dá)載體,轉(zhuǎn)化大腸桿菌中篩選陽性重組子,并用IPTG誘導(dǎo)表達(dá)、SDS-PAGE電泳檢測(cè),發(fā)現(xiàn)pP_(RO)EXHTc/c-src為高效的表達(dá)重組質(zhì)粒,而在pET系列的重組質(zhì)粒中c-src基因得不到明顯表達(dá)。工程菌通過正交實(shí)驗(yàn)優(yōu)化的表達(dá)條件是:30℃、0.1mM IPTG、5h,然后大量表達(dá)得到(His)_6/c-Src融合蛋白,在全菌蛋白表達(dá)含量中可以占20%以上。最后用(His)_6-Tag特異性親和的Ni~(2+)-NTA層析柱分離純化,得到的高純度c-Src融合蛋白,與經(jīng)變性透析處理的包涵體蛋白分別測(cè)定酪氨酸蛋白磷酸化活性。結(jié)果證明,表達(dá)的c-Src融合蚩白具有一定的激酶活性,短小的(His)_6-Tag并不影響其結(jié)構(gòu)和功能。這是國內(nèi)首次在大腸桿菌原核表達(dá)體系中克隆表達(dá)得到完整的人c-Src蛋白激酶,為后期的進(jìn)一步研究以及抗癌藥物篩選奠定了基礎(chǔ)。
[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，

本文編號(hào)：1600738