新型抗腫瘤轉(zhuǎn)移多肽（β肽）的基因工程制備及其生物學(xué)效應(yīng)
發(fā)布時(shí)間：2019-04-18 23:43
【摘要】：腫瘤細(xì)胞的高轉(zhuǎn)移特性與腫瘤細(xì)胞的高粘附特性有關(guān)。阻斷腫瘤細(xì)胞的粘附可能是預(yù)防腫瘤轉(zhuǎn)移和復(fù)發(fā)的最佳途徑。本研究在前人工作的基礎(chǔ)上,設(shè)計(jì)了三個(gè)β肽(DLYYLMDLSYSMK)的重復(fù)序列(DLYYLMDLSYSMKGGDLYYLMD LSYSMKGGDLYYLMDLSYSMK,β3),嘗試用基因工程的方法構(gòu)建pET-His-β3表達(dá)載體,然后在大腸桿菌表達(dá)系統(tǒng)中表達(dá),并進(jìn)一步研究基因工程表達(dá)出的β3(簡(jiǎn)稱基工β3)的抗腫瘤細(xì)胞粘附和遷移侵襲的活性及其對(duì)腫瘤細(xì)胞分泌的基質(zhì)金屬蛋白酶的影響,以期為抗腫瘤轉(zhuǎn)移藥物的生產(chǎn)探索新的途徑。 根據(jù)大腸桿菌的偏愛密碼子人工合成了β3的基因序列。并同時(shí)采用6種融合蛋白表達(dá)載體表達(dá)β3,以盡快篩選出高效表達(dá)載體。結(jié)果可見,各種表達(dá)載體的表達(dá)情況不同。在pGEX4T-1中,β3可以得到明顯表達(dá),表達(dá)產(chǎn)物GST-β3為包涵體,表達(dá)量約為細(xì)菌總不溶性蛋白量的40%。利用其載體上編碼的GST蛋白用Glutathione Sepharose 4B作為親和介質(zhì)可純化出一定量的GST-β3融合蛋白。而用pMBP-P表達(dá)載體、pTrc-CKS表達(dá)載體和pET-DsbA表達(dá)載體,β3均未得到滿意表達(dá)。在pET22b(+)中,β3獲得了一定的表達(dá)。表達(dá)量可達(dá)細(xì)菌總蛋白的5.5%。從表達(dá)產(chǎn)物的分子量大小來判斷,載體的pel B分泌信號(hào)并未被切除。 用pET-His表達(dá)載體,β3可以得到一定的表達(dá),表達(dá)產(chǎn)物His-β3也是以包涵體的形式存在的。表達(dá)量約占細(xì)胞總蛋白的4%,占細(xì)胞總不溶性蛋白的10%。在變性條件下用金屬螯合瓊脂糖凝膠6B FF柱親和層析,可以從每升細(xì)菌培養(yǎng)液中得到純度為92.2%的His-β3融合蛋白20毫克。提示pET-His表達(dá)系統(tǒng)是高效表達(dá)β3的合適的表達(dá)系統(tǒng)。基工β3(序列為MGSSHHHHHHSSGLVPRGSDL YYLMDLSYSMKGGDLYYLMDLSYSMKGGDLYYLMDLSYSMKAS),分子量約為5.5KD。 用纖連蛋白(fibronectin,FN)作為細(xì)胞外基質(zhì)成分(extracellular matrix,ECM),研究多肽對(duì)腫瘤細(xì)胞與FN粘附的影響。結(jié)果發(fā)現(xiàn):基工β3、化學(xué)合成的β3肽(簡(jiǎn)稱化合β3)、化學(xué)合成的β2肽(簡(jiǎn)稱化合β2)、化學(xué)合成的β1肽(簡(jiǎn)稱化合β1)和GRGDS對(duì)HCCLM6細(xì)胞與FN粘附具有特異的抑制作用,呈現(xiàn)
[Abstract]:The high metastasis characteristics of tumor cells are related to the high adhesion characteristics of tumor cells. Blocking the adhesion of tumor cells may be the best way to prevent tumor metastasis and recurrence. In this study, on the basis of previous work, we designed three repeat sequences of 尾-peptide (DLYYLMDLSYSMK) (DLYYLMDLSYSMKGGDLYYLMD LSYSMKGGDLYYLMDLSYSMK, 尾 3), tried to construct pET-His- 尾 3 expression vector by genetic engineering method, and then expressed it in E. coli expression system. Furthermore, the anti-adhesion, migration and invasion activity of 尾 _ 3 expressed by gene engineering and its effect on matrix metalloproteinases (MMP) secreted by tumor cells were further studied, and the effects of 尾 _ 3 expressed by gene engineering on the adhesion, migration and invasion of tumor cells were also studied. In order to explore a new way for the production of anti-tumor metastasis drugs. The 尾 3 gene sequence was synthesized according to the preferred codon of E. coli. At the same time, 6 fusion protein expression vectors were used to express 尾 3 in order to screen the high efficiency expression vector as soon as possible. The results showed that the expression of different expression vectors was different. In pGEX4T-1, 尾 3 can be expressed obviously. The expression product GST- 尾 3 is an inclusion body, and the expression level is about 40% of the total insoluble protein content of bacteria. A certain amount of GST 尾 3 fusion protein was purified by using Glutathione Sepharose 4B as affinity medium. Using pMBP-P expression vector, pTrc-CKS expression vector and pET-DsbA expression vector, 尾 3 was not expressed satisfactorily. 尾 3 was expressed in pET22b (). The expression level could reach 5.5% of the total bacterial protein. Judging from the molecular weight of the expressed product, the pel B secretion signal of the vector was not excised. By using pET-His expression vector, 尾 3 can be expressed, and the expression product His- 尾 3 also exists in the form of inclusion body. About 4% of the total cell protein and 10% of the total insoluble protein were expressed. Under denatured conditions, 20 mg of His- 尾 3 fusion protein with purity of 92.2% was obtained by metal chelating agarose gel 6B FF column affinity chromatography. These results suggest that the pET-His expression system is a suitable expression system for high efficiency expression of 尾 3. The molecular weight of MGSSHHHHHHSSGLVPRGSDL YYLMDLSYSMKGGDLYYLMDLSYSMKGGDLYYLMDLSYSMKAS), is about 5.5KD. Fibronectin (fibronectin,FN) was used as extracellular matrix component (extracellular matrix,ECM) to study the effect of polypeptide on the adhesion of tumor cells to FN. The results showed that 尾 3, 尾 3 peptide (尾 3), 尾 2 peptide (尾 2 peptide), 尾 1 peptide (尾 1) and GRGDS had a specific inhibitory effect on the adhesion of HCCLM6 cells to FN.
【學(xué)位授予單位】：復(fù)旦大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2005
【分類號(hào)】：Q78
本文編號(hào)：2460393
[Abstract]:The high metastasis characteristics of tumor cells are related to the high adhesion characteristics of tumor cells. Blocking the adhesion of tumor cells may be the best way to prevent tumor metastasis and recurrence. In this study, on the basis of previous work, we designed three repeat sequences of 尾-peptide (DLYYLMDLSYSMK) (DLYYLMDLSYSMKGGDLYYLMD LSYSMKGGDLYYLMDLSYSMK, 尾 3), tried to construct pET-His- 尾 3 expression vector by genetic engineering method, and then expressed it in E. coli expression system. Furthermore, the anti-adhesion, migration and invasion activity of 尾 _ 3 expressed by gene engineering and its effect on matrix metalloproteinases (MMP) secreted by tumor cells were further studied, and the effects of 尾 _ 3 expressed by gene engineering on the adhesion, migration and invasion of tumor cells were also studied. In order to explore a new way for the production of anti-tumor metastasis drugs. The 尾 3 gene sequence was synthesized according to the preferred codon of E. coli. At the same time, 6 fusion protein expression vectors were used to express 尾 3 in order to screen the high efficiency expression vector as soon as possible. The results showed that the expression of different expression vectors was different. In pGEX4T-1, 尾 3 can be expressed obviously. The expression product GST- 尾 3 is an inclusion body, and the expression level is about 40% of the total insoluble protein content of bacteria. A certain amount of GST 尾 3 fusion protein was purified by using Glutathione Sepharose 4B as affinity medium. Using pMBP-P expression vector, pTrc-CKS expression vector and pET-DsbA expression vector, 尾 3 was not expressed satisfactorily. 尾 3 was expressed in pET22b (). The expression level could reach 5.5% of the total bacterial protein. Judging from the molecular weight of the expressed product, the pel B secretion signal of the vector was not excised. By using pET-His expression vector, 尾 3 can be expressed, and the expression product His- 尾 3 also exists in the form of inclusion body. About 4% of the total cell protein and 10% of the total insoluble protein were expressed. Under denatured conditions, 20 mg of His- 尾 3 fusion protein with purity of 92.2% was obtained by metal chelating agarose gel 6B FF column affinity chromatography. These results suggest that the pET-His expression system is a suitable expression system for high efficiency expression of 尾 3. The molecular weight of MGSSHHHHHHSSGLVPRGSDL YYLMDLSYSMKGGDLYYLMDLSYSMKGGDLYYLMDLSYSMKAS), is about 5.5KD. Fibronectin (fibronectin,FN) was used as extracellular matrix component (extracellular matrix,ECM) to study the effect of polypeptide on the adhesion of tumor cells to FN. The results showed that 尾 3, 尾 3 peptide (尾 3), 尾 2 peptide (尾 2 peptide), 尾 1 peptide (尾 1) and GRGDS had a specific inhibitory effect on the adhesion of HCCLM6 cells to FN.
【學(xué)位授予單位】：復(fù)旦大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2005
【分類號(hào)】：Q78
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