新型抗肿瘤转移多肽（β肽）的基因工程制备及其生物学效应

发布时间：2019-04-18 23:43

【摘要】：肿瘤细胞的高转移特性与肿瘤细胞的高粘附特性有关。阻断肿瘤细胞的粘附可能是预防肿瘤转移和复发的最佳途径。本研究在前人工作的基础上,设计了三个β肽(DLYYLMDLSYSMK)的重复序列(DLYYLMDLSYSMKGGDLYYLMD LSYSMKGGDLYYLMDLSYSMK,β3),尝试用基因工程的方法构建pET-His-β3表达载体,然后在大肠杆菌表达系统中表达,并进一步研究基因工程表达出的β3(简称基工β3)的抗肿瘤细胞粘附和迁移侵袭的活性及其对肿瘤细胞分泌的基质金属蛋白酶的影响,以期为抗肿瘤转移药物的生产探索新的途径。根据大肠杆菌的偏爱密码子人工合成了β3的基因序列。并同时采用6种融合蛋白表达载体表达β3,以尽快筛选出高效表达载体。结果可见,各种表达载体的表达情况不同。在pGEX4T-1中,β3可以得到明显表达,表达产物GST-β3为包涵体,表达量约为细菌总不溶性蛋白量的40%。利用其载体上编码的GST蛋白用Glutathione Sepharose 4B作为亲和介质可纯化出一定量的GST-β3融合蛋白。而用pMBP-P表达载体、pTrc-CKS表达载体和pET-DsbA表达载体,β3均未得到满意表达。在pET22b(+)中,β3获得了一定的表达。表达量可达细菌总蛋白的5.5%。从表达产物的分子量大小来判断,载体的pel B分泌信号并未被切除。用pET-His表达载体,β3可以得到一定的表达,表达产物His-β3也是以包涵体的形式存在的。表达量约占细胞总蛋白的4%,占细胞总不溶性蛋白的10%。在变性条件下用金属螯合琼脂糖凝胶6B FF柱亲和层析,可以从每升细菌培养液中得到纯度为92.2%的His-β3融合蛋白20毫克。提示pET-His表达系统是高效表达β3的合适的表达系统。基工β3(序列为MGSSHHHHHHSSGLVPRGSDL YYLMDLSYSMKGGDLYYLMDLSYSMKGGDLYYLMDLSYSMKAS),分子量约为5.5KD。用纤连蛋白(fibronectin,FN)作为细胞外基质成分(extracellular matrix,ECM),研究多肽对肿瘤细胞与FN粘附的影响。结果发现:基工β3、化学合成的β3肽(简称化合β3)、化学合成的β2肽(简称化合β2)、化学合成的β1肽(简称化合β1)和GRGDS对HCCLM6细胞与FN粘附具有特异的抑制作用,呈现
[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.
【学位授予单位】：复旦大学
【学位级别】：博士
【学位授予年份】：2005
【分类号】：Q78

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