【作者】 史晓儒；

【导师】 张政； 王转花；

【作者基本信息】 山西大学 ， 生物化学与分子生物学， 2005， 硕士

【摘要】 细胞凋亡在维持机体处于稳定状态中起着重要作用,细胞凋亡异常与人体多种恶性肿瘤的发生发展过程密切相关。细胞内蛋白酶及其抑制剂可能在细胞凋亡的启动方面起着关键性作用,以选择性地诱导肿瘤细胞凋亡为目标的凋亡干预技术可能成为治疗恶性肿瘤的基本策略。 本项研究以山西寿阳荞麦为材料,提取植株叶片的总RNA反转录成cDNA,经过多重PCR扩增,得到荞麦胰蛋白酶抑制剂基因。根据原核表达载体基因序列设计带有双酶切位点的引物,PCR扩增后连接到pGEM-Teasy载体中,经鉴定正确后,用BamH Ⅰ和Hind Ⅲ双酶切得到目的基因,与经过同样双酶切的原核表达载体pQE31连接,将构建好的原核表达载体转化到大肠杆菌M15中诱导表达。对表达条件优化后,确定OD₆₀₀在0.5-0.6,IPTG浓度为0.5 mmol/L,37℃诱导表达4小时为最佳表达系统。表达产物经过HiTrap Chelating HP column亲和纯化,用含0.5 mol/L咪唑的洗脱液洗脱。纯化的目的蛋白经Tris-Tricine SDS-PAGE和Western blot鉴定后,分析目的蛋白的表达量、回收率及抑制活性。 研究结果显示,我们成功地构建了pQE31-BTI原核表达载体,经表达、纯化获得纯度较高的目的蛋白。重组蛋白具有较高的胰蛋白酶抑制剂活性,专一性抑制活性约为77.16 U_I/mg纯化蛋白,抑制活性回收率达到88.56%。 已有报道表明,多种蛋白酶抑制剂能够抑制肿瘤细胞的生长,是一种潜在的、有应用前景的抗肿瘤药物。本研究将表达纯化的重组蛋白进行诱导肿瘤细胞凋亡的实验。通过MTT法测定和流式细胞仪初步分析表明:重组荞麦胰蛋白酶抑制剂能够抑制IM9细胞的生长,抑制作用达到27.56%,当IM9细胞同时表达Bcl-2蛋白时,该重组蛋白对细胞仍有14.46%的抑制作用,说明本项研究通过基因克隆、表达纯化获得的重组BTI对IM9细胞和IM9/Bcl-2细胞都有很强的抑制生长的作用。这一研究结果为今后深入开展荞麦胰蛋白酶抑制剂抑制肿瘤细胞生长并诱导其发生凋亡的机理及药物开发研究均具有重要的理论意义与实践意义。更多还原

【Abstract】 Apoptosis is a cell initiative death course, that caused by cell’s internal and external environment changes, death signal touching off and the regulation of gene. This course plays an important role for dispelling the aging cell in the organism or having potential cell that unusually grew, and keeping the organism being in homeostasis. The disorder of internal apoptosis regulation and control mechanism will cause human body many kinds of illnesses. The formation of tumors and the senile dementia belong to this kind of typical illness. A lot of researchers at present focus on study on protease and protease inhibitor in the cell and they think these moleculars might play a key role in apoptosis.This paper regarded Shouyang buckwheat as studied materials. TotalRNA which was isolated from the leaves of plants was reversely transcribedinto cDNA. Buckwheat trypsin inhibitor gene was gotten through numerousPCR with designed primers which had enzyme cut site according to thesequence of expressive vector. Then PCR product was connected topGEM-Teasy vector after amplifying. After verification, the purpose genewas cut from the plasmid of pGEM-T-BTI by BamH I and Hind Ⅲ and wasconnected with the expressive vector pQE31 that was cut by the sameenzymes. The constructed plasmid was transformed into the host cell ofE.coli M15[pREP4]. Through optimizing the expression condition, it wasconsidered appropriate to expressive the target protein with OD60o=0.5-0.6, 0.5mmol/L IPTG, 37°C, and express for 4 hours. The expressed product could be purified through HiTrap Chelating HP column, and be eluted by the elution buffer contianing 0.5mol/L imidazole. Target protein was identified by Tris-Tricine SDS-PAGE and Western blot, and the recovery rate and yield were estimated by means of the analysis of BTI activity.The result of study showed that we constructed successfully pQE31-BTI expression vector, and purified the expressed product. After identification, the target protein had been dried in frozen state. The inhibitory activity of recombinant BTI was about 77.16 Ui/mg. The recovery rate could be up to 88. 56%.Because the protease inhibitor can suppress the growth of the tumour cell, it is regard as a kind of potential antineoplastic factor. The recombinant protein could induce apoptosis of tumour cell. At first its biological activation of suppressing tumour cell to grow was determined with MTT assay, and then its inhibitory rate growing to tumour cell was determined with flow cytometry.The recombinant BTI could suppress growth of IM9 cell through MTT assay. Result showed that recombinant BTI had strong restrain function of growth to IM9 cell and IM9/ Bcl-2. Up to 27.56% IM9 cell was restrained, and when IM9 cell expresses Bcl-2 at the same time, 14.46% IM9/ Bcl-2 cells were strongly restrained. It was shown that the recombinant BTI could更多还原