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摘要：昆虫Kazal型丝氨酸蛋白酶抑制剂常表现出调节机体免疫应答、参与凝血、抑制肿瘤细胞的增殖和迁移等方面的活性。但到目前为止，与马陆来源的丝氨酸蛋白酶抑制剂的相关报道非常少。在本研究中，克隆了一个马陆来源的Kazal型丝氨酸蛋白酶抑制剂（Serpin-YM），该丝氨酸蛋白酶抑制剂长为228bp，其中前19个氨基酸为信号肽。然后利用大肠杆菌原核表达系统成功表达出了一个27KD的融合蛋白，并利用镍离子亲和柱层析和透析进行纯化。最后对Serpin-YM的抑癌活性进行测定，发现Serpin-YM能够抑制HeLa细胞的增殖，并且在5μM时就表现出显著的抑制作用，Serpin-YM有潜力开发为癌症治疗药物。37296
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毕业论文关键词：约安巨马陆；丝氨酸蛋白酶抑制剂；原核表达；细胞活力
PROKARYOTIC EXPRESSION AND CELL VIABILITY OF A NOVEL SERPIN-YM IN THE PROSPIROBOLUS JOANNSI
Abstract: Insect-derived Kazal-type serine protease inhibitors can regulate the immune response of organisms and inhibit the migration and proliferation of tumor cells. It is also involved in blood clotting. But so far, there has no reports for Prospirobolus-derived serine protease inhibitors. In this study, we clone a kind of Prospirobolus-derived Kazal-type serine protease inhibitors named Serpin-YM. The cloned sequence included 228 nucleotides and the first 20 amino acid residues makes up the signal peptide. The predicted protein which is 27KD has been successfully expressed by using prokaryotic expression system. And the protein is purified by High Affinity Ni-charged Resin(GenScript) and dialysis. Finally, we find that Serpin-YM has activity on anti-cancer. It can inhibit the proliferation of HeLa cells. When the concentration of Serpin-YM is up to 5μM, the inhibitory effect becomes remarkable. Serpin-YM has the potential to become a kind of cancer treatment drugs.
Key words: Prospirobolus joannsi; Serpin; Prokaryotic expression; Cell viability
目 录
摘要： 2
关键词： 2
Abstract: 2
Key words: 2
1 引言 3
2 材料与方法 4
2.1 实验动物和细胞 4
2.2 菌株和载体 4
2.3 主要试剂 4
2.4 主要实验仪器和设备 5
2.5 Serpin-YM基因克隆 5
2.5.1 样品总RNA的提取 5
2.5.2 反转录获取cDNA 5
2.5.3 Serpin-YM基因扩增 5
2.6 Serpin-YM重组表达质粒pET32a-Serpin-YM的构建 6
2.6.1 目的片段与T载体的连接 6
2.6.2 T载体连接产物转化大肠杆菌感受态DH5∂ 6
2.6.3 克隆质粒pTG19-T与表达载体pET-32a的双酶切及纯化 7
2.6.4 目的基因与表达载体pET-32a的连接与转化 7
2.7 Serpin-YM重组蛋白的诱导表达 7
2.7.1 重组表达质粒pET32a-Serpin-YM转化BL21感受态 7
2.7.2 重组蛋白诱导表达并验证 7
2.7.3 重组蛋白的纯化 8
2.7.4 融合蛋白Serpin-YM透析脱盐 8
2.7.5 考马斯亮蓝法测蛋白浓度 8
2.8 Serpin-YM重组蛋白对HeLa细胞的影响 9
3 结果 9
3.1 目的片段的获取及分析 9
3.1.1 RNA的提取 9
3.1.2 目的基因片断PCR扩增结果 9
3.2 诱导表达结果 10
3.2.1 克隆质粒pTG19-T构建结果 10
3.2.2 重组表达质粒pET32a-Serpin-YM构建结果 11
3.2.3 Serpin-YM重组蛋白诱导表达及可溶性分析 11