【作者】 赵秀玲；

【导师】 张耀洲； 金勇丰；

【作者基本信息】 浙江大学 ， 基础兽医学， 2002， 硕士

【摘要】 人表皮生长因子(hEGF)是从人尿中提取的由53个氨基酸组成的小分子活性多肽，是促进细胞增殖和分化至关重要的生长因子。为大量获得EGF，本研究利用家蚕杆状病毒表达系统，以家蚕作为生物反应器，在家蚕细胞和幼虫中表达人表皮生长因子。 从pET22-EGF质粒中分离出末端带His-tag的EGF基因，对位融合于多角体蛋白N端116个氨基酸基因序列的下游(命名为Ph-EGF)，并在两段基因间设计了凝血酶Xa蛋白酶切位点，经过酶切、测序等鉴定正确后，克隆至pBacPAK8中，使Ph-EGF融合基因置于多角体蛋白(polyhedrin，ph)基因启动子控制之下，构建成重组转移载体pBacPh-EGF。重组转移载体DNA与经Bsu36I酶切线性化的修饰型病毒Bm-BacPAK DNA共转染家蚕BmN细胞，两轮空斑筛选和纯化，获得重组病毒rBmBacPh-EGF，经PCR扩增、DNA点杂交等方法鉴定证实重组病毒中已正确插入Ph-EGF融合基因。 将重组病毒rBmBacPh-EGF以10MOI感染BmN细胞，72小时后收集培养细胞和上清；培养上清和经超声波处理的细胞样品ELISA检测发现胞内样品中存在能与EGF抗体免疫反应的产物，粗略估计表达量约6～7μg／2×10~6个细胞(相当于EGF标准品)；重组病毒rBmBacPh-EGF穿刺接种5龄家蚕幼虫，每隔24h收集蚕血淋巴，经ELISA检测发现第4天表达量最高，根据EGF标准曲线计算蚕血淋巴的表达量约32μg／ml；ELISA定性实验还发现正常蚕血也存在与EGF抗体间交叉反应的物质。蚕血淋巴SDS-PAGE和Western blotting分析发现在19kD的位置有特异性条带，与预期表达的融合蛋白大小相一致。 用小鼠Balb／c 3T3成纤维细胞和MTT法测定表达产物的促细胞增殖作用，发现重组病毒感染家蚕细胞72小时的胞内样品与正常家蚕细胞裂解物，以及重组病毒感染4天的蚕血淋巴与正常蚕血淋巴均具有相似的促细胞增殖作用，甚至野生型病毒感染的细胞裂解物和蚕血淋巴也有一定的细胞促生长作用，提示家蚕系统本身可能含有能促进细胞生长、类似于EGF的细胞因子。 本研究还建立了动物模型，大鼠乙醇灌胃后的胃粘膜经病理学观察和 病理组织学检查，发现重组病毒感染蚕蛹5 天的样品灌胃后对乙醇诱导 溃疡的预防性抑制率达50％左右，但正常蚕蛹样品也有一定的预防乙醇 诱导损伤、保护胃粘膜作用。更多还原

【Abstract】 Epidermal growth factor (EOF), a single-chain polypeptide of 53-amino acid, which was firstly isolated from the submaxillary gland of mice in 1962, has wide potent applications. For mass production of EGF, Bombyx mori baculovirus expression vector system (BEVS) was adopted in this experiment to express recombinant EGF.hEGF gene with His-tag at the end, which was derived from pET22 -EGF, was in-frame fused to the carboxy-terminal of polyhedrin (Ph) gene, which included the amino-terminal 116aa coding region. The polyhedrin-EGF fusion gene (named Ph-EGF) was then cut out with EcoRV and EcoRI, and was cloned between EcoRV and EcoRI sites of pBacPAK.8 (the result plasmid was named pBacPh-EGF and the Ph-EGF fusing gene was right under the control of Ph promoter). The pBacPh-EGF structure was verified with restriction enzymes digestion and PCR.The pBacPh-EGF plasmid DNA was used to co-transfect BmN cell with the modified Bombyx mori baculovirus Bm-BacPAK DNA, which was first linearized by Aocl. After two rounds of plaque isolation, the recombinant virus (named BmBacPh-EGF) was further verified with PCR and Dot hybridization.The recombinant BmBacPh-EGF virus was used to infect BmN culture cells at a MOI of 10. The cells and the culture supernatant were collected 72 hours post-infection. For detection of recombinant Ph-EGF expression, SDS-PAGE and ELISA analysis were applied. No trace of any newly expressed protein band was noticed in supernant as well as in the cells by SDS-PAGE, except the verification of the substitution of beta-galactosidase gene (the lose of galactosidase protein band), which is a selective marker of the wild-type virus. ELISA test results suggested the expression of EGF in cells, but not in culture supernant. The quantitative calculation suggested the expressed EGF was about 6-7 U g (as EGF standard) per flask (2><106cells) in the cellular extract. The larvae of 5th instar silkworm were inoculated with the recombinant virus and grew a further 5 days. Hemolymph fluid of the infected larvae was collected every day. The ELISA assay showed a maximum expression hi day 4, which was consensus to other genes expressed in the BEVS. SDS-PAGE and Western blotting analysis showed a newly protein band, which can specifically react to EGF antibody, with a molecular weight about 19 kD as expected, was expressed in the larvae hemolymph fluid.Biological activity was determined by EGF dependent Balb/c 3T3 cell line and with MTT colorimetric assay. Extracts of the recombinant virus-infected and mock-infected cells, haemolymph of the recombinant virus infected and mock-infected silkworm larvae could all support the proliferation of Balb/c 3T3 cell. This phenomena implied that there were some EGF-like growth factors in the haemolymph of normal silkworm larvae, which could enhance the proliferation of the cell line.The animal study was designed to determine whether Ph-EGF fusion protein was protective to ethanol induced injury of gastric mucosa in rats. Thirty-two SD rats weighting 200-250g starved for 24hour were divided into four groups which received pupae infected with recombinant BmBacPh-EGF virus, normal pupae, Ranitidine, 0.9% saline respectively for 3days before received absolute ethanol. The results showed that both pupae infected with BmBacPh-EGF virus and normal pupae protect the gastric mucosa against ethanol induced damage.更多还原