【作者】 颜新敏；

【导师】 孙怀昌；

【作者基本信息】 扬州大学 ， 预防兽医， 2004， 硕士

【摘要】 人表皮生长因子(hEGF)是一种很强的促细胞有丝分裂原，通过与表皮细胞等细胞表面受体结合，导致受体自身酪氨酸激酶的磷酸化，进而激活下游一系列信号级联反应，最终表现为广泛的促细胞增殖作用，因此被广泛应用于医学临床。 为了建立表达hEGF的鸡输卵管瞬时生物反应器，本研究根据发表的hEGF编码区序列设计引物，用PCR从人肝脏混合单链cDNAs中扩增出与山羊β-乳球蛋白信号肽序列融合的基因片断，序列测定结果显示，所克隆的hEGF编码区序列与已发表的序列完全相同，引入的山羊β-乳球蛋白信号肽序列正确。将此基因片断插入到原核表达载体pGEX-6p-1中，重组载体转化的大肠杆菌在IPTG的诱导下能表达预计大小的融合蛋白。将上述基因片断克隆入真核表达载体pcDAN-3中，用获得的重组质粒pcDNA-hEGF免疫家兔，经Western Blotting检测证明，获得的抗血清能识别大肠杆菌表达的GST-hEGF融合蛋白。 将上述hEGF编码序列克隆到鸡输卵管定位表达载体的鸡卵清蛋白基因5-和3-调控区之间，获得的重组质粒pcDNAOV-hEGF用PEI包埋后，以1mg／只的剂量通过翅静脉注射产蛋鸡，用上述基因免疫获得的抗血清进行的Western Blotting检测结果显示，载体注射鸡的蛋清中能检测到约6KDa的重组蛋白。经MTT法细胞增殖试验证明，含表达产物的蛋清对NIH／3T3细胞生长具有促进或抑制作用，蛋清浓度低于5％时表现生长促进作用，大于5％时表现为生长抑制作用。以添加2.5％蛋清进行的表达的动态试验，结果表明一次注射1mg重组载体鸡的蛋清中重组hEGF的表达至少维持一周。更多还原

【Abstract】 Human epidermal growth factor is powerful mitogen for a wide variety of cells in culture and in vivo. It can bind to the extracellular domain of its receptor, which is a kind of tyrosine kinase. leading to itself phosphorylation and cells proliferation.To develop the recombinant hEGF. a pair of primers were synthesized according to previously published hEGF coding sequence with goat p-lactoglobulin signal peptide sequence fused to the 5-end for secretive expression, a cDNA of correct size was amplified from pooled human liver single-stranded cDNAs by high fidelity PCR. Sequence analysis showed that the hEGF coding region was identical to that previously published and the goat p-lactoglobulin signal peptide sequence was introduced correctly. The cDNA was subcloned into prokaryotic expression vector pGEX-6p-l and the resulting recombinant plasmid was transformed to E .coli. BL21 (DE3) for expression. After IPTG induction, an additional band of 32KDa was detected by SOS-PAGE. The cDNA was then subcloned into eukaryotic expression vector pcDNA-3 and the recombinant vector was injected into rabbits. After six times of injection, an antiserum to hEGF was obtained, which was able to recognize GST-hEGF fusion protein expressed in E .coli.The hEGF cDNA was subcloned into hen oviduct-specific expression vector pOV. The recombinant vector was mixed with 25kDa polyethyleneimine and injected into egg-dropping hens via wing vein route. Western Blotting showed that a 6KDa recombinant protein was detectable in egg white from vector-injected hens by the rabbit antiserum. Cell proliferation assay using MTT method showed the recombinant hEGF-containing egg white had promotion or inhibition effect on NIH/3T3 cells, depending on the amounts of egg white used. Based on the cell proliferation assay, the expression of hEGF in vector-injected hen egg white lasted for at least one week. These data indicate the correctness of cloned hEGF cDNA and the feasibility of the construct for production of recombinant hEGF by hen oviduct bioreactor.更多还原