【作者】 张波；

【导师】 韩跃武；

【作者基本信息】 兰州大学 ， 生物化学与分子生物学， 2010， 硕士

【摘要】 目的：利用Bac-to-Bac杆状病毒表达系统表达抗菌肽Dermaseptin S4 6串联体,并对其进行纯化和鉴定。为进一步研究抗菌肽Dermaseptin S4结构与功能的关系奠定基础。方法：1.根据抗菌肽Dermaseptin S4的氨基酸序列及真核生物偏爱密码子,推出Dermaseptin S4编码基因的序列,并将Dermaseptin S4基因通过连接肽(GATAGGTAGCG)将六个基因片段连接在一起,形成Dermaseptin S4 6串联体基因,并在六串联体的两侧分别加上了Xbal和SalⅠ的限制酶切位点,设计出目的基因,并通过化学合成方法将Dermaseptin S4 6串联体基因合成。2.将抗菌肽基因Dermaseptin S4六串联体克隆到BAC-TO-BACTM重组杆状病毒表达系统的pFASTBac1载体中,构建了重组转座载体pFASTBac-6Dermaseptin S4,抽提重组质粒测序鉴定。3.将鉴定正确的重组质粒转化到DH10Bac大肠杆菌感受态细胞,经蓝白筛选,挑取阳性重组菌落,抽提重组质粒以通用引物RV作为引物进行PCR鉴定。4.获得重组成功的Bacmid DNA,并转染昆虫草地夜蛾(Bombyx mandafina)St9细胞出现病变后,收集含有重组病毒颗粒的培养上清,重新感染草地夜蛾SD单层细胞,收集SD细胞培养上清和细胞再进行SDS-PAGE及Western blot5.用ProBondTM纯化系统(Invitrogen公司产品)采用非变性条件对抗菌肽Dermaseptin S4蛋白进行纯化。结果：1.重组表达质粒经双酶切、PCR及测序鉴定,证明成功构建了pFASTBac-6Dermaseptin S4质粒。2.12%聚丙烯酰胺凝胶电泳可见表达的目的蛋白带,分子量大小为27000，与理论值相符,灰度扫描显示,表达量约占细胞总蛋白的20.1%.Western blot显示目的蛋白均具有良好的反应原性。结论：成功构建了抗菌肽Dermaseptin S4 6串联体,并在昆虫细胞中经杆状病毒表达系统得到成功表达,并进一步对目的蛋白进行了纯化,为下一步蛋白结构与功能的关系奠定基础。更多还原

【Abstract】 Objective:We express and purify the antimicrobial peptide Dermaseptin S4 (DS4) in Bac-to-Bac baculovirus expression system.It layed the foundation in order to study the structure and function of antimicrobial peptides Dermaseptin S4Methods:1.According to amino acid sequences of antimicrobial peptides Dermaseptin S4 and eukaryotic preferred codons genes sequence,we inducted the genes of Dermaseptin S4,and we connected 6 Dermaseptin S4 with 5 connecting peptides.2.the fragment was cloned into the pFASTBac1 vector of BAC-TO-BAC recombinant baculovirus expression system to construct a recombinant expression vector.3.The plasmid pFASTBac-Dermaseptin S4 (DS4) was then transformed into DH10BACTM competent cell. High molecular weight DNA, The recombinant bacmid was prepared from the ovemight cultures of selected Eschefichia coli DH10BACTM colonies. We extracted recomplasmid and identified it with universal primer RV as primers for PCR cation.4.By transfecting spodoptem fragiperda 9 (SD)cells with the recombinant bacmid, the recombinant baculovirus from the supernatant was obtained, which could further infect SD cells and express the target protein.Identify the expressed protein by SDS-PAGE and Western blot.5.We purified the protein of Dermaseptin S4 with ProBondTM purification system on non-denaturing conditions.Results:PCR amp lification and DNA sequencing analys is confirmed that the gene was correctly inserte d into the vector. Relative molecular masses of 27000 were expressed by SDS-PAGE and accounted for 20.1%。Conclus:The correct tandem of Dermaseptin S4 gene is constructed and expressed in insect cells by baculovirus expression system successfully.We Purified the protein furtherly and we made the basis to study the structure and function of the protein again.更多还原