应用Bac-to-Bac杆状病毒表达体系在sf9细胞中重组表达保守性多巴胺能神经营养因子

发布时间：2018-05-16 22:24

  本文选题：杆状病毒表达系统 + 保守性多巴胺能神经营养因子　； 参考：《安徽医科大学》2011年硕士论文

【摘要】：目的:应用bac-to-bac杆状病毒表达体系在sf9昆虫细胞中表达重组CDNF蛋白。 方法:应用Trizol法提取小鼠组织总RNA,RT-PCR扩增得到小鼠CDNF基因全长(564bp)片段,将CDNF基因插入至转座载体pFastBacHTb中,构建重组质粒pFastBacHTb-CDNF,然后转化到E.coli DH5α感受态细胞中,以LB/Amp平板筛选阳性重组子,并经PCR、酶切及测序验证以保证重组载体的正确及可靠性;将杆状病毒转移载体pFasBacHTb-CDNF转化到DH10Bac E.coli(自带杆粒及辅助质粒)感受态细胞中,通过转座作用将目的片段整合到杆粒中,抗性及蓝白斑筛选法筛选出重组杆状病毒载体Bacmid-CDNF,使用CDNF特异引物及PUC/M13杆粒测序引物对重组杆粒进行验证;参照CELLFECTINⅡ脂质体转染剂操作说明,将重组Bacmid-CDNF杆粒转染sf9昆虫细胞,并在光学显微镜下观察细胞形态学变化,以判断转染的成功性;转染成功后,收集P1代病毒液,继续感染sf9细胞,可获取P2代和P3代病毒液,应用P3代病毒诱导重组CDNF蛋白的表达,并应用Western blot对重组蛋白的表达进行验证。 结果: (1)RT-PCR结果经琼脂糖凝胶电泳显示,成功获取564bp大小的CDNF目的基因片段,重组质粒pFastBacHTb-CDNF经PCR、酶切及基因测序验证均正确无误;(2)重组转移载体pFastBacHTb-CDNF转化DH10Bac感受态细胞后,阳性bacmid-CDNF重组子经CDNF特异引物及pUC/M13杆粒测序引物PCR验证可分别获取561bp及3000bp大小目的片段,均与理论大小相符,表明杆粒构建成功;(3)重组杆粒通过Cellfectin转染剂转染sf9昆虫细胞,转染48~72后,sf9细胞细胞直径逐渐增大,细胞渐脱离贴壁培养状态,随之,细胞渐渐肿胀,种种迹象均表明,杆粒转染成功,收获杆状病毒液;(4)P3代杆状病毒液感染sf9细胞,诱导重组CDNF蛋白表达,经Western blot验证,目的蛋白成功诱导表达,并符合预计大小(21KD)。 结论:通过bac-to-bac杆状病毒表达系统成功诱导CDNF重组蛋白的表达。
[Abstract]:Objective: to express recombinant CDNF protein in sf9 insect cells by using bac-to-bac baculovirus expression system. Methods: the full-length CDNF gene fragment of mouse CDNF was amplified by RT-PCR with Trizol method. The CDNF gene was inserted into the transposable vector pFastBacHTb to construct the recombinant plasmid pFastBacHTb-CDNF.Then the recombinant plasmid pFastBacHTb-CDNFS was transformed into E.coli DH5 伪 competent cells, and the positive recombinant plasmid was screened by LB/Amp plate. In order to ensure the correctness and reliability of the recombinant vector, the baculovirus transfer vector (pFasBacHTb-CDNF) was transformed into DH10Bac E.coli (self-contained stem grains and auxiliary plasmids) competent cells, and the target fragment was integrated into the rod grains by transposition. The recombinant baculovirus vector Bacmid-CDNFwas screened by resistance and blue-white spot screening, and the recombinant baculovirus vector Bacmid-CDNFwas verified by CDNF specific primer and PUC/M13 core sequencing primer, and the recombinant Bacmid-CDNF rod was transfected into sf9 insect cells according to the operation of CELLFECTIN 鈪，

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