發(fā)布時(shí)間：2018-05-16 22:24

  本文選題：桿狀病毒表達(dá)系統(tǒng) + 保守性多巴胺能神經(jīng)營(yíng)養(yǎng)因子　； 參考：《安徽醫(yī)科大學(xué)》2011年碩士論文

【摘要】：目的:應(yīng)用bac-to-bac桿狀病毒表達(dá)體系在sf9昆蟲細(xì)胞中表達(dá)重組CDNF蛋白。 方法:應(yīng)用Trizol法提取小鼠組織總RNA,RT-PCR擴(kuò)增得到小鼠CDNF基因全長(zhǎng)(564bp)片段,將CDNF基因插入至轉(zhuǎn)座載體pFastBacHTb中,構(gòu)建重組質(zhì)粒pFastBacHTb-CDNF,然后轉(zhuǎn)化到E.coli DH5α感受態(tài)細(xì)胞中,以LB/Amp平板篩選陽性重組子,并經(jīng)PCR、酶切及測(cè)序驗(yàn)證以保證重組載體的正確及可靠性;將桿狀病毒轉(zhuǎn)移載體pFasBacHTb-CDNF轉(zhuǎn)化到DH10Bac E.coli(自帶桿粒及輔助質(zhì)粒)感受態(tài)細(xì)胞中,通過轉(zhuǎn)座作用將目的片段整合到桿粒中,抗性及藍(lán)白斑篩選法篩選出重組桿狀病毒載體Bacmid-CDNF,使用CDNF特異引物及PUC/M13桿粒測(cè)序引物對(duì)重組桿粒進(jìn)行驗(yàn)證;參照CELLFECTINⅡ脂質(zhì)體轉(zhuǎn)染劑操作說明,將重組Bacmid-CDNF桿粒轉(zhuǎn)染sf9昆蟲細(xì)胞,并在光學(xué)顯微鏡下觀察細(xì)胞形態(tài)學(xué)變化,以判斷轉(zhuǎn)染的成功性;轉(zhuǎn)染成功后,收集P1代病毒液,繼續(xù)感染sf9細(xì)胞,可獲取P2代和P3代病毒液,應(yīng)用P3代病毒誘導(dǎo)重組CDNF蛋白的表達(dá),并應(yīng)用Western blot對(duì)重組蛋白的表達(dá)進(jìn)行驗(yàn)證。 結(jié)果: (1)RT-PCR結(jié)果經(jīng)瓊脂糖凝膠電泳顯示,成功獲取564bp大小的CDNF目的基因片段,重組質(zhì)粒pFastBacHTb-CDNF經(jīng)PCR、酶切及基因測(cè)序驗(yàn)證均正確無誤;(2)重組轉(zhuǎn)移載體pFastBacHTb-CDNF轉(zhuǎn)化DH10Bac感受態(tài)細(xì)胞后,陽性bacmid-CDNF重組子經(jīng)CDNF特異引物及pUC/M13桿粒測(cè)序引物PCR驗(yàn)證可分別獲取561bp及3000bp大小目的片段,均與理論大小相符,表明桿粒構(gòu)建成功;(3)重組桿粒通過Cellfectin轉(zhuǎn)染劑轉(zhuǎn)染sf9昆蟲細(xì)胞,轉(zhuǎn)染48~72后,sf9細(xì)胞細(xì)胞直徑逐漸增大,細(xì)胞漸脫離貼壁培養(yǎng)狀態(tài),隨之,細(xì)胞漸漸腫脹,種種跡象均表明,桿粒轉(zhuǎn)染成功,收獲桿狀病毒液;(4)P3代桿狀病毒液感染sf9細(xì)胞,誘導(dǎo)重組CDNF蛋白表達(dá),經(jīng)Western blot驗(yàn)證,目的蛋白成功誘導(dǎo)表達(dá),并符合預(yù)計(jì)大小(21KD)。 結(jié)論:通過bac-to-bac桿狀病毒表達(dá)系統(tǒng)成功誘導(dǎo)CDNF重組蛋白的表達(dá)。
[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 偽 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，

本文編號(hào)：1898679