本文選題：流感病毒　切入點(diǎn)：NP蛋白　出處：《中國農(nóng)業(yè)科學(xué)》2016年22期 　論文類型：期刊論文

【摘要】：【目的】構(gòu)建肺組織細(xì)胞Calu-3及A549的高質(zhì)量酵母cDNA文庫并篩選與流感病毒NP蛋白相互作用的宿主因子,為深入研究流感病毒NP蛋白功能、病毒復(fù)制及致病機(jī)制奠定基礎(chǔ)。【方法】提取等量Calu-3及A549細(xì)胞的總RNA,混合后反轉(zhuǎn)錄生成cDNA,利用長(zhǎng)距離PCR(LD-PCR)擴(kuò)增合成dscDNA,用CHROMA SPINTM+TE-400純化柱純化dsDNA,按照Clontech公司的Make Your Own"MatePlate"Library System操作程序,將帶有同源臂的dscDNA與線性化p GADT7-Rec共同轉(zhuǎn)化Y187酵母感受態(tài)細(xì)胞,涂布SD/-Leu平板后于30℃培養(yǎng)4d左右,收集所有菌落,混勻分裝即為Calu-3和A549細(xì)胞cDNA的酵母文庫,并對(duì)文庫庫容、滴度及多樣性進(jìn)行分析。利用Eco RⅠ和Bam HⅠ雙酶切將A/Auhui/2/2005(H5N1)NP定向插入pGBKT7載體,構(gòu)建高致病性流感病毒NP的誘餌質(zhì)粒p GBKT7-NP,經(jīng)驗(yàn)證該質(zhì)粒無自激活活性,并進(jìn)一步采用構(gòu)建完成的Calu-3/A549細(xì)胞酵母文庫進(jìn)行雜交篩選,篩選得到的正確閱讀的獵物質(zhì)粒與誘餌質(zhì)粒共轉(zhuǎn)Y2H Gold酵母菌,分別以BD-P53/AD-T7作為陽性對(duì)照和BD-Lam/AD-T7作為陰性對(duì)照,挑取最終在SD/-Trp/-Leu/-Ade/-His/X-α-gal/Aro A(SD/-4/X/A)固體培養(yǎng)板上生長(zhǎng)良好且變藍(lán)的菌落,即為候選與目標(biāo)蛋白互作陽性的蛋白,提取酵母質(zhì)粒,進(jìn)行測(cè)序分析、序列比對(duì)和Gene Ontology分析�！窘Y(jié)果】提取兩種細(xì)胞RNA 28S與18S條帶清晰,5S條帶暗淡,表明所提RNA質(zhì)量較高,基本無降解;對(duì)提取的RNA反轉(zhuǎn)錄純化獲得dscDNA,dscDNA條帶呈彌散狀,片段大小分布于500—2 000bp之間,說明不同豐度及大小的RNA均成功反轉(zhuǎn)錄;構(gòu)建的dscDNA文庫庫容為1.5×10~7,滴度為2.2×10~8cfu/m L,重組率為88%,PCR鑒定文庫插入片段,條帶大小不一、多樣性好;利用誘餌質(zhì)粒與文庫進(jìn)行雙雜交篩選,回交驗(yàn)證后得到11個(gè)與NP蛋白互作的宿主因子。經(jīng)Gene Ontology分析顯示,11個(gè)宿主因子參與的生物過程包括:細(xì)胞凋亡、胚胎發(fā)育、可變剪接、轉(zhuǎn)錄調(diào)節(jié)及細(xì)胞增殖等;涉及的分子功能包括:GTP結(jié)合活性、金屬離子結(jié)合活性、DNA結(jié)合活性及轉(zhuǎn)錄因子活性�！窘Y(jié)論】成功構(gòu)建同時(shí)含有Calu-3和A549兩種人源肺細(xì)胞cDNA的酵母文庫,文庫覆蓋cDNA更全面,為后期篩選與流感病毒其它蛋白互作的宿主蛋白奠定基礎(chǔ),篩選得到與NP蛋白存在相互作用的宿主因子為進(jìn)一步深入研究NP功能提供了可靠的前期數(shù)據(jù)。
[Abstract]:[objective] to construct a high quality yeast cDNA library of lung tissue cells Calu-3 and A549 and to screen host factors interacting with NP protein of influenza virus in order to study the function of NP protein of influenza virus. [methods] Total RNAs of Calu-3 and A549 cells were extracted, and then reverse transcription was used to produce cDNA. dscDNA was amplified by long distance PCRLD-PCR. CHROMA SPINTM TE-400 was used to purify dsDNA. according to the Make Your Own "MatePlate" Library System procedure of Clontech company, dscDNA was purified by PCR. DscDNA with homologous arm and linearized p GADT7-Rec were co-transformed into Y187 yeast receptive cells. The cells were coated with SD/-Leu plate and cultured at 30 鈩，

本文編號(hào)：1611627