【摘要】：根據(jù)已報(bào)道的RDE-4基因序列,設(shè)計(jì)一對(duì)引物,從含C.elegance的cDNA文庫中,通過PCR擴(kuò)增得到大小為1158nt的RDE-4基因,并把它重組到細(xì)菌表達(dá)載體pET-21b(+)中,該載體C-端具有6個(gè)組氨酸標(biāo)記多肽。酶切鑒定及序列測(cè)定表明,重組表達(dá)質(zhì)粒pET-21b-RDE-4連接區(qū)域符合設(shè)計(jì)要求,具有正確的開放閱讀框架,插入片段含有385個(gè)氨基酸的完整編碼區(qū),其核苷酸序列與報(bào)道的RDE-4基因的同源性為100%。重組表達(dá)載體導(dǎo)入宿主菌E.coli BL21(DE3),用終濃度為3mmol/L IPTG誘導(dǎo),37℃培養(yǎng)6小時(shí)后,RDE-4基因的表達(dá)量達(dá)到最高,每毫升菌液含目的蛋白的量約為2mg。通過大量培養(yǎng),利用親和純化和FPLC純化技術(shù)將RDE-4基因蛋白純化、濃縮,并用PEG等條件養(yǎng)晶,進(jìn)行條件篩選。全長基因蛋白晶形不好,進(jìn)而用胰蛋白酶對(duì)RDE-4基因蛋白限制性酶切,通過NI-ATK親和柱層析,判斷目的蛋白的穩(wěn)定結(jié)構(gòu)域在N-端。根據(jù)以上分析結(jié)果,重新設(shè)計(jì)N-端引物,亞克隆RDE-4(1-280Aa,315Aa)基因片段,并將它們插入GEX-6P-1原核載體,該載體N-端帶有GST融合標(biāo)簽,構(gòu)建pGEX-6P-1-RDE-4(1-280Aa,315Aa)表達(dá)載體。重組表達(dá)載體導(dǎo)入E.coli BL21(DE3),用IPTG誘導(dǎo)高效表達(dá)。通過GST-column親和層析,純化RDE-4(1-280Aa)基因片段蛋白,并用PPase在柱上對(duì)其進(jìn)行限制性酶切,一般過夜后洗脫、10KD孔徑的超濾管濃縮至1mL,注射器被動(dòng)注入FPLC上樣孔,通過凝膠分子篩進(jìn)一步純化,獲得高濃度和純度蛋白,濃度為50mg/mL。用懸吊液滴蒸汽擴(kuò)散法和40%PEG-600,0.1MCHES,pH6.0[Cryoll screen kit,Enedd Biostructure]加入stock Options~(TM) pH screen(Hampton Research)蛋白質(zhì)養(yǎng)晶條進(jìn)行養(yǎng)晶,一星期,晶體成規(guī)則八面體,通過X-Ray衍射,收到晶體電子密度圖及相關(guān)數(shù)據(jù)。本論文研究為進(jìn)一步研究RNAi的作用機(jī)制奠定基礎(chǔ),為研究在此過程中相關(guān)蛋白之間的相互作用提供結(jié)構(gòu)依據(jù)。
[Abstract]:According to the reported RDE-4 gene sequence, a pair of primers were designed. The RDE-4 gene of 1158nt was amplified by PCR from the cDNA library containing C.elegance. The RDE-4 gene was recombined into the bacterial expression vector pET-21b (), which had six histidine labeled peptides at the C-terminal. The restriction endonuclease analysis and sequencing showed that the pET-21b-RDE-4 ligation region of the recombinant plasmid met the design requirements and had a correct open reading frame. The inserted fragment contained a complete coding region of 385 amino acids. Its nucleotide sequence is 100% homology with the reported RDE-4 gene. The recombinant expression vector was introduced into the host strain E.coli BL21 (DE3). After 6 hours of incubation at 37 鈩，

本文編號(hào)：2194514