【摘要】：原發(fā)性扭轉(zhuǎn)痙攣是一組由于軀干、肢體、頸部或顏面肌肉協(xié)調(diào)功能失調(diào)而出現(xiàn)各種姿勢(shì)的異�；蛑w的扭轉(zhuǎn)，已有14種類(lèi)型的扭轉(zhuǎn)痙攣被定位。常染色體顯性早發(fā)性扭轉(zhuǎn)痙攣定位于9q34，是一種最常見(jiàn)、最為嚴(yán)重的扭轉(zhuǎn)痙攣，1989年Ozelius等將該基因命名為DYT1，1997年又克隆出該基因，它編碼332個(gè)氨基酸組成的DYT1扭轉(zhuǎn)蛋白A(Torsin A)，該蛋白是腺苷三磷酸酶AAA家族的成員，一種ATP結(jié)合蛋白，它與許多細(xì)胞活動(dòng)有關(guān)。DYT1基因突變可引起9q連鎖的扭轉(zhuǎn)痙攣。 在實(shí)驗(yàn)中，我們從人扭轉(zhuǎn)蛋白A的編碼序列中設(shè)計(jì)引物，以人肝cDNA文庫(kù)作模板，成功地?cái)U(kuò)增到編碼DYT1蛋白的基因片段，該片段全長(zhǎng)939bp。將所得片段與pMD18-T載體連接，轉(zhuǎn)化到JM109大腸桿菌中，從轉(zhuǎn)化平板上挑出菌落，用堿裂法提取質(zhì)粒，，通過(guò)PCR和酶切分析，成功地篩選到陽(yáng)性克隆，測(cè)序結(jié)果與文獻(xiàn)報(bào)道結(jié)果一致。 提取質(zhì)粒，用BamH I和Xho I酶切，回收目的片段，分別克隆到原核表達(dá)載體pET28a(+)和pGEX-6P-1中，轉(zhuǎn)化JM109受體菌，從JM109受體菌中提出質(zhì)粒，再轉(zhuǎn)化到BL21(DE3)菌中，篩選出陽(yáng)性克隆，成功地構(gòu)建了人扭轉(zhuǎn)蛋白A原核表達(dá)載體。將該工程菌接種于含有適當(dāng)抗生素的LB培養(yǎng)基中，37℃振蕩培養(yǎng)過(guò)夜，次日將此菌液按1：100的比例接種于250mL LB液體培養(yǎng)基中，37℃振蕩培養(yǎng)，當(dāng)細(xì)菌長(zhǎng)至適當(dāng)?shù)拿芏葧r(shí)，加入IPTG(終濃度為1mM)誘導(dǎo)，繼續(xù)培養(yǎng)約5h，離心收集菌體，進(jìn)行SDS-PAGE，結(jié)果在兩種載體中均得到了高效表達(dá)。 在pET28a(+)中表達(dá)的DYTI蛋白為包含體(IBS)，經(jīng)過(guò)IBS制備、蛋 白質(zhì)復(fù)性、金屬鰲合層析純化、superdex75凝膠過(guò)濾純化，得到了純的DYTI 蛋白;在pGEX一6P一1中表達(dá)的DYTI蛋白與GST的融合蛋白也為IBS，但此融 合蛋白復(fù)性不好，只得到極少量的融合蛋白。 關(guān)鍵詞:人;OYTI基因;扭轉(zhuǎn)蛋白A;cDNA;克隆;原核表達(dá);蛋白質(zhì)純化
[Abstract]:Primary torsion spasm is a group of abnormal posture or limb torsion due to the malcoordination of torso, limb, neck or facial muscles. Fourteen types of torsion spasm have been located. Autosomal dominant early torsion spasm, located at 9q34, is one of the most common and severe torsion spasms. In 1989, Ozelius et al named the gene DYT1, 1997 and cloned the gene. It encodes a 332-amino acid DYT1 torsion protein A (Torsin A), a member of the AAA family of adenosine triphosphatase, a ATP binding protein, which is associated with many cellular activities and causes 9q-linked torsion spasm by mutations in the .DYT1 gene. In the experiment, we designed primers from the coding sequence of human torsion protein A and used human liver cDNA library as template to amplify the gene fragment encoding DYT1 protein successfully. The fragment was ligated with pMD18-T vector and transformed into JM109 Escherichia coli. The colony was isolated from the transformed plate and the plasmid was extracted by alkali cleavage method. The positive clones were successfully screened by PCR and enzyme digestion. The results of sequencing were consistent with those reported in the literature. The plasmids were extracted and digested with BamH I and Xho I, and the target fragments were cloned into prokaryotic expression vectors pET28a () and pGEX-6P-1, respectively. The plasmids were transformed into JM109 receptor bacteria, then transformed into BL21 (DE3) bacteria, and the positive clones were screened out. The prokaryotic expression vector of human torsion protein A was successfully constructed. The engineered bacteria were inoculated in LB medium containing appropriate antibiotics for the night at 37 鈩

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