【摘要】：衣原體(Chlamydia)是一種可導(dǎo)致多種疾病的病原微生物,它屬嚴(yán)格胞內(nèi)寄生,沒有基因水平的遺傳操作系統(tǒng)。于其它生物體如大腸桿菌中表達衣原體蛋白質(zhì),并在體外鑒定表達蛋白質(zhì)的生物學(xué)功能是目前衣原體分子生物學(xué)研究的可行方法。 RNase H 是一類特異性內(nèi)切 RNA/DNA 雜合體中 RNA 鏈的酶,其活性依賴于二價金屬離子,酶切產(chǎn)物具有 5′-磷酸末端和 3′-羥基末端。RNase H 普遍存在于病毒、原核生物、真核生物。單個的細菌和真核細胞通常含有兩個不同的 RNase H,它們彼此之間表現(xiàn)出較少的序列相似性。依據(jù)氨基酸序列特征已經(jīng)將 RNase H 分為兩個家族,1 型 RNase H 和 2 型 RNase H。 衣原體(Chlamydia pneumoniae)AR39 全基因組序列分析表明衣原體 AR39 沒有編碼 RNase HI 的基因,但有兩個序列不同的編碼 RNase HII 的基因:CP0645 和 CP0782。我們分別將 CP0645和 CP0782 編碼的酶命名為 CpRNase HIIa 和 CpRNase HIIb。以衣原體全基因組為模板,PCR獲得衣原體 AR39 的 CP0645 和 CP0782 基因。采用常規(guī)的基因重組技術(shù),即限制性內(nèi)切酶酶切和 T4-DNA 連接酶連接,將這兩個基因克隆到大腸桿菌表達質(zhì)粒 pET28a 中。DNA 序列分析證實重組質(zhì)粒含有相應(yīng)的衣原體基因,其讀碼框架正確。參照 Novagen 公司提供的操作手冊,表達并純化了重組的 CpRNase HIIa 和 CpRNase HIIb。在大腸桿菌 BL21(DE3)細胞內(nèi),表達的 CpRNase HIIa 中可溶形式占 20%,而表達的 CpRNase HIIb 可溶形式占 65%。 變性和非變性的鎳-樹脂(Ni-NTA)親和層析純化技術(shù)分別用于這兩種重組蛋白質(zhì)的純化。非變性的鎳-樹脂(Ni-NTA)親和層析純化能夠獲得有活性的蛋白質(zhì),而變性的鎳-樹脂(Ni-NTA)親和層析純化獲得的蛋白質(zhì)沒有活性,需要進一步復(fù)性才具有活性。簡單的透析復(fù)性,通過透析溶液的尿素濃度梯度逐步遞減,使變性的 CpRNase HIIa 和 CpRNase HIIb 復(fù)性,所獲得的酶比活性與用非變性純化獲得的蛋白質(zhì)相當(dāng)。
[Abstract]:Chlamydia (Chlamydia) is a pathogenic microorganism that can cause many diseases. It is a feasible method to express chlamydia protein in other organisms such as Escherichia coli and to identify its biological function in vitro. RNase H is a kind of enzyme of RNA chain in specific endonuclear RNA/DNA heterozygotes. Its activity is dependent on divalent metal ions. The products of RNase H have the terminal of 5- phosphoric acid and the terminal of 3- hydroxyl group. RNase H is ubiquitous in viruses and prokaryotes. Eukaryotes. Single bacteria and eukaryotic cells usually contain two different RNase Hs, which exhibit less sequence similarity. According to the amino acid sequence characteristics, RNase H has been divided into two families: RNase H and RNase H. The whole genome sequence analysis of Chlamydia (Chlamydia pneumoniae) AR39 showed that Chlamydia AR39 did not encode RNase HI gene, but there were two different genes encoding RNase HII: CP0645 and CP0782. We named the enzyme encoding CP0645 and CP0782 as CpRNase HIIa and CpRNase HIIb. respectively. The CP0645 and CP0782 genes of chlamydia AR39 were obtained using chlamydia genomes as template. By using conventional gene recombination techniques, namely restriction endonuclease digestion and T4-DNA ligase ligation, the two genes were cloned into E. coli expression plasmid pET28a. DNA sequence analysis confirmed that the recombinant plasmid contained the corresponding chlamydia gene. The frame of reading code is correct. According to the operating manual provided by Novagen, the recombinant CpRNase HIIa and CpRNase HIIb. were expressed and purified. In Escherichia coli BL21 (DE3) cells, the soluble form of CpRNase HIIa was 20%, while the soluble form of CpRNase HIIb was 65%. Denatured and undenatured nickel resin (Ni-NTA) affinity chromatography techniques were used to purify the two recombinant proteins respectively. Non-denatured nickel-resin (Ni-NTA) affinity chromatography can obtain the active protein, while the denatured Ni-resin (Ni-NTA) affinity chromatography has no activity and needs further renaturation to be active. After simple dialysis renaturation, the denatured CpRNase HIIa and CpRNase HIIb were renatured gradually by the urea concentration gradient of dialysis solution, and the specific enzyme activity was similar to that of the protein purified by non-denaturation.
【學(xué)位授予單位】：河南大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2005
【分類號】：Q78;Q55

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中國碩士學(xué)位論文全文數(shù)據(jù)庫 前1條

1 裴冬麗;衣原體RNase H Ⅱ編碼基因的克隆與表達及酶的純化與性質(zhì)鑒定[D];河南大學(xué);2005年

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本文編號：2150907