衣原体RNase H Ⅱ编码基因的克隆与表达及酶的纯化与性质鉴定
发布时间：2018-07-28 16:38
【摘要】：衣原体(Chlamydia)是一种可导致多种疾病的病原微生物,它属严格胞内寄生,没有基因水平的遗传操作系统。于其它生物体如大肠杆菌中表达衣原体蛋白质,并在体外鉴定表达蛋白质的生物学功能是目前衣原体分子生物学研究的可行方法。 RNase H 是一类特异性内切 RNA/DNA 杂合体中 RNA 链的酶,其活性依赖于二价金属离子,酶切产物具有 5′-磷酸末端和 3′-羟基末端。RNase H 普遍存在于病毒、原核生物、真核生物。单个的细菌和真核细胞通常含有两个不同的 RNase H,它们彼此之间表现出较少的序列相似性。依据氨基酸序列特征已经将 RNase H 分为两个家族,1 型 RNase H 和 2 型 RNase H。 衣原体(Chlamydia pneumoniae)AR39 全基因组序列分析表明衣原体 AR39 没有编码 RNase HI 的基因,但有两个序列不同的编码 RNase HII 的基因:CP0645 和 CP0782。我们分别将 CP0645和 CP0782 编码的酶命名为 CpRNase HIIa 和 CpRNase HIIb。以衣原体全基因组为模板,PCR获得衣原体 AR39 的 CP0645 和 CP0782 基因。采用常规的基因重组技术,即限制性内切酶酶切和 T4-DNA 连接酶连接,将这两个基因克隆到大肠杆菌表达质粒 pET28a 中。DNA 序列分析证实重组质粒含有相应的衣原体基因,其读码框架正确。参照 Novagen 公司提供的操作手册,表达并纯化了重组的 CpRNase HIIa 和 CpRNase HIIb。在大肠杆菌 BL21(DE3)细胞内,表达的 CpRNase HIIa 中可溶形式占 20%,而表达的 CpRNase HIIb 可溶形式占 65%。 变性和非变性的镍-树脂(Ni-NTA)亲和层析纯化技术分别用于这两种重组蛋白质的纯化。非变性的镍-树脂(Ni-NTA)亲和层析纯化能够获得有活性的蛋白质,而变性的镍-树脂(Ni-NTA)亲和层析纯化获得的蛋白质没有活性,需要进一步复性才具有活性。简单的透析复性,通过透析溶液的尿素浓度梯度逐步递减,使变性的 CpRNase HIIa 和 CpRNase HIIb 复性,所获得的酶比活性与用非变性纯化获得的蛋白质相当。
[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.
【学位授予单位】：河南大学
【学位级别】：硕士
【学位授予年份】：2005
【分类号】：Q78;Q55
本文编号：2150907
[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.
【学位授予单位】：河南大学
【学位级别】：硕士
【学位授予年份】：2005
【分类号】：Q78;Q55
【相似文献】
中国硕士学位论文全文数据库 前1条
1 裴冬丽;衣原体RNase H Ⅱ编码基因的克隆与表达及酶的纯化与性质鉴定[D];河南大学;2005年
，本文编号：2150907
本文链接：https://www.wllwen.com/yixuelunwen/binglixuelunwen/2150907.html
最近更新
教材专著
