结核分枝杆菌Rv3813c的克

发布时间：2018-09-01 11:00

【摘要】：Cole 等人在 1998 年发表了结核分枝杆菌 (Mycobacteriumtuberculosis) H37Rv 菌株的基因组序列和基因组注释,Camus 等人在2002 年对 H37Rv 菌株的基因组注释进行了修正。H37Rv 菌株的基因组信息为我们深入了解结核病原菌的致病机理及研发新的抗结核药物提供了坚实的基础。H37Rv 菌株基因组的全长为 4411532 bp,含有4044 个基因,按功能分为 11 类,其中 272 个基因编码未知功能的蛋白质,1051 个基因编码具有保守序列的假定蛋白质 (Conservedhypothetical protein)。细胞壁是结核分枝杆菌赖以生存和增殖的结构基础,可作为研发抗结核新药的靶标,因此,鉴定新的与细胞壁代谢有关的蛋白质及酶,有助于我们深入了解细胞壁的生物合成及分解代谢规律及确定新的药物作用靶标。聚阿拉伯糖聚半乳糖 (Arabinogalactan, AG) 是分枝杆菌细胞壁特有的结构,由 D-Galf 形成的聚半乳糖和 D-Araf 形成的聚阿拉伯糖构成。目前,尚未完全了解聚阿拉伯糖及聚半乳糖生物合成过程中的一些细节。参与聚阿拉伯糖合成的糖基供体是聚十异戊二烯磷酸阿拉伯糖 (Decaprenyl-phospho-arabinose,DPA),但对 DPA 形成的详细过程缺乏认识,DPA 合成途径可能有转移酶、磷酸酶和差向异构酶参与。Huang 等人已鉴定出结核分枝杆菌的 Rv3806c 基因产物具有转移酶活性。马郁芳教授从结核分枝杆菌中克隆了编码差向异构酶的侯选基因并正在鉴定其功能。因此,对磷酸酶功能的鉴定便于我们全面了解 2 结核分枝杆菌中 DPA 形成的机制。 Rv3813c 基因编码保守的假定蛋白质 (Conserved hypothetical protein),利用 NCBI 的 Conserved Domain Search Tool 和 Conserved Domain Architecture Retrieval Tool 分析 Rv3813c 编码的蛋白质,发现 Rv3813c 蛋白质具有水解酶或磷酸酶的保守的结构域,因此,本论文的目的是 (1) 用 PCR 方法从结核分枝杆菌 H37Rv 基因组中扩增 Rv3813c 基因;(2) 克隆 Rv3813c 基因并测定其核苷酸序列;(3) 构建表达质粒 pET29b-Tb Rv3813c;(4) 优化 Rv3813c 蛋白质在大肠杆菌 BL21(DE3) 中的表达;(5) 纯化 Rv3813c 蛋白质并对纯化的 Rv3813c 蛋白质进行功能鉴定。本论文所获得结果如下: 1. 用 PCR 方法扩增 Rv3813c 基因从结核分枝杆菌 H37Rv 菌株基因组数据库 (http: //genolist.pasteur.fr/Tuberculist/) 中获得 Rv3813c 基因的核苷酸序列, 根据此序列设计 PCR 引物,并在上游引物 Rv3813c-1 和下游引物 Rv3813c-2 的 5’ 端中分别引入 NdeI 和XhoI 位点,利用 Vent DNA 聚合酶成功地从 H37Rv 菌株基因组 DNA 中扩增了 Rv3813c 基因。 2. 克隆 Rv3813c 基因和测定其核苷酸序列将 Rv3813c PCR 产物克隆到 pSTBlue1 克隆质粒中,构建出 pSTBlue1- Rv3813c 重组质粒。对 pSTBlue1-Rv3813c (5) 的 Rv3813c 基因进行 DNA 序列测定,结果表明用 PCR 方法扩增的 Rv3813c 基因与 H37Rv 菌株基因组数据库中的 Rv3813c 基因有完全一致的核苷酸序列,即所克隆的 Rv3813c 基因中不存在任何碱基突变。 3. 构建表达质粒 pET29b-Tb Rv3813c 用 NdeI 和 XhoI 双酶切 pSTBlue1-Rv3813c (5) 而获得 Rv3813c 基因,将纯化 Rv3813c 基因克隆到 pET29b 的 NdeI 和 XhoI 位点,构建出 pET29b- Rv3813c 表达质粒,Rv3813c 基因表达的 Rv3813c 蛋白质,其 C 端与来自表达质粒的组氨酸标签 (6 个组氨酸) 形成融合蛋白,便于 Rv3813c 蛋白质的鉴定及纯化。 3 4. Rv3813c 蛋白质在大肠杆菌 BL21(DE3) 中的表达 (1) 用表达质粒 pET29b-Tb Rv3813c (5) 转化 BL21(DE3)。 (2) 在 37°C 条件下用 1 mM IPTG 诱导 pET29b-Rv3813c/BL21 (DE3),用 SDS-PAGE 电泳及 Western blot 方法检测和鉴定 Rv3813c 基因的表达产物。结果显示,Rv3813c 基因能高表达出 Rv3813c 蛋白质。 5. Rv3813c 蛋白质的纯化及酶活性测定 (1) 用组氨酸-镍亲和层析凝胶 (HIS-Select HF Nickel Affinity Gel) 纯化 Rv3813c 蛋白质,用 SDS-PAGE 电泳及 Western blot 方法检测并鉴定每管洗脱液 (共 5 管) 中的 Rv3813c 蛋白质。结果显示,第一管洗脱液中含有极少量的杂蛋白,其余 4 管洗脱液中没有出现杂蛋白,说明组氨酸-镍亲和层析方法能有效地纯化 Rv3813c 蛋白质。对纯化的 Rv3813c 蛋白质进行脱盐和脱咪唑后,测定其酶活性。 (2) 测定磷酸酶活性:pNPP 法用 0.45 μg Rv3813c 蛋白质与 1 mM pNPP 在 200 μl 反应体系中进行反应,在 550 nm 处测定其光吸收值,并计算出磷酸酶活性。酶活性 = 18.3 x 2 x 10-7 x 109 x Absorbance/0.45 μg/30 min = 7.45 x 103 nmol/mg/min (3) 测定磷酸酶活性:利用 DP、PRPP 及转移酶测定磷酸酶活性将纯化的 Rv3813c 蛋白质与 DP (聚十异戊二烯磷酸)、14C 标记的 PRPP (磷酸核糖焦磷酸) 和高表达的转移酶 (Rv3806c) 进行孵育, 用薄板层析方法分析所生成的产物。结果显示,反应体系中由转
[Abstract]:Cole et al. published the genome sequence and annotation of Mycobacterium tuberculosis H37Rv strain in 1998. Camus et al. revised the genome annotation of H37Rv strain in 2002. The genome information of H37Rv strain provides us with insight into the pathogenesis of tuberculosis and new research and development. Antituberculosis drugs provide a solid foundation. The genome of H37Rv strain is 441 1532 BP in length and contains 4044 genes. It is divided into 11 groups according to its function. Of these, 272 genes encode proteins with unknown function and 1051 genes encode conserved hypothetical protein.
Cell wall is the structural basis for the survival and proliferation of Mycobacterium tuberculosis, and can be used as a target for the development of new anti-tuberculosis drugs. Therefore, identifying new proteins and enzymes related to cell wall metabolism is helpful for us to understand the rules of cell wall biosynthesis, degradation and metabolism and to identify new drug targets.
Arabinogalactan (AG) is a unique structure of the cell wall of Mycobacterium. It is composed of D-Galf-formed polygalactose and D-Araf-formed polyarabinose. Decaprenyl-phospho-arabinose (DPA) is a polyisoprene phospho-arabinose (DPA), but the detailed process of DPA formation is poorly understood. DPA synthesis pathways may involve transferases, phosphatases and differential isomerases. Huang et al. have identified Rv3806c gene product of Mycobacterium tuberculosis as having transferase activity. The candidate genes encoding the differential isomerase have been cloned from Mycobacterium nucleus and their functions are being identified.
Two
The mechanism of DPA formation in Mycobacterium tuberculosis.
The Rv3813c gene encodes a conservative putative protein (Conserved hypothetical).
Protein), using NCBI's Conserved Domain Search Tool and Conserved
Domain Architecture Retrieval Tool analysis of Rv3813c encoded proteins, found
Rv3813c protein has a conserved domain of hydrolases or phosphatase, so this paper
The purpose is (1) to amplify the H37Rv genome from Mycobacterium tuberculosis by PCR.
Rv3813c gene; (2) Rv3813c gene was cloned and its nucleotide sequence was determined; (3) construction.
Construct expression plasmid pET29b-Tb Rv3813c; (4) optimize Rv3813c protein in colon tube
Expression of bacteria BL21 (DE3); (5) purification of Rv3813c protein and purification.
The function of Rv3813c protein was identified.
The results obtained in this paper are as follows:
1. amplification of Rv3813c gene by PCR.
From the genomic database of Mycobacterium tuberculosis H37Rv (http:)
The nucleotide sequence of Rv3813c gene was obtained from //genolist.pasteur.fr/Tuberculist/.
Based on this sequence, PCR primers were designed, and primers Rv3813c-1 and downstream primers were used.
NdeI and XhoI sites were introduced into the 5 'end of Rv3813c-2, respectively, using Vent DNA.
Polymerase has successfully amplified the Rv3813c gene from genomic DNA of H37Rv strain.
2. cloning of Rv3813c gene and determination of its nucleotide sequence
The Rv3813c PCR product was cloned into pSTBlue1 clone plasmid and constructed.
PSTBlue1- Rv3813c recombinant plasmid. Rv3813c for pSTBlue1-Rv3813c (5)
The DNA sequence of the gene was analyzed, and the results showed that Rv3813c was amplified by PCR.
Because the Rv3813c gene is completely consistent with the H37Rv genome database.
There is no nucleotide mutation in the Rv3813c gene, which is cloned.
3. construction of expression plasmid pET29b-Tb Rv3813c
NdeI and XhoI were digested by pSTBlue1-Rv3813c (5).
Rv3813c gene, the purified Rv3813c gene was cloned into NdeI and pET29b.
XhoI locus, pET29b- Rv3813c expression plasmid was constructed and Rv3813c gene expression was constructed.
The Rv3813c protein and its C end were labeled with histidine from the expression plasmid (6 groups).
The fusion protein was formed to facilitate the identification and purification of Rv3813c protein.
Three
Expression of 4. Rv3813c protein in Escherichia coli BL21 (DE3)
(1) BL21 (DE3) was transformed by expression plasmid pET29b-Tb Rv3813c (5).
(2) induction of pET29b-Rv3813c/BL21 with 1 mM IPTG at 37 C.
(DE3) detection and identification of Rv3813c by SDS-PAGE electrophoresis and Western blot.
The results showed that Rv3813c gene could express Rv3813c protein highly.
Quality.
Purification of 5. Rv3813c protein and determination of its enzyme activity
(1) use histidine nickel affinity chromatography gel (HIS-Select HF Nickel Affinity Gel).
Purified Rv3813c protein was detected by SDS-PAGE electrophoresis and Western blot method.
The Rv3813c protein of each eluent (a total of 5 tubes) was identified.
There was a small amount of miscellaneous protein in the eluent, and there was no miscellaneous egg in the other 4 tubes.
It shows that histidine nickel affinity chromatography can effectively purify Rv3813c protein.
The purified Rv3813c protein was desalted and deimidazole to determine its enzyme activity.
(2) determination of phosphatase activity: pNPP method
The reaction system was 0.45 micron g Rv3813c and 1 mM pNPP at 200 L.
The reaction was performed at 550 nm and the phosphatase activity was calculated.
Enzyme activity = 18.3 x 2 x 10-7 x 109 x Absorbance/0.45 g/30 min
= 7.45 x 103 nmol/mg/min
(3) determination of phosphatase activity: Determination of phosphatase activity by DP, PRPP and transferase
The purified Rv3813c protein was labeled with DP (poly ten isoprenoid phosphate) and 14C.
PRPP (ribose pyrophosphate) and high expression transferase (Rv3806c) were incubated.
The products were analyzed by thin layer chromatography.
【学位授予单位】：大连医科大学
【学位级别】：硕士
【学位授予年份】：2005
【分类号】：Q78

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