抗耻垢分枝杆菌GlmU蛋白抗体的制备及glmU反义RNA表达的检测

发布时间：2018-01-06 12:33

  本文关键词：抗耻垢分枝杆菌GlmU蛋白抗体的制备及glmU反义RNA表达的检测　出处：《大连医科大学》2007年硕士论文　论文类型：学位论文

  更多相关文章： 耻垢分枝杆菌 GlmU 多克隆抗体 四环素诱导 反义RNA

【摘要】： 分枝杆菌具有特殊的细胞壁结构,其核心部分由肽聚糖、聚阿拉伯糖半乳糖和分枝菌酸组成,其中分枝菌酸和聚阿拉伯糖半乳糖通过衔接双糖(L-鼠李糖-D-N-乙酰葡糖胺)共价连接到肽聚糖大分子上。UDP-N-乙酰葡糖胺是N-乙酰葡糖胺的糖基供体,而glmU基因编码的GlmU蛋白具有葡糖胺-1-磷酸乙酰基转移酶活性和N-乙酰葡糖胺-1-磷酸尿苷转移酶活性,参与UDP-N-乙酰葡糖胺的生物合成过程。本实验室张文利的研究结果表明,glmU基因为分枝杆菌生长必需基因,因此,GlmU可作为研发抗结核新药的作用靶标。 为了深入研究GlmU酶活性对分枝杆菌细胞壁结构和组成的影响并建立筛选GlmU酶抑制剂的细胞模型,本实验室已构建了glmU反义RNA表达载体pMind-glmU-AS,glmU反义RNA在耻垢分枝杆菌(Mycobacterium smegmatis)的表达受四环素的调控。为了优化四环素浓度对glmU反义RNA表达的调节,需要用抗GlmU抗体检测GlmU在耻垢分枝杆菌中的表达量。 本论文的目的是:(1)用PCR法从耻垢分枝杆菌mc2155菌株基因组DNA中扩增glmU基因,构建pMD18-glmU克隆载体;(2)将glmU基因亚克隆到pET29b表达载体中,在宿主菌BL21(DE3)中诱导表达GlmU重组蛋白。用亲和层析法纯化重组GlmU蛋白,并用Western Blotting方法鉴定GlmU重组蛋白;(3)用纯化的GlmU蛋白免疫小鼠以制备抗GlmU的多克隆抗体;(4)用不同剂量的四环素诱导glmU反义RNA在耻垢分枝杆菌中的表达,用抗GlmU抗体检测GlmU在耻垢分枝杆菌中的表达量。 本论文所获得结果如下: 1. glmU基因的扩增和pMD18-glmU克隆载体的构建 用结核分枝杆菌GlmU的氨基酸序列从TIGR的耻垢分枝杆菌mc2155菌株基因组数据库中获得mc2155菌株的glmU基因的核苷酸序列(1449 bp)。根据该序列设计一对PCR引物,并在上游与下游引物的5’端分别引入Nde I和XhoI限制性内切酶位点。用保真度高的LA Taq DNA聚合酶,以mc2155菌株基因组DNA为模板扩增了glmU基因,并将glmU基因连接到pMD18-T载体,构建了pMD18-glmU克隆载体。对glmU基因进行DNA测序测定,结果表明利用PCR技术扩增出正确的mc~2155 glmU基因。 2.表达载体pET29b-glmU的构建 用Nde I和Xho I双酶切pMD18-glmU质粒,回收和纯化glmU基因,将其连接到pET29b表达载体的Nde I和Xho I位点,构建了pET29b-glmU表达质粒。GlmU蛋白的C端与质粒上的组氨酸标签形成融合蛋白。 3. GlmU蛋白在大肠杆菌BL21(DE3)中表达和纯化 将pET29b-glmU质粒转化到BL21(DE3)感受态细胞中,用IPTG诱导携带pET29b-glmU质粒的BL21(DE3)表达重组蛋白。用超声方法破碎诱导后的BL21(DE3),对上清和沉淀组分进行SDS-PAGE和Western blotting分析,结果表明GlmU蛋白在BL21(DE3)中可溶性表达。 采用组氨酸-Ni~(2+)亲和层析技术纯化GlmU蛋白,对纯化的GlmU蛋白进行蛋白质定量(考马斯亮蓝法),第1 ml GlmU蛋白的浓度为0.635 mg/ml。SDS-PAGE和Western blotting分析结果表明GlmU蛋白的纯度高。 4.抗GlmU蛋白的多克隆抗体的制备 将纯化的GlmU蛋白质溶液经过特殊处理后制备成免疫用抗原,免疫BalB/C小鼠,制备抗血清,通过间接法酶联免疫吸附试验测得抗血清的抗体滴度为1:51200,采用抗血清及偶联碱性磷酸酶的马抗小鼠IgG二抗对耻垢分枝杆菌总蛋白中GlmU进行Western Blotting分析,结果表明制备的GlmU多克隆抗体特异性高。 5. glmU反义RNA的诱导表达与GlmU蛋白的检测 分别用5 ng/ml、10 ng/ml和20 ng/ml的四环素诱导携带pMind-glmU-AS表达载体的mc~2155菌株表达反义RNA,绘制细菌生长曲线,结果表明20 ng/ml浓度的四环素可抑制mc~2155/pMind-glmU-AS的生长,但Western Blotting分析结果显示经四环素诱导和未诱导的mc~2155/pMind-glmU-AS中,GlmU蛋白表达量无明显的变化。 结论:在本研究中,我们构建了pET29b-glmU表达载体,在大肠杆菌BL21(DE3)中表达出大量的可溶性耻垢分枝杆菌GlmU蛋白。用纯化的GlmU蛋白免疫BalB/C小鼠,成功地制备了抗GlmU蛋白的多克隆抗体,并将抗GlmU蛋白抗体应用到检测四环素诱导表达glmU反义RNA的系统中,初步研究了GlmU蛋白表达水平与耻垢分枝杆菌生长速率之间的关系,这一研究成果为我们进一步研究GlmU酶蛋白活性与分枝杆菌细胞壁结构和组成的关系奠定了基础。
[Abstract]:Mycobacterial cell wall structure is special, its core part is composed of peptidoglycan, poly Arabia sugar galactose and mycolic acids, including mycolic acid and poly Arabia sugar galactose by the disaccharide linker (L- -D-N- rhamnose GlcNAc) covalently attached to the peptidoglycan macromolecular.UDP-N- acetyl glucose glycosyl amine is N- acetyl glucosamine donor, and glmU gene encoding the GlmU protein with -1- glucosamine phosphate acetyltransferase activity of N- enzyme and -1- uridine diphosphate N-acetylglucosamine transferase activity, biosynthesis in UDP-N- acetylglucosamine. The laboratory of Zhang Wenli's results show that glmU gene is essential for mycobacterial growth. Therefore, GlmU can be used as the development of new anti tuberculosis drug targets.
In order to study the effect of GlmU enzyme on the mycobacterial cell wall structure and composition and establish a cell model for screening GlmU inhibitors, the laboratory has been constructed glmU antisense RNA expression vector pMind-glmU-AS, glmU antisense RNA in Mycobacterium smegmatis (Mycobacterium smegmatis) expression regulated by tetracycline. In order to adjust the optimal concentration of tetracycline the expression of glmU antisense RNA expression by GlmU, need to detect the anti GlmU antibody in Mycobacterium smegmatis.
The purpose of this paper is: (1) glmU gene was amplified from Mycobacterium smegmatis strain mc2155 genome DNA by PCR method, the pMD18-glmU clone vector; (2) the glmU gene was cloned into pET29b expression vector, in E.coli BL21 (DE3) induced by recombinant protein GlmU with purified recombinant GlmU protein. Affinity chromatography, using Western and Blotting method for identification of recombinant GlmU protein; (3) polyclonal antibody in mice immunized with GlmU purified to prepare anti GlmU; (4) glmU antisense RNA in Mycobacterium smegmatis expression with different doses of tetracycline induced expression in Mycobacterium smegmatis using GlmU to detect anti GlmU antibody.
The results obtained in this paper are as follows:
Amplification of 1. glmU gene and construction of pMD18-glmU cloning vector
Obtain the nucleotide sequence of glmU gene of mc2155 strain TIGR from Mycobacterium smegmatis strain mc2155 genome database with the amino acid sequence of Mycobacterium tuberculosis GlmU (1449 BP). According to the design of a pair of PCR primers and the sequence in the upstream and downstream primer 5 'end were introduced by Nde I and XhoI restriction sites. With high fidelity LA Taq DNA polymerase, mc2155 strain genomic DNA as template to amplify the glmU gene, and glmU gene was inserted into pMD18-T vector, constructed the pMD18-glmU cloning vector of the glmU gene. DNA sequencing determination shows that amplified mc~2155 glmU gene correctly by using PCR technology.
Construction of 2. expression vector pET29b-glmU
The pMD18-glmU plasmid was digested by Nde I and Xho I. The glmU gene was recovered and purified, and it was connected to Nde I and Xho I site of pET29b expression vector. The fusion protein was constructed from the ends of the expression plasmid and the histidine tag on the plasmid.
Expression and purification of 3. GlmU protein in Escherichia coli BL21 (DE3)
The pET29b-glmU plasmid was transformed into BL21 (DE3) competentcells, induced with pET29b-glmU plasmid with IPTG BL21 (DE3) expression of recombinant protein. After the induction of BL21 broken by ultrasonic method (DE3), both supernatant and pellet fractions were analyzed by SDS-PAGE and Western blotting, the results showed that GlmU protein in BL21 (DE3). Soluble expression.
-Ni~ (2+) by histidine affinity purified GlmU protein chromatography for protein quantitation of purified GlmU protein (Kaumas Bradford method), the concentration of first ml GlmU protein is 0.635 mg/ml.SDS-PAGE and Western blotting analysis showed that the purity of GlmU protein.
Preparation of polyclonal antibody against 4. anti GlmU protein
The purified GlmU protein solution after special treatment to prepare immune antigen, immune BalB/C mice antiserum titer by indirect enzyme-linked immunosorbent assay measured was 1:51200, using antisera conjugated with alkaline phosphatase and horse anti mouse IgG two anti Western Blotting analysis of GlmU Mycobacterium smegmatis total bacterial protein, the results show that the prepared GlmU polyclonal antibody with high specificity.
Induced expression of 5. glmU antisense RNA and detection of GlmU protein
With 5 ng/ml mc~2155, 10 ng/ml and 20 ng/ml strains of tetracycline inducible expression vector carrying pMind-glmU-AS antisense RNA expression, the growth curves were drawn. Results showed that 20 ng/ml tetracycline concentration could inhibit the growth of mc~2155/pMind-glmU-AS, but the Western Blotting analysis showed that the tetracycline induced and non induced mc~2155/pMind-glmU-AS, GlmU protein expression the obvious change.
Conclusion: in this study, we constructed pET29b-glmU expression vector in Escherichia coli BL21 (DE3) in the expression of a large number of soluble Mycobacterium smegmatis GlmU protein. The purified GlmU protein to immunize BalB/C mice successfully prepared polyclonal antibody against GlmU protein, and GlmU antibody was applied to system for detecting tetracycline inducible expression of glmU antisense RNA, preliminary study on the relationship between the expression level of GlmU protein from Mycobacterium smegmatis growth rate, the research results have laid the foundation for our further study of GlmU protein and activity of mycobacterial cell wall structure and composition of the relationship.

【学位授予单位】：大连医科大学
【学位级别】：硕士
【学位授予年份】：2007
【分类号】：R392
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本文编号：1387901