本文關鍵詞：抗恥垢分枝桿菌GlmU蛋白抗體的制備及glmU反義RNA表達的檢測　出處：《大連醫(yī)科大學》2007年碩士論文　論文類型：學位論文

  更多相關文章： 恥垢分枝桿菌 GlmU 多克隆抗體 四環(huán)素誘導 反義RNA

【摘要】： 分枝桿菌具有特殊的細胞壁結(jié)構(gòu),其核心部分由肽聚糖、聚阿拉伯糖半乳糖和分枝菌酸組成,其中分枝菌酸和聚阿拉伯糖半乳糖通過銜接雙糖(L-鼠李糖-D-N-乙酰葡糖胺)共價連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體,而glmU基因編碼的GlmU蛋白具有葡糖胺-1-磷酸乙�；D(zhuǎn)移酶活性和N-乙酰葡糖胺-1-磷酸尿苷轉(zhuǎn)移酶活性,參與UDP-N-乙酰葡糖胺的生物合成過程。本實驗室張文利的研究結(jié)果表明,glmU基因為分枝桿菌生長必需基因,因此,GlmU可作為研發(fā)抗結(jié)核新藥的作用靶標。 為了深入研究GlmU酶活性對分枝桿菌細胞壁結(jié)構(gòu)和組成的影響并建立篩選GlmU酶抑制劑的細胞模型,本實驗室已構(gòu)建了glmU反義RNA表達載體pMind-glmU-AS,glmU反義RNA在恥垢分枝桿菌(Mycobacterium smegmatis)的表達受四環(huán)素的調(diào)控。為了優(yōu)化四環(huán)素濃度對glmU反義RNA表達的調(diào)節(jié),需要用抗GlmU抗體檢測GlmU在恥垢分枝桿菌中的表達量。 本論文的目的是:(1)用PCR法從恥垢分枝桿菌mc2155菌株基因組DNA中擴增glmU基因,構(gòu)建pMD18-glmU克隆載體;(2)將glmU基因亞克隆到pET29b表達載體中,在宿主菌BL21(DE3)中誘導表達GlmU重組蛋白。用親和層析法純化重組GlmU蛋白,并用Western Blotting方法鑒定GlmU重組蛋白;(3)用純化的GlmU蛋白免疫小鼠以制備抗GlmU的多克隆抗體;(4)用不同劑量的四環(huán)素誘導glmU反義RNA在恥垢分枝桿菌中的表達,用抗GlmU抗體檢測GlmU在恥垢分枝桿菌中的表達量。 本論文所獲得結(jié)果如下: 1. glmU基因的擴增和pMD18-glmU克隆載體的構(gòu)建 用結(jié)核分枝桿菌GlmU的氨基酸序列從TIGR的恥垢分枝桿菌mc2155菌株基因組數(shù)據(jù)庫中獲得mc2155菌株的glmU基因的核苷酸序列(1449 bp)。根據(jù)該序列設計一對PCR引物,并在上游與下游引物的5’端分別引入Nde I和XhoI限制性內(nèi)切酶位點。用保真度高的LA Taq DNA聚合酶,以mc2155菌株基因組DNA為模板擴增了glmU基因,并將glmU基因連接到pMD18-T載體,構(gòu)建了pMD18-glmU克隆載體。對glmU基因進行DNA測序測定,結(jié)果表明利用PCR技術擴增出正確的mc~2155 glmU基因。 2.表達載體pET29b-glmU的構(gòu)建 用Nde I和Xho I雙酶切pMD18-glmU質(zhì)粒,回收和純化glmU基因,將其連接到pET29b表達載體的Nde I和Xho I位點,構(gòu)建了pET29b-glmU表達質(zhì)粒。GlmU蛋白的C端與質(zhì)粒上的組氨酸標簽形成融合蛋白。 3. GlmU蛋白在大腸桿菌BL21(DE3)中表達和純化 將pET29b-glmU質(zhì)粒轉(zhuǎn)化到BL21(DE3)感受態(tài)細胞中,用IPTG誘導攜帶pET29b-glmU質(zhì)粒的BL21(DE3)表達重組蛋白。用超聲方法破碎誘導后的BL21(DE3),對上清和沉淀組分進行SDS-PAGE和Western blotting分析,結(jié)果表明GlmU蛋白在BL21(DE3)中可溶性表達。 采用組氨酸-Ni~(2+)親和層析技術純化GlmU蛋白,對純化的GlmU蛋白進行蛋白質(zhì)定量(考馬斯亮藍法),第1 ml GlmU蛋白的濃度為0.635 mg/ml。SDS-PAGE和Western blotting分析結(jié)果表明GlmU蛋白的純度高。 4.抗GlmU蛋白的多克隆抗體的制備 將純化的GlmU蛋白質(zhì)溶液經(jīng)過特殊處理后制備成免疫用抗原,免疫BalB/C小鼠,制備抗血清,通過間接法酶聯(lián)免疫吸附試驗測得抗血清的抗體滴度為1:51200,采用抗血清及偶聯(lián)堿性磷酸酶的馬抗小鼠IgG二抗對恥垢分枝桿菌總蛋白中GlmU進行Western Blotting分析,結(jié)果表明制備的GlmU多克隆抗體特異性高。 5. glmU反義RNA的誘導表達與GlmU蛋白的檢測 分別用5 ng/ml、10 ng/ml和20 ng/ml的四環(huán)素誘導攜帶pMind-glmU-AS表達載體的mc~2155菌株表達反義RNA,繪制細菌生長曲線,結(jié)果表明20 ng/ml濃度的四環(huán)素可抑制mc~2155/pMind-glmU-AS的生長,但Western Blotting分析結(jié)果顯示經(jīng)四環(huán)素誘導和未誘導的mc~2155/pMind-glmU-AS中,GlmU蛋白表達量無明顯的變化。 結(jié)論:在本研究中,我們構(gòu)建了pET29b-glmU表達載體,在大腸桿菌BL21(DE3)中表達出大量的可溶性恥垢分枝桿菌GlmU蛋白。用純化的GlmU蛋白免疫BalB/C小鼠,成功地制備了抗GlmU蛋白的多克隆抗體,并將抗GlmU蛋白抗體應用到檢測四環(huán)素誘導表達glmU反義RNA的系統(tǒng)中,初步研究了GlmU蛋白表達水平與恥垢分枝桿菌生長速率之間的關系,這一研究成果為我們進一步研究GlmU酶蛋白活性與分枝桿菌細胞壁結(jié)構(gòu)和組成的關系奠定了基礎。
[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.

【學位授予單位】：大連醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2007
【分類號】：R392
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本文編號：1387901