發(fā)布時(shí)間：2018-12-08 16:48

【摘要】： 目的結(jié)核分枝桿菌長(zhǎng)期處于持續(xù)感染狀態(tài)已成為目前在全世界范圍內(nèi)消滅結(jié)核病的最大阻礙之一。異檸檬酸裂解酶(ICL)是結(jié)核分枝桿菌在體內(nèi)持續(xù)感染時(shí)所進(jìn)行的乙醛酸途徑中的關(guān)鍵酶,對(duì)結(jié)核分枝桿菌在巨噬細(xì)胞內(nèi)生存及持續(xù)感染起著重要的作用。編碼異檸檬酸裂解酶的基因有兩個(gè):icl和aceA,我們已經(jīng)成功地克隆表達(dá)了icl基因,并對(duì)其表達(dá)的重組酶進(jìn)行了深入的研究。本研究擬克隆結(jié)核分枝桿菌異檸檬酸裂解酶的aceA基因,并構(gòu)建表達(dá)重組體,通過表達(dá)條件的優(yōu)化,獲得AceA可溶性蛋白。純化該蛋白,并對(duì)其結(jié)構(gòu)、分子量及性質(zhì)等進(jìn)行研究,為以異檸檬酸裂解酶為靶點(diǎn)的抗結(jié)核藥物篩選奠定基礎(chǔ)。 方法根據(jù)aceA基因編碼序列設(shè)計(jì)引物,并在引物中設(shè)計(jì)HindⅢ和BamHI酶切位點(diǎn),以MTB基因組為模板,PCR擴(kuò)增編碼異檸檬酸裂解酶的aceA基因,并將其克隆入pUC18質(zhì)粒進(jìn)行擴(kuò)增,再亞克隆入原核表達(dá)載體pET28a(+),構(gòu)建pET28a(+)-aceA重組體;用重組體轉(zhuǎn)化大腸桿菌BL21(DE3)plysS菌,在IPTG誘導(dǎo)下進(jìn)行表達(dá);表達(dá)產(chǎn)物經(jīng)SDS-PAGE鑒定,再經(jīng)鎳離子螯合型瓊脂糖凝膠親和層析柱純化,以獲得純化的結(jié)核分枝桿菌異檸檬酸裂解酶重組蛋白;以時(shí)實(shí)波長(zhǎng)掃描對(duì)重組蛋白的酶活性進(jìn)行測(cè)定分析,通過圓二色及質(zhì)譜分析對(duì)其結(jié)構(gòu)及分子質(zhì)量進(jìn)行測(cè)定。 結(jié)果以MTB基因組DNA為模板,以Pfu Tag DNA聚合酶擴(kuò)增得到2.3kb的片段,并克隆入pUC18質(zhì)粒;經(jīng)測(cè)序證實(shí)與GenBank中的aceA基因序列一致。進(jìn)而構(gòu)建出pET28a(+)-aceA表達(dá)質(zhì)粒,該重組質(zhì)粒能在大腸桿菌BL21(DE3)plysS中高效表達(dá);以0.1mM的IPTG誘導(dǎo)6hrs后重組蛋白表達(dá)量最高。表達(dá)蛋白以可溶性非包涵體形式存在于胞漿中,經(jīng)Ni-NTA瓊脂糖親和柱一步純化得到了90KD左右的純化蛋白產(chǎn)物。通過酶學(xué)方法檢測(cè)該重組蛋白證明其具有異檸檬酸裂解酶活性;重組蛋白經(jīng)高效液相色譜及質(zhì)譜鑒定,測(cè)得相對(duì)分子質(zhì)量為89660Da。經(jīng)圓二色儀分析,重組ICL的二級(jí)結(jié)構(gòu)中相對(duì)有32.4 %的α螺旋,27.5%β轉(zhuǎn)角,40.1 %無規(guī)卷曲。 結(jié)論本研究成功地克隆表達(dá)并純化了具有生物學(xué)活性的結(jié)核分枝桿菌異檸檬酸裂解酶AceA蛋白,并對(duì)其分子質(zhì)量,結(jié)構(gòu)及酶活性進(jìn)行了研究。該研究對(duì)于結(jié)核病藥物靶點(diǎn)的研究有著重要的價(jià)值,為結(jié)核病的治療及抗結(jié)核藥物的開發(fā)奠定了基礎(chǔ)。
[Abstract]:Objective Mycobacterium tuberculosis has become one of the biggest obstacles to the worldwide eradication of tuberculosis. Isocitrate lyase (ICL) is a key enzyme in glyoxylic acid pathway of Mycobacterium tuberculosis, which plays an important role in the survival and persistent infection of Mycobacterium tuberculosis in macrophages. There are two genes encoding isocitrate lyase: icl and aceA,. We have successfully cloned and expressed icl gene and studied its recombinant enzyme. In this study, the aceA gene of isocitrate lyase of Mycobacterium tuberculosis was cloned, and the expression recombinant was constructed. The soluble protein of AceA was obtained by optimizing the expression conditions. The protein was purified, and its structure, molecular weight and properties were studied, which laid a foundation for the screening of antituberculous drugs targeting isocitrate lyase. Methods the primers were designed according to the coding sequence of aceA gene, and Hind 鈪，

本文編號(hào)：2368668