CAJ | 학술논문

获得耻垢分枝杆菌 MurB 纯化蛋白，制备小鼠源性 MurB 多克隆抗血清。首先，利用 PCR 技术扩增耻垢分枝杆菌murB基因，连接至 pColdII 载体并转化至 E．coli BL21（DE3）菌株；然后，低温下以 IPTG 诱导 MurB 蛋白的表达，并利用 Ni-His 亲和层析技术纯化 MurB 蛋白；最后，将 MurB 纯化蛋白联合免疫佐剂注射 BalB／c 小鼠，经过初次免疫和2次加强免疫后分离抗血清，并分别利用 ELISA 和 Western blot 技术检测制备的抗血清对 MurB 蛋白的效价和特异性。结果显示：已获得具有理想浓度和纯度的 MurB 纯化蛋白；已制备具有较高效价的小鼠源性 MurB 多克隆抗血清，该抗血清能够特异性识别耻垢分枝杆菌可溶性蛋白组分中的 MurB 蛋白。因此，制备的 MurB 多克隆抗血清能够应用于分枝杆菌 MurB 蛋白的检测分析，为 MurB 功能研究奠定了基础。
획득치구분지간균 MurB 순화단백，제비소서원성 MurB 다극륭항혈청。수선，이용 PCR 기술확증치구분지간균murB기인，련접지 pColdII 재체병전화지 E．coli BL21（DE3）균주；연후，저온하이 IPTG 유도 MurB 단백적표체，병이용 Ni-His 친화층석기술순화 MurB 단백；최후，장 MurB 순화단백연합면역좌제주사 BalB／c 소서，경과초차면역화2차가강면역후분리항혈청，병분별이용 ELISA 화 Western blot 기술검측제비적항혈청대 MurB 단백적효개화특이성。결과현시：이획득구유이상농도화순도적 MurB 순화단백；이제비구유교고효개적소서원성 MurB 다극륭항혈청，해항혈청능구특이성식별치구분지간균가용성단백조분중적 MurB 단백。인차，제비적 MurB 다극륭항혈청능구응용우분지간균 MurB 단백적검측분석，위 MurB 공능연구전정료기출。
To obtain purified M.smegmatis MurB protein and to prepare anti-MurB polyclonal antiserum from mice are the main purposes of this work.Firstly,themurB gene of M.smegmatis was amplified though PCR technology and cloned to pColdII vec-tor,then transformed to E.coli BL21(DE3)strain.Secondly,the expression of MurB was induced by IPTG at a low tempera-ture.The MurB protein was purified by Ni-His affinity chromatography.Finally,the purified MurB protein emulsified with im-munoadjuvant was injected to BalB/c mice though initial immunity and two times of supplemental immunity.The antiserum was prepared,then the titer and specificity of the antiserum were detected by ELISA and Western blot,respectively.The results show that the purified MurB protein with ideal concentration and purity is obtained and the polyclonal antiserum of MurB is pre-pared from mice.The MurB antiserum has a high titer and can specifically recognize the MurB protein in the soluble protein frac-tion of M.smegmatis.Therefore,the polyclonal antiserum against M.smegmatis MurB protein can be used in the detection of mycobacterial MurB,which will lay foundations for MurB functional study in the future.