【摘要】：目的表達并純化克羅諾桿菌Ibp A蛋白,檢測其免疫原性。方法提取克羅諾桿菌菌株CMCC45402的基因組,經(jīng)PCR獲得ibp A基因片段,連接至表達載體p ET30a(+),構(gòu)建重組質(zhì)粒p ET30a(+)-ibp A。將重組質(zhì)粒轉(zhuǎn)化E.coli BL21(DE3)菌株,用不同終濃度的IPTG(0.1、0.2、0.5、0.8、1.0 mmol/L)分別于30和37℃誘導表達不同時間(2.5、4、5 h),進行SDS-PAGE及Western blot分析。表達產(chǎn)物經(jīng)Ni凝膠親和層析純化后,經(jīng)腹腔免疫BALB/c小鼠,每2周加強免疫1次,共2次,末次免疫1周后,經(jīng)小鼠眼球采血,分離血清,ELISA法檢測血清效價。同時檢測熱激對Ibp A蛋白表達水平的影響。結(jié)果重組質(zhì)粒p ET30a(+)-ibp A經(jīng)雙酶切鑒定證明構(gòu)建正確。最佳IPTG誘導終濃度為0.2 mmol/L,最佳誘導溫度為30℃,最佳誘導時間為5 h。誘導表達產(chǎn)物相對分子質(zhì)量約24 000,主要以可溶性形式存在,可與抗His-Tag單克隆抗體特異性結(jié)合;純化蛋白的純度可達96%;小鼠免疫血清效價均可達1:25 000以上。用小鼠血清可檢測到熱激后克羅諾桿菌Ibp A的表達。結(jié)論本實驗成功構(gòu)建了ibp A基因的高效原核表達系統(tǒng),獲得了高純度重組蛋白,制備了高效價抗血清,為后續(xù)ibp A基因和克羅諾桿菌熱抗性關(guān)系的研究奠定了基礎。
[Abstract]:Objective to express and purify the Ibp A protein of Clostridium sp., and to detect its immunogenicity. Methods Genome of CMCC45402 was extracted and ibp A gene fragment was obtained by PCR. The recombinant plasmid p ET30a ()-ibp A was constructed by ligating into expression vector p-ET30a (), to construct recombinant plasmid p ET30a ()-ibp A. The recombinant plasmid was transformed into E.coli BL21 (DE3) strain and expressed in different concentrations of IPTG (0.1, 0.2, 0.5, 0.8, 1.0 mmol/L) at 30 鈩，

本文編號：2442079