摘要
目的对氯霉素乙酰基转移酶(Chloramphenicol acetyltransferase,CAT)基因进行表达和纯化。方法通过PCR扩增编码CAT氨基酸序列的基因片断,并将之克隆入pGEX-2T载体。IPTG诱导蛋白融合表达,产物经琼脂糖亲和层析树脂纯化。免疫印迹鉴定纯化蛋白的抗原性。结果筛选得到的重组子诱导后表达相对分子质量约为52 600的CAT融合蛋白。树脂纯化及酶切后均得到高纯度的蛋白样品。纯化蛋白能与抗CAT抗体特异结合。结论本研究获得了CAT基因的融合表达蛋白,并对其完成纯化,为制备抗血清和抗体打下基础。
Objective To express and purify chloramphenicol acetyltransferase (CAT) gene. Methods The gene sequence which codes natural CAT portion was amplified with polymerase chain reaction (PCR). The fragment was cloned into prokaryotic expression vector pGEX-2T. The recombinant protein expression was induced by IPTG and the products were purified by agarose gel affinity chromatography resin. The antigenicity of purified recombinant CAT was identified by western blot. Results The recombinant CAT with a molecular weight of 52.6 kDAa was obtained in GST expression system. SDS-PAGE assay showed that the purified and digested product has high purity. The purified protein had the ability to reactive specifically with anti-CAT antibody. Conclusion The CAT fusion protein is expressed and purified, which lay a basis for preparation of antiserum and antibody.
出处
《免疫学杂志》
CAS
CSCD
北大核心
2004年第z1期163-164,169,共3页
Immunological Journal
基金
广东省自然科学基金重点资助项目(020100)
广东省自然科学基金资助项目(010637)
关键词
氯霉素乙酰基转移酶
融合蛋白
表达
纯化
鉴定
<Keyword>loramphenicol acetyltransferase
Fusion protein
Expression
Purification
Identification
作者简介
潘玉先(1965-),女,河南商丘市人,主管技师,本科,主要从事病毒学方面的研究.Tel: (020)61643592; E-mail: linche @ pub. guangzhou. gd. cn 车小燕,通讯作者
