摘要
目的利用大肠埃希菌表达系统表达宫颈癌相关BLCAP基因,并优化表达条件。方法利用PCR技术从逆转录病毒重组载体pL(BLCAP)SN中扩增宫颈癌相关BLCAP基因,将其插入到原核表达载体pET-32(a)中,从而构建原核表达重组质粒pET-32(a)-BLCAP,随后将阳性重组质粒转化到表达宿主菌中,通过IPTG诱导表达并优化表达条件,所表达的带有His标签目的融合蛋白经Ni2+亲和层析纯化回收,并采用SDS-PAGE和Western印迹对目的蛋白进行分析和鉴定。结果构建的重组表达质粒经PCR、酶切和DNA测序鉴定与预期的结果一致,含有重组质粒的表达宿主菌经过IPTG诱导表达了分子量约为28ku的融合蛋白,并经优化确定了最佳的诱导表达条件。结论成功构建了pET-32(a)-BLCAP原核表达质粒,表达并经纯化得到了BLCAP目的蛋白,为研究该蛋白的性质及其制备针对该蛋白的抗体奠定了基础。
Objective To express cervical carcinoma associated BLCAP gene in E. coli and determine its optimal expression conditions. Methods The cervical carcinoma associated BLCAP gene was amplified from retrovirus recombinant pL （BLCAP） SN by PCR and was inserted into a prokaryotic expression vector pET-32 （a） to construct the prokaryotic expression plasmid pET-32 （a）-BLCAP. The positive recombinant plasmid was transformed into expression host strains, and then was induced by IPTG, with the expression conditions optimized. The expressed His-tagged protein was purified by Ni^2＋ affinity chromatography column and analyzed and identified by SDS-PAGE and Western blotting. Results The recombinant expression plasmid was identified by PCR method, digested with restricted endoenzymes and subjected to DNA sequencing. It was found to be consistent with the predicted result. The host cells containing the recombinant plasmid expressed fusion protein of 28 ku after being induced by IPTG and the optimal inducing conditions were determined. Conclusion The prokaryotic expression plasmid pET-32 （a）-BLCAP was successfully constructed and the target recombinant protein BLCAP was expressed and purified, which lays a foundation for the further study and preparation of antibodies against BLCAP protein.
出处
《医学分子生物学杂志》
CAS
CSCD
2007年第1期15-19,共5页
Journal of Medical Molecular Biology
基金
国家自然科学基金(No:30250008)
湖北省自然科学基金(No:301130735)~~
