摘要
目的构建人类磷脂酰肌醇蛋白聚糖3(glypican-3,GPC3)重组真核表达载体,并进行蛋白表达,为后续研究提供基础。方法以人类肝脏c DNA文库为模板扩增出GPC3基因序列,将扩增产物与p MD-18 simple T载体进行连接构建出T-GPC3克隆载体,用EcoRⅠ、Bam HⅠ对T-GPC3克隆载体和pc DNA4.1空载体进行双酶切,从T-GPC3克隆载体上获取GPC3基因片段,用T4连接酶将GPC3基因片段连接到pc DNA4.1酶切后的载体上,构建出pc DNA4.1-GPC3真核表达载体;将构建好的载体再次测序,并进行蛋白表达及WB的鉴定。结果扩增GPC3的PCR产物长度为1 740 bp,质粒测序结果经与NCBI网站比对后序列完全一致,说明pc DNA4.1载体中正确插入了目的片段。Western blotting分析发现有相对分子质量大小约为66 k D的蛋白条带,蛋白经鉴定后亦为GPC3蛋白。结论成功地构建了真核表达载体pc DNA-GPC3并获得了GPC3蛋白。
Objective To construct the eukaryotic expression vector of the human glypican- 3（ GPC3） gene,obtain protein expression,and provide basis for further study. Methods To amplify the human GPC3 gene from human liver c DNA library,and then combine the amplification product with p MD- 18 simple T vector to construct T- GPC3 clone. The target gene GPC3 part from T- GPC3 vector and pc DNA4. 1 vector were digested with EcoRⅠ and Bam HⅠ restrictive enzymes. Then the amplified product was linked to digested pc DNA4. 1 vector using T4 ligase. The recontructed product was sequenced and performed for protein expression and Western blotting identificantion. Results The length of PCR,the amplification product of GPC3 was1 740 bp,and the sequencing result was same as the sequence on the NCBI web,which suggested that the target fragment was successfully inserting pc DNA4 carrier. Western blotting analysis showed that the relative molecular mass was about 66 k D protein bands,and the protein was also expressed as GPC3 protein. Conclution The eukaryotic expression vector pc DNA- GPC3 was successfully constructed and the GPC3 protein was obtained.
出处
《中国卫生检验杂志》
CAS
2015年第14期2367-2369,共3页
Chinese Journal of Health Laboratory Technology
基金
北京市科学技术委员会资助课题(Z131107002213-071)
首都医科大学基础-临床科研合作基金资助(13JL14)
