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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 a recombinant eukaryotic expression vector of human glypican-3 (GPC3) for protein expression, which will provide the basis for further research. Methods Human liver c DNA library was used as a template to amplify the sequence of GPC3 gene. The amplified product was ligated with p MD-18 simple T vector to construct T-GPC3 cloning vector. EcoRI, pcDNA4.1 empty vector double digestion, GPC3 gene fragment was obtained from the T-GPC3 cloning vector, the GPC3 gene fragment was ligated to the pcDNA4.1 digested vector T4 T4 ligase to construct pcDNA4.1 -GPC3 eukaryotic expression vector; the constructed vector was sequenced again, and protein expression and WB identification. Results The length of PCR product amplified by GPC3 was 1 740 bp. The result of plasmid sequencing was exactly the same as that of NCBI website, which indicated that the target fragment was correctly inserted into pcDNA4.1 vector. Western blotting analysis showed that the molecular weight of about 66 kD protein bands, proteins were also identified as GPC3 protein. Conclusion The eukaryotic expression vector pcDNA-GPC3 was successfully constructed and the GPC3 protein was obtained.