ACBD3基因片段真核表达载体的构建与表达

发布时间：2018-06-02 13:30

本文选题：ACBD + 真核表达载体　；参考：《中国病原生物学杂志》2017年08期

【摘要】：目的构建ACBD3基因片段真核表达载体并表达蛋白,为研究其与肺炎衣原体包涵体膜蛋白Cpn0308/Cpn0147相互作用的分子机制奠定基础。方法根据与肺炎衣原体包涵体膜蛋白Cpn0308/Cpn0147作用的配体核苷酸序列设计引物,以HeLa细胞cDNA为模板,通过PCR获得ACBD3基因片段,与pcDNA3.1+/Flag质粒经双酶切后在T4连接酶作用下连接,构建表达载体pcDNA3.1+/Flag-ACBD3,经菌落PCR、双酶切及质粒测序鉴定后转染HeLa细胞,采用Western blot与间接免疫荧光法检测目的蛋白表达。结果 PCR扩增目的基因片段约883bp,与预期相符。经菌落PCR、双酶切验证及测序分析重组质粒构建成功。间接免疫荧光法检测重组质粒转染后的HeLa细胞,观察到表达的蛋白位于细胞胞浆;SDS-PAGE检测表达蛋白分子质量单位(Mr)为33.5×10~3,与预期相符。结论成功构建了pcDNA3.1+/Flag-ACBD3真核表达载体并实现目的蛋白的表达,为进一步研究其生物学功能及其与肺炎衣原体包涵体膜蛋白Cpn0308/Cpn0147之间的相互作用奠定了基础。
[Abstract]:Objective to construct the eukaryotic expression vector of ACBD3 gene fragment and express the protein in order to study the molecular mechanism of its interaction with chlamydia pneumoniae inclusion body membrane protein (Cpn0308/Cpn0147). Methods according to the ligand nucleotide sequence of chlamydia pneumoniae inclusion body membrane protein (Cpn0308/Cpn0147), the ACBD3 gene fragment was obtained by PCR using HeLa cell cDNA as template. The ACBD3 gene fragment was digested with pcDNA3.1 / Flag plasmid and ligated with T4 ligand. The expression vector pcDNA3.1 / Flag-ACBD3 was constructed and identified by colony PCR, double enzyme digestion and plasmid sequencing. The expression of the target protein was detected by Western blot and indirect immunofluorescence assay. Results the target gene fragment amplified by PCR was 883 BP, which was consistent with the expectation. The recombinant plasmid was successfully constructed by colony PCR, double enzyme digestion and sequencing analysis. Indirect immunofluorescence assay was used to detect the HeLa cells transfected with recombinant plasmids. It was observed that the expressed protein was located in the cytoplasmic SDS-PAGE and the molecular weight unit of the expressed protein was 33.5 脳 10 ~ (-3), which was consistent with the expectation. Conclusion the eukaryotic expression vector of pcDNA3.1 / Flag-ACBD3 was successfully constructed and the expression of target protein was realized, which laid a foundation for further study of its biological function and its interaction with chlamydia pneumoniae inclusion body membrane protein Cpn0308/Cpn0147.
【作者单位】：河北北方学院病原生物学与免疫研究所临床检验诊断学重点实验室;
【基金】：河北省自然科学基金项目(No.C2014405041)
【分类号】：R374

【相似文献】