【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。更多还原

【Abstract】 Objectives To construct a eukaryotic expression vector of an ACBD3 gene fragment and to obtain an expressed product in order to lay the foundation for the further study of the interaction between the ACBD3 protein and the Chlamydia pneumoniae inclusion proteins Cpn0308/Cpn0147. Methods Primers were designed in accordance with the nucleotide sequence encoding a ligand that interacted with the inclusion membrane proteins Cpn0308/Cpn0147.The cDNA of HeLa cells was used as a template,the ACBD3 gene fragment was obtained using PCR,and the PCR product was recombined with a pcDNA3.1+/Flag empty vector via double digestion and T4 ligase ligation.The recombinant expression vector pcDNA3.1+/Flag-ACBD3 was identified using colony-PCR,double digestion,and sequencing.PcDNA3.1+/Flag-ACBD3 was transfected into HeLa cells with LipofectamineTM2000 transfection reagent.Western blotting and indirect immunofluorescence were used to detect expression of the target protein. Results A Agarose gel electrophoresis indicated that the specific band of about 883 bp was amplified,which was consistent with expectations.The results of colony-PCR and double digestion indicated that the recombinant plasmid was successfully constructed,and the results of plasmid sequencing were consistent with sequence of the ACBD3 gene in the NCBI database.Fluorescence microscopy indicated that the protein was expressed in the cytoplasm of cells,and Western blotting indicated that the expressed protein was 33.5×10~3 in size,which was consistent with expectations. Conclusion The eukaryotic expression vector pcDNA3.1+/Flag-ACBD3 was successfully constructed and the target protein was correctly expressed.This work is of significance to further study of ACBD3 and its interaction with the Chlamydia pneumoniae inclusion proteins Cpn0308/Cpn0147.更多还原