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目的构建pBudCE4.1-AMA1-ROP18 DNA重组质粒,并对其进行克隆及鉴定。方法采用PCR法对目的基因进行扩增,构建pBudCE4.1-AMA1-ROP18重组质粒,转染至人包皮成纤维细胞(HFF)进行表达并用SDS-PAGE和β-actin进行鉴定。结果经测序、双酶切和PCR鉴定,重组质粒pBudCE4.1-AMA1-ROP18构建正确;经RT-PCR和SDS-PAGE鉴定,AMA1、ROP18基因均在哺乳动物细胞中正确转录与表达。结论成功构建pBudCE4.1-AMA1-ROP18重组质粒,并可在HFF细胞中稳定表达。
Objective To construct pBudCE4.1-AMA1-ROP18 DNA recombinant plasmid and clone and identify it. Methods The recombinant plasmid pBudCE4.1-AMA1-ROP18 was amplified by PCR and transfected into human foreskin fibroblasts (HFF) for expression. SDS-PAGE and β-actin were used for identification. Results The recombinant plasmid pBudCE4.1-AMA1-ROP18 was constructed correctly by sequencing, double enzyme digestion and PCR. AMA1 and ROP18 were correctly expressed and expressed in mammalian cells by RT-PCR and SDS-PAGE. Conclusion The recombinant plasmid pBudCE4.1-AMA1-ROP18 was successfully constructed and stably expressed in HFF cells.