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目的构建弓形虫pcDNA3-ROP2-p30-HSP70真核表达重组质粒。方法根据HSP70基因序列,设计合成HSP70基因的引物,PCR扩增HSP70基因片段,克隆入pMD18-T载体,再经酶切、连接等,亚克隆至pcDNA3-ROP2-p30表达重组质粒中,并进行酶切、PCR和测序鉴定。结果 PCR扩增出长度为916bp的HSP70基因片段,将其克隆到pMD18-T中,成功亚克隆获得pcDNA3-ROP2-p30-HSP70表达重组质粒。测序结果显示,pcDNA3-ROP2-p30-HSP70表达重组质粒包含了HSP70蛋白基因完整序列。结论成功构建弓形虫pcDNA3-ROP2-p30-HSP70真核表达重组质粒,为下一步核酸疫苗的研究奠定了基础。
Objective To construct the eukaryotic expression plasmid pcDNA3-ROP2-p30-HSP70. Methods According to the sequence of HSP70 gene, primers for HSP70 gene were designed and synthesized. The HSP70 gene fragment was amplified by PCR, cloned into pMD18-T vector, subcloned into pcDNA3-ROP2-p30 recombinant plasmid by digestion, Digestion, PCR and sequencing identification. Results A 916 bp fragment of HSP70 was amplified by PCR and cloned into pMD18-T. The recombinant plasmid pcDNA3-ROP2-p30-HSP70 was successfully subcloned. The sequencing results showed that the pcDNA3-ROP2-p30-HSP70 expression recombinant plasmid contains the complete sequence of the HSP70 protein gene. Conclusion The eukaryotic expression plasmid pcDNA3-ROP2-p30-HSP70 was successfully constructed, which laid the foundation for the further study of nucleic acid vaccine.