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目的体外扩增弓形虫棒状体分泌抗原2(ROP2)靶基因,构建真核表达载体pc-DNA3-ROP2。方法收集、纯化RH株弓形虫速殖子,提取基因组DNA;根据基因库ROP2基因序列设计合成1对引物,应用PCR扩增ROP2基因片段,回收纯化后克隆入TA载体质粒pUCm-T;用限制性内切酶EcoRⅠ、HindⅢ双酶切该重组子,将切下的ROP2基因在T4DNA连接酶作用下插入真核细胞表达载体质粒pc-DNA3,并进一步作双酶切、PCR及测序鉴定。结果以弓形虫基因组DNA为模板,PCR扩增出1.7kbROP2基因片段,克隆于pUCm-T载体中,再将ROP2基因亚克隆于真核表达载体质粒pc-DNA3,经筛选鉴定,构建pc-DNA3-ROP2重组质粒;测序结果显示,重组质粒包含了ROP2蛋白基因读码框内的完整序列,能完整表达ROP2的抗原蛋白。结论弓形虫ROP2基因片段,经TA克隆及亚克隆,构建弓形虫pc-DNA3-ROP2重组质粒。
Objective To amplify the target gene of Toxoplasma gondii secreted antigen 2 (ROP2) in vitro and construct eukaryotic expression vector pc-DNA3-ROP2. Methods Toxoplasma gondii RH strains were collected and purified for genomic DNA extraction. One pair of primers were designed and synthesized according to ROP2 gene sequence of R RH gene. The ROP2 gene fragment was amplified by PCR and cloned into TA vector plasmid pUCm-T. The recombinants were digested by EcoRⅠand HindⅢ. The excised ROP2 gene was inserted into eukaryotic expression plasmid pc-DNA3 under the action of T4 DNA ligase and further identified by double enzyme digestion, PCR and sequencing. Results The Toxoplasma gondii genomic DNA was used as a template to amplify the 1.7kbROP2 gene fragment and cloned into pUCm-T vector. The ROP2 gene was subcloned into the eukaryotic expression vector pc-DNA3. The recombinant plasmid pc-DNA3 -ROP2 recombinant plasmid. The sequencing results showed that the recombinant plasmid contains the complete sequence in the reading frame of ROP2 protein gene, which can completely express the antigen protein of ROP2. Conclusion Toxoplasma gondii ROP2 gene fragment was cloned by TA and subcloned to construct Toxoplasma gondii pc-DNA3-ROP2 recombinant plasmid.