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目的 对恶性疟原虫热休克蛋白基因进行体外扩增 ,构建 HSP86基因打靶载体 ,利用基因敲除技术进行恶性疟原虫热休克蛋白基因功能的研究。方法 恶性疟原虫体外培养 ,基因组 DNA提取 ,目的基因扩增 ,载体与目的片段连接、阳性克隆筛选及酶切鉴定。结果 经酶切鉴定 PCR产物及插入片段大小与预期值相符 ,进一步测序鉴定证实 PCR产物及插入片段为所需目的基因片段。插入片段经酶切鉴定证实插入方向正确。结论 成功构建了用于恶性疟原虫基因打靶的插入型及置换型载体 (p HCl- HSP86 int和 p HCl- HSP86 rep) ,该打靶载体的成功构建为随后将进行的恶性疟原虫细胞内基因转染和进一步的功能研究奠定了基础
Objective To amplify the heat shock protein gene of Plasmodium falciparum in vitro and construct HSP86 gene targeting vector to study the function of heat shock protein gene of Plasmodium falciparum by using gene knockout technique. Methods Plasmodium falciparum was cultured in vitro, genomic DNA was extracted, the target gene was amplified, the vector was ligated with the target fragment, the positive clones were screened and identified by restriction enzyme digestion. Results The size of PCR products and inserted fragments were confirmed by restriction enzyme digestion. The results of further sequencing proved that the PCR products and inserted fragments were the desired gene fragments. The inserted fragment was confirmed by restriction enzyme digestion in the correct direction. Conclusion The insertion and substitution vectors (p HCl-HSP86 int and p HCl-HSP86 rep) for gene targeting of Plasmodium falciparum were successfully constructed. The target vector was successfully constructed into the plasmodium falciparum intracellular gene transfer Dyeing and further functional research laid the foundation