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目的构建并鉴定弓形虫RH株rop16_(I/III)缺陷虫株。方法利用CRISPR-Cas9技术进行构建基因缺陷虫株。运用E-CRISPR数据库设计gRNA,并使用定点突变试剂盒突变pSAG1::Cas9-U6::sgUPRT质粒上的gRNA,构建pSAG1::Cas9-U6::sgrop16质粒。此外将rop16上游序列、乙胺嘧啶抗性基因、rop16下游序列3个片段连接成donorDNA,克隆于pUC19质粒上,PCR扩增donor DNA片段。pSAG1::Cas9-U6::sgrop16质粒和donor DNA片段电穿孔转染弓形虫,电转后悬液接种于HFF-1细胞中,3μmol/L乙胺嘧啶筛选电转后的虫株。PCR和Western blotting鉴定克隆化筛选虫株。吉姆萨染色分别比较RH株和RHΔrop16株对HFF-1细胞的增殖与入侵。并比较RH株和RHΔrop16株分别感染昆明小鼠后小鼠的生存和死亡率。结果经测序比对,成功构建了pSAG1::Cas9-U6::sgrop16质粒和pUC19-donorDNA质粒。PCR鉴定结果显示,DHFR编码(编码乙胺嘧啶抗性基因)序列成功插入至靶点位置,Western blotting分析结果未见RHΔrop16株有Rop16_(I/III)蛋白表达。吉姆萨染色后计数结果表明,RH株感染的细胞内每个纳虫泡内速殖子的平均数显著高于RHΔrop16虫株。毒力试验结果显示,RH株感染的小鼠在第7d即出现死亡,而rop16_(I/III)缺陷株在第9d出现死亡,但两种弓形虫株感染动物在第10d均全部死亡,两组间无统计学差异。结论利用CRISPR-Cas9技术成功构建了rop16_(I/III)缺陷的弓形虫RH虫株,rop16_(I/III)基因敲除对弓形虫RH株毒力无明显影响。
Objective To construct and identify Toxoplasma RH strain rop16_ (I / III) deficient strains. Methods CRISPR-Cas9 technology was used to construct gene-defective insect strains. The gRNA was designed using the E-CRISPR database and the pSAG1 :: Cas9-U6 :: sgrop16 plasmid was constructed by mutating the gRNA on the pSAG1 :: Cas9-U6 :: sgUPRT plasmid using a site-directed mutagenesis kit. In addition, the rop16 upstream sequence, pyrimethamine resistance gene, rop16 downstream sequences of three fragments connected donorDNA, cloned in pUC19 plasmid, PCR amplification of the donor DNA fragment. The pSAG1 :: Cas9-U6 :: sgrop16 plasmid and donor DNA fragment were transfected into Toxoplasma gondii by electroporation. The electroporation suspension was inoculated into HFF-1 cells and the electroporation strain was screened with 3 μmol / L pyrimethamine. Identification of clones by PCR and Western blotting. Giemsa staining respectively compared the proliferation and invasion of HFF-1 cells by RH strain and RHΔrop16 strain. The survival and mortality of mice infected with RH strain and RHΔrop16 strain were compared. Results The pSAG1 :: Cas9-U6 :: sgrop16 plasmid and pUC19-donorDNA plasmid were successfully constructed by sequencing. The results of PCR showed that the sequence of DHFR (encoding pyrimethamine resistance gene) was successfully inserted into the target site. No Rop16_ (I / III) protein was expressed in RHΔrop16 strain by Western blotting. The results of Giemsa staining showed that the mean number of tachyzoites per tubule in RH-infected cells was significantly higher than that of RHΔrop16 strain. The results of virulence test showed that mice infected with RH strain died on the 7th day and the rop16_ (I / III) strain died on the 9th day. However, both Toxoplasma gondii infected animals died on the 10th day, No significant difference between groups. Conclusion The Toxoplasma gondii RH strain with rop16_ (I / III) deficiency was successfully constructed by CRISPR-Cas9 technique. The knockout of rop16_ (I / III) had no significant effect on Toxoplasma RH strain virulence.