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目的 构建并鉴定弓形虫RH株rop16Ⅰ/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RH△rop16株对HFF-1细胞的增殖与入侵.并比较RH株和RH△roop16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RH△rop16株有Rop16Ⅰ/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RH△rop16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rop16Ⅰ/Ⅲ缺陷株在第9d出现死亡,但两种弓形虫株感染动物在第10 d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rop16Ⅰ/Ⅲ缺陷的弓形虫RH虫株,rop16Ⅰ/Ⅲ基因敲除对弓形虫RH株毒力无明显影响.“,”Toxoplasma gondii RH rop16Ⅰ/Ⅲ deficient strain was constructed based on CRSPR/Cas9 technology.E-CRISP database was used to design gRNA;mutation of gRNA in pSAG1:∷Cas9-U6∷ sgUPRT plasmid was performed by using sitedirected mutagenesis to construct pSAG1∷Cas9-U6∷sgrop16 plasmid.The pUC19-donorDNA plasmid was constructed and fragments were amplified by PCR.The plasmid pSAG1:Cas9-U6:sgrop16Ⅰ/Ⅲ and donor DNA fragments were electroporated into T.gondii RH strain.Follow electroporation,suspension was inoculated into human foreskin fibroblast cells (HFF-1).The 3 μmol/L pyrimethamine was used to screen the electroporated parasite and monoclone was detected by PCR and Western blotting.The proliferation and invasion of RH△ropo16 strain were observed in HFF-1 cells by Giemsa staining.Twenty KM mice were infected with 200 tachyzoite of wild type or RH△rop16 parasite,respectively.The animal survival was recorded.The results showed that pSAG1:Cas9-U6:sgrop16 plasmid and pUC19-donor DNA plasmid were successfully constructed and confirmed by DNA sequencing.PCR identification proved that DHFR coding scquence was successfully inserted to the target position.Western blotting analysis revealed deficient expression of ROP16 in the RH△rop16 strain.Giemsa staining indicated that the number of parasites per parasite phorous vacuole of wild type Toxoplasma-infected cells was more than that of the RH△rop16 infected cells.Virulence examination indicated that,the wild type strain infected mice began to die on day 7 post-infection where as Arop16Ⅰ/Ⅲ strain,on day 9.However,all animals infected with both strains died on day 10 post-infection.The Arop16Ⅰ/Ⅲ strain of T.gondii was successfully constructed by the CRISPR-Cas9 technology and no difference was noted between wild type and RHArop16 strain in their virulence to mice.