目的利用CRISPR/Cas9基因编辑系统敲除小鼠源肝癌细胞H22中的GP73基因,构建H22细胞GP73基因敲除的稳定细胞株。方法根据CRISPR/Cas9靶点设计原则,设计2条特异性识别GP73基因启动子的上下游sgRNA,利用载体p X459质粒,构建2对重组真核表达质粒。经酶切和测序鉴定后,将重组质粒转染至H22细胞内,使用嘌罗霉素加压筛选稳定敲除GP73的H22细胞株,利用免疫印迹检测重组质粒对内源GP73的敲除效果。MTT实验检测GP73被敲除后对细胞增殖能力的影响,再利用划痕实验检测细胞的迁移能力。结果免疫印迹结果说明敲除GP73基因的小鼠源H22细胞株内无GP73蛋白的表达;并且GP73被敲除后,H22细胞的增殖能力和迁移能力减慢。结论通过CRISPR/Cas9系统获得了靶向GP73基因的重组质粒,并且筛选出了稳定干扰GP73表达的细胞株,从而为探讨GP73在肝癌发生中的作用奠定基础。 OBJECTIVE: To knockdown the GP73 gene in mouse hepatoma H22 cell line by using the CRISPR / Cas9 gene editing system to construct a stable cell line with GP73 knockout in H22 cells. Methods According to the principle of CRISPR / Cas9 target design, two upstream and downstream sgRNAs were designed to specifically recognize GP73 promoter. Two pairs of recombinant eukaryotic expression plasmids were constructed by using plasmid pX459. After identification by restriction enzyme and sequencing, the recombinant plasmids were transfected into H22 cells, and the H22 cells stably knocked out by GP73 were screened with puromycin. The knockdown effect of recombinant plasmids on GP73 was detected by Western blotting. MTT assay GP73 was knocked out after the impact on cell proliferation, and then use the scratch test to detect cell migration. Results The result of western blotting showed that there was no GP73 protein expression in mouse-derived H22 cell line which knocked out GP73 gene. And GP73 knocked out the ability of H22 cells to proliferate and migrate slowly. Conclusion The recombinant plasmids targeting GP73 gene were obtained by CRISPR / Cas9 system and the cell lines stably interfering with the expression of GP73 were screened out, which laid the foundation for the study on the role of GP73 in the pathogenesis of hepatocellular carcinoma.