利用CRISPR/Cas9技术构建MDH2敲除细胞株及抗呕吐毒素效应研究

doi:10.13560/j.cnki.biotech.bull.1985.2022-1429

生物技术通报 ›› 2023, Vol. 39 ›› Issue (7): 307-315.doi: 10.13560/j.cnki.biotech.bull.1985.2022-1429

利用CRISPR/Cas9技术构建MDH2敲除细胞株及抗呕吐毒素效应研究

施炜涛¹^,²(), 姚春鹏³, 魏文康², 王蕾², 房元杰², 仝钰洁¹, 马晓姣¹, 蒋文¹, 张晓爱²(), 邵伟¹()

1.新疆农业大学动物科学学院，乌鲁木齐 830052
2.广东省农业科学院农业生物基因研究中心广东省农作物种质资源保存与利用重点实验室，广州 510640
3.广东省农业科学院蔬菜研究所广东省蔬菜新技术研究重点实验室，广州 510640

收稿日期:2022-11-18 出版日期:2023-07-26 发布日期:2023-08-17
通讯作者: 张晓爱，女，博士，副研究员，硕士生导师，研究方向：微生物；E-mail: zhangxiaoai@gdaas.cn；
邵伟，男，博士，副教授，研究方向：动物生产与管理；E-mail: dksw@xjau.edu.cn
作者简介:施炜涛，男，硕士研究生，研究方向：基因编辑与毒素；E-mail: shiweitao19980303@163.com
基金资助:
国家自然科学基金项目(31872127);国家自然科学基金项目(32002287);广东省农科院学科团队项目(202110TD);广东省农科院学科团队项目(202131TD)

Establishment of MDH2 Knockout Cell Line Using CRISPR/Cas9 Technology and Study of Anti-deoxynivalenol Effect

SHI Wei-tao¹^,²(), YAO Chun-peng³, WEI Wen-Kang², WANG Lei², FANG Yuan-jie², TONG Yu-jie¹, MA Xiao-jiao¹, JIANG Wen¹, ZHANG Xiao-ai²(), SHAO Wei¹()

1. College of Animal Science, Xinjiang Agricultural University, Urumqi 830052
2. Agro-biological Gene Research Center, Guangdong Academy of Agricultural Sciences, Guangdong Key Lab of Crop Germplasm Resources Preservation and Utilization, Guangzhou 510640
3. Vegetable Research Institute, Guangdong Academy of Agricultural Sciences, Guangdong Key Laboratory for New Technology Research of Vegetables， Guangzhou 510640

Received:2022-11-18 Published:2023-07-26 Online:2023-08-17

摘要/Abstract

摘要：

试验旨在利用CRISPR/Cas9基因编辑技术构建稳定敲除苹果酸脱氢酶2（malate dehydrogenase 2，MDH2）基因的IPEC-J2单克隆细胞株，并研究敲除MDH2基因后抗呕吐毒素效应。针对MDH2基因序列设计sgRNA插入PX459载体中，构建PX459-sgRNA-MDH2重组质粒；将质粒通过电转导入IPEC-J2细胞中，并加入嘌呤霉素筛选阳性细胞；利用有限稀释法筛选单克隆细胞并进行基因型测序鉴定、荧光定量PCR和蛋白质印迹法验证，获得MDH2基因敲除的单克隆细胞株；最后通过CCK8试剂盒和细胞凋亡与坏死检测试剂盒检测细胞存活情况，测定MDH2敲除细胞株对呕吐毒素的抗性。测序结果显示MDH2基因敲除载体构建成功。利用荧光定量PCR及蛋白免疫印迹法验证了获得的细胞系为MDH2基因敲除的单克隆细胞株。CCK8细胞活力实验结果显示，与野生型细胞相比，敲除MDH2可使IPEC-J2细胞在不同浓度呕吐毒素（4、2、1和0.5 μg/mL）处理5 d时细胞活力分别极显著提高18.67%、19.59%、26.36%和27.01%。流式细胞仪实验结果显示，与野生型细胞相比，敲除MDH2使不同浓度呕吐毒素处理5 d的细胞死亡率分别极显著降低30.33%、15.81%、16.00%和14.70%。利用CRISPR/Cas9系统对IPEC-J2细胞中的MDH2基因进行敲除并筛选获得了MDH2-KO单克隆细胞株，并通过细胞活力和细胞死亡检测，证明该细胞株具有抗呕吐毒素毒性效应的效果，为揭示呕吐毒素诱导宿主细胞死亡的毒性机制提供借鉴，为防治呕吐毒素提供新策略。

关键词: CRISPR/Cas9系统, 呕吐毒素, MDH2基因, 细胞存活, 细胞死亡

Abstract:

The aim of this study is to establish the IPEC-J2 cell line with MDH2 gene knockout by CRISPR/Cas9 genome editing technology and investigate whether MDH2 gene knockout confers resistance to deoxynivalenol. sgRNA sequences targeting MDH2 gene were designed and cloned into PX459 vector; the recombinant plasmid PX459-sgRNA-MDH2 was introduced into the IPEC-J2 cells by electroporation，and puromycin was added to screen KO cell colony generations. The MDH2 KO single cell colony generations were isolated by limited dilution method and validated by genotype sequencing, quantitative PCR and Western blotting to obtain MDH2-KO monoclonal cell colony generation. Finally, the cell viability was detected by CCK8 kit and apoptosis and necrosis assay kit to determine the resistance of MDH2 knockout cell colony generation to deoxynivalenol. The sequencing results showed that the MDH2 knockout vector was successfully constructed. The obtained cell colony generations were validated as MDH2 knockout monoclonal cell colony generations by RT-qPCR and immunoblotting. The CCK8 cell viability assay showed that knockout of MDH2 increased the viability of IPEC-J2 cells by 18.67%, 19.59%, 26.36%, and 27.01% at different concentrations of deoxynivalenol（4, 2, 1, and 0.5 μg/mL）for 5 d compared with wild-type cells. Flow cytometry assays showed that knockout of MDH2 reduced the mortality rate of IPEC-J2 cells by 30.33%, 15.81%, 16.00%, and 14.70% at different concentrations of deoxynivalenol respectively compared with wild-type cells for 5 d. We obtained the MDH2-KO monoclonal cell colony generation by editing the MDH2 gene in IPEC-J2 cells using the CRISPR/Cas9 system. The resistance of MDH2-KO cell colony generation to deoxynivalenol was demonstrated by cell viability and cell death assays, which provids insight into the toxic mechanism of deoxynivalenol-induced cell death and offers a new strategy for the prevention and treatment of deoxynivalenol.

Key words: CRISPR/Cas9 system, deoxynivalenol, MDH2 gene, cell survival, cell death

施炜涛, 姚春鹏, 魏文康, 王蕾, 房元杰, 仝钰洁, 马晓姣, 蒋文, 张晓爱, 邵伟. 利用CRISPR/Cas9技术构建MDH2敲除细胞株及抗呕吐毒素效应研究[J]. 生物技术通报, 2023, 39(7): 307-315.

SHI Wei-tao, YAO Chun-peng, WEI Wen-Kang, WANG Lei, FANG Yuan-jie, TONG Yu-jie, MA Xiao-jiao, JIANG Wen, ZHANG Xiao-ai, SHAO Wei. Establishment of MDH2 Knockout Cell Line Using CRISPR/Cas9 Technology and Study of Anti-deoxynivalenol Effect[J]. Biotechnology Bulletin, 2023, 39(7): 307-315.

图/表 10

表1 MDH2靶向位点及sgRNA寡核苷酸序列

Table 1 MDH2 targeting sites and sgRNA oligonucleotide sequences

引物Primer	序列 Sequence（5' -3'）
sgRNA-MDH2-F	CACCGCCCGTCATTGGCGGCCACGC
sgRNA-MDH2-R	AAACGCGTGGCCGCCAATGACGGGC

表2 MDH2基因PCR测序引物

Table 2 Primers for PCR sequencing of MDH2 gene

引物Primer	序列Sequence（5'-3'）
PCR-F	CCGTCTTCAGTCCGTCGTG
PCR-R	TAACCAGCGGTGCTCCAGTG

表3 MDH2基因RT-qPCR引物

Table 3 RT-qPCR primers for MDH2 gene

引物Primer	序列Sequence（5'-3'）
RT-qPCR-F	GGCCACGCCGGGAAG
RT-qPCR-R	GATGCCCTTTTTCCCCAGCA

图1 重组质粒PX459-sgRNA-MDH2测序峰图

Fig. 1 Sequencing peak of recombinant plasmid PX459-sgRNA-MDH2

图2 MDH2-KO细胞系CRISPR靶位点测序峰图

Fig. 2 Sequencing peaks of CRISPR target sites in MDH2-KO cell line

图3 MDH2野生型IPEC-J2细胞系CRISPR靶位点测序峰图

Fig. 3 Sequencing peaks of CRISPR target sites in MDH2 wild-type IPEC-J2 cell line

图4 野生型IPEC-J2细胞与MDH2-KO序列比对图

Fig. 4 Sequence comparison of wild-type IPEC-J2 cells with MDH2-KO

图5 MDH2-KO基因的表达检测 A: RT-qPCR检测MDH2的表达水平。B: Western blot鉴定MDH2蛋白的表达水平

Fig. 5 Expression assay of MDH2-KO gene A: RT-qPCR to detect the expression of MDH2. B: Western blot to identify the expressionof MDH2 protein

图6 CCK8检测MDH2基因敲除对IPEC-J2细胞抗DON毒素效应的影响

Fig. 6 CCK8 detects the effect of MDH2 knockdown on the anti-DON toxin effect in IPEC-J2 cells

图7 流式检测DON处理后IPEC-J2和MDH2-KO的细胞死亡率

Fig. 7 Flow assay of cell mortality of IPEC-J2 and MDH2-KO after DON treatment

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利用CRISPR/Cas9技术构建MDH2敲除细胞株及抗呕吐毒素效应研究

Establishment of MDH2 Knockout Cell Line Using CRISPR/Cas9 Technology and Study of Anti-deoxynivalenol Effect

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