POR基因敲除、回补及过表达LO2细胞株的构建及作为AFB1染毒模型的初步应用

doi:10.3969/j.issn.1004-1524.20230325

浙江农业学报 ›› 2024, Vol. 36 ›› Issue (2): 272-283.DOI: 10.3969/j.issn.1004-1524.20230325

POR基因敲除、回补及过表达LO2细胞株的构建及作为AFB₁染毒模型的初步应用

王琳¹^,²(), 袁建林¹^,², 缪昌¹^,², 马玉晗¹^,², 曹三杰¹^,², 赵勤¹^,²^,^*()

1.四川农业大学动物医学院猪病研究中心,四川成都 611130
2.农业农村部兽用药物与兽医诊断技术四川科学观测站,四川成都 611130

收稿日期:2023-03-14 出版日期:2024-02-25 发布日期:2024-03-05
作者简介:王琳(1998—),女,四川广安人,硕士研究生,研究方向为预防兽医学。E-mail:3462582036@qq.com
通讯作者: *赵勤,E-mail:zhao.qin@sicau.edu.cn
基金资助:
四川省“十四五”川猪重大科技专项(2021ZDZX0010)

Construction of POR gene knockout, complementation and overexpression LO2 cell lines and preliminary application as AFB₁ exposed model

WANG Lin¹^,²(), YUAN Jianlin¹^,², MIAO Chang¹^,², MA Yuhan¹^,², CAO Sanjie¹^,², ZHAO Qin¹^,²^,^*()

1. Research Center for Swine Diseases, College of Veterinary Medicine, Sichuan Agricultural University, Chengdu 611130, China
2. Sichuan Science-Observation Experimental Station of Veterinary Drugs and Veterinary Diagnostic Technique, Ministry of Agriculture and Rural Affairs, Chengdu 611130, China

Received:2023-03-14 Online:2024-02-25 Published:2024-03-05

摘要/Abstract

摘要：

为了构建POR基因稳定敲除(POR^KO)、回补(POR^CO)、过表达(POR^OE)的LO2细胞株,以便为后续深入开展POR基因在AFB₁致毒性机理中的功能研究提供细胞模型。本研究首先利用CRISPR/Cas9技术设计2条靶向POR基因的sgRNA并克隆至lentiCRISPR v2载体,进行慢病毒包装与感染,筛选出LO2 POR^KO单克隆细胞后进行测序及Western blot鉴定。其次用同源重组方法构建pcDNA3.1(+)/myc-His B-POR重组质粒,经对POR^KO细胞和野生型细胞转染分别构建LO2 POR^CO及LO2 POR^OE细胞株,以G-418筛选后进行Western blotting鉴定。再用4 μmol·L^-1及8 μmol·L^-1 AFB₁分别对野生型、LO2 POR^KO、LO2 POR^CO、LO2 POR^OE细胞株染毒48 h,观察细胞形态并测定细胞存活率。结果表明,靶向exon5的sgRNA-2可成功敲除POR基因,得到LO2 POR^KO细胞株;成功构建表达POR重组蛋白的LO2 POR^CO及LO2 POR^OE细胞株。对AFB₁处理后的各细胞存活率测定结果表明,相较于野生型LO2细胞43.66%±0.58%和27.90%±0.46%的存活率,LO2 POR^KO细胞存活率具有极显著性差异,均为100%(P<0.000 1);LO2 POR^CO细胞存活率仍有显著性差异,分别为57.07%±0.74%和42.54%±0.68%(P<0.05);LO2 POR^OE细胞存活率则显著降低,分别为24.58%±0.92%和17.99%±0.81%(P<0.01)。综上所述,本研究成功构建了LO2 POR^KO、LO2 POR^CO、LO2 POR^OE细胞株,并以AFB₁染毒处理分别研究了各细胞株的存活率和细胞形态学变化,发现POR基因对AFB₁所致细胞毒性的影响巨大。研究结果为后续深入开展POR基因在AFB₁致毒性机理中的功能研究奠定了基础。

关键词: POR, CRISPR/Cas9, LO2细胞, AFB₁

Abstract:

To construct three LO2 cell lines separately with POR gene stable knockout (POR^KO), complementation (POR^CO), and overexpression (POR^OE), and provide cell models for further in-depth research on the function of the POR gene in the mechanism of AFB₁toxicity. Firstly, two sgRNAs targeting the POR gene were designed using CRISPR/Cas9 technology and cloned into the lentiCRISPR v2 vector, and then lentivirus packaging and virus infection were performed. After screening LO2 POR^KO monoclonal cells, they were sequenced and identified by Western blot. Secondly, the pcDNA3.1(+)/myc-His B-POR recombinant plasmid was constructed by homologous recombination method, and the LO2 POR^CO and LO2 POR^OE cell lines were respectively constructed through cell transfection and were identified by Western blot after screening with G-418. After the wild-type LO2, LO2 POR^KO, LO2 POR^CO, and LO2 POR^OE cell lines were exposed with 4 μmol·L^-1 and 8 μmol·L^-1 AFB₁for 48 h, then the cell morphology was observed, and the cell viability was determined. The results showed that sgRNA-2 could successfully knock out the POR gene to obtain the LO2 POR^KO cell line, and the LO2 POR^CO and LO2 POR^OE cell lines were successfully constructed. After each cell line was exposed to 4 μmol·L^-1 and 8 μmol·L^-1 AFB₁for 48 h, compared with the wild-type LO2 cell viability rate of 43.66%±0.58% and 27.90%±0.46%, the cell viability rate of LO2 POR^KO cells was extremely significant, both 100% (P<0.000 1); the cell viability rate of LO2 POR^CO cells was still significantly different, respectively 57.07%±0.74% and 42.54%±0.68% (P<0.05); the cell viability rate of LO2 POR^OE cells was significantly lower, respectively 24.58%±0.92% and 17.99%±0.81% (P<0.01). In conclusion, this paper successfully constructed LO2 POR^KO, LO2 POR^CO, and LO2 POR^OE cell lines, and studied the cell viability rate and cell morphology changes of normal LO2, LO2 POR^KO, LO2 POR^CO, and LO2 POR^OE cell lines with AFB₁exposure, and discovered that the POR gene has a huge impact on AFB₁-induced cytotoxicity. This paper laid a foundation for the subsequent in-depth research on the function of the POR gene in the mechanism of AFB₁toxicity.

Key words: POR, CRISPR/Cas9, LO2 cells, AFB₁

中图分类号:

S851.31

王琳, 袁建林, 缪昌, 马玉晗, 曹三杰, 赵勤. POR基因敲除、回补及过表达LO2细胞株的构建及作为AFB₁染毒模型的初步应用[J]. 浙江农业学报, 2024, 36(2): 272-283.

WANG Lin, YUAN Jianlin, MIAO Chang, MA Yuhan, CAO Sanjie, ZHAO Qin. Construction of POR gene knockout, complementation and overexpression LO2 cell lines and preliminary application as AFB₁ exposed model[J]. Acta Agriculturae Zhejiangensis, 2024, 36(2): 272-283.

图/表 11

表1 sgRNA及相应引物序列

Table 1 sgRNA and corresponding primer sequences

靶位点 Target	靶位点序列 Target sequence (5'→3')	引物序列 Primer sequence(5'→3')
sgRNA-POR-1	AGAGATCGACAACGCCCTGG	F: ACCGAGAGATCGACAACGCCCTGG R: AAACCCAGGGCGTTGTCGATCTCTC
sgRNA-POR-2	GCCAGAGATCGACAACGCCC	F:GACCGCCAGAGATCGACAACGCCC R:AAACGGGCGTTGTCGATCTCTGGC

表2 POR基因敲除鉴定引物序列

Table 2 POR gene deletion identification primer sequence

靶位点 Target	敲除鉴定引物序列 Primer sequence(5'→3')
sgRNA-POR-1	hPOR-KO1-F: AACCTTGAACAGGCTCAGTCAT hPOR-KO1-R: ATACATGGATCTGGAGTCCCC
sgRNA-POR-2	hPOR-KO2-F: AGCCCCTCCGTGTTGTTA hPOR-KO2-R: AGGGGACATTCTCGTAGTGC

表3 POR同源扩增引物序列

Table 3 POR homologous amplified primer sequence

引物名称 Primer name	引物序列 Primer sequence(5'→3')
POR-F	ctaccggactcagatctcgagATGATCAACATGGGAGACTCCC
POR-R	gtaccgtcgactgcagaattcCTAGCTCCACACGTCCAGGG

图1 lentiCRISPR v2-sgRNA-POR重组质粒构建 A,lentiCRISPR v2质粒信息部分示意图及sgRNA插入位置;B,sgRNA序列及靶位点示意图;C,lentiCRISPR v2-sgRNA重组质粒测序结果。

Fig.1 Construction of lentiCRISPR v2-sgRNA-POR recombinant plasmid A, lentiCRISPR v2 plasmid information partial schematic diagram and sgRNA insertion location; B, sgRNA sequence and target site diagram; C, lentiCRISPR v2-sgRNA recombinant plasmid sequencing results.

图2 LO2 PORKO单克隆细胞系的鉴定 A,LO2 PORKO细胞测序峰图;B,LO2 PORKO细胞序列比对图。

Fig.2 Identification of LO2 PORKO monoclonal cell lines A, LO2 PORKO cell sequencing peak map; B, LO2 PORKO cell sequence comparison map.

图3 pcDNA3.1(+)/myc-His B-POR重组真核表达质粒构建 A,pcDNA3.1(+)/myc-His B质粒部分信息示意图及POR片段插入位点;B,以LO2细胞cDNA为模板扩增的POR同源片段,1为DNA marker,2为目的片段;C,菌落PCR鉴定电泳图,1为DNA marker,2~4为阳性菌落;D,阳性菌落测序比对结果。

Fig.3 Construction of pcDNA3.1(+)/myc-His B-POR recombinant eukaryotic expression plasmid A, pcDNA3.1(+)/myc-His B plasmid partial information schematica and insertion site of POR target fragment; B, POR homologous fragment amplified in LO2 cell cDNA as template,1 is DNA marker,2 is the target fragment; C, PCR electrophoretic map of colony identification, 1 is DNA marker, 2-4 is positive colony; D, Sequencing results of positive colonies.

图4 各细胞系WB鉴定结果以LO2细胞为对照,WB检测LO2 PORKO、LO2 PORCO及LO2 POROE细胞中POR蛋白表达情况,并对LO2 PORKO细胞中氨基酸序列进行比对分析。

Fig.4 Results of WB identification of cell lines With LO2 cells as the control, WB detected the expression of POR protein in LO2 PORKO, LO2 PORCO and LO2 POROE cells, and the amino acid sequences in LO2 PORKO cells were analyzed.

图5 AFB1对野生型LO2细胞剂量效应曲线

Fig.5 The toxic dose-effect curve of AFB1on the relative cell viability of wild-type LO2 cell lines

表4 IC值的统计结果

Table 4 The results of IC values

毒素IC值 Toxin IC value		浓度的95%置信限度 95% confidence limit for concentration/(μmol·L^-1)			毒素IC值 Toxin IC value		浓度的95%置信限度 95% confidence limit for concentration/(μmol·L^-1)
毒素IC值 Toxin IC value		估算 Estimated value	上限 Upper limit	下限 Lower limit	毒素IC值 Toxin IC value		估算 Estimated value		上限 Upper limit		下限 Lower limit
AFB₁	IC₁₀	1.57	0.00	4.44	AFB₁	IC₁₀	0.22	0.02		0.65
24 h	IC₁₅	4.97	0.10	9.37	48 h	IC₁₅	0.41	0.05		1.03
	IC₂₀	11.43	3.34	25.27		IC₂₀	0.66	0.12		1.46
	IC₂₅	23.11	12.68	270.59		IC₂₅	0.97	0.22		1.94
	IC₃₀	42.74	21.06	4.14×10³		IC₃₀	1.36	0.37		2.50
	IC₃₅	74.72	30.47	5.41×10⁴		IC₃₅	1.86	0.61		3.15
	IC₄₀	126.03	42.02	6.14×10⁵		IC₄₀	2.48	0.96		3.92
	IC₄₅	208.04	56.62	6.37×10⁶		IC₄₅	3.27	1.49		4.86
	IC₅₀	339.97	75.45	6.33×10⁷		IC₅₀	4.28	2.26		6.05
	IC₆₀	917.09	133.59	6.62×10⁹		IC₆₀	7.41	5.02		9.89
	IC₇₀	2 704.23	247.31	1.06×10¹²		IC₇₀	13.45	10.07		20.06
	IC₈₀	1.01×10⁴	521.74	5.18×10¹⁴		IC₈₀	27.86	18.96		59.05
	IC₉₀	7.36×10⁴	1.60×10³	5.80×10¹⁸		IC₉₀	83.30	43.69		336.79
	IC₉₉	2.60×10⁴	4.30×10⁴	5.52×10³⁰		IC₉₉	2.12×10³	465.01		6.44×10⁴

图6 AFB1作用48 h对细胞形态特征的影响(100×)

Fig.6 Effect of morphological characteristics on cells after 48 h treated with AFB1under optical microsope (100×)

图7 AFB1处理对细胞存活率的影响 AFB1处理48 h后野生型LO2、LO2 PORKO、LO2 PORCO及LO2 POROE细胞存活率。各浓度下不同细胞株组与各自阴性对照组比较,**,P<0.01;***,P<0.001;ns, P>0.05;各浓度下不同细胞株组与同等浓度AFB1作用下的LO2细胞组相比,*,P<0.05;**,P<0.01;****,P<0.000 1。

Fig.7 Effect of AFB1treatment on cell viability The cell viability of LO2, LO2 PORKO, LO2 PORCO and POROE cells treated with AFB1for 48 hours. Different cell lines at different concentrations were compared with negative control groups, **, P<0.01; ***, P<0.001; ns, P>0.05. Compared with LO2 cell group under the same concentration of AFB1, *, P<0.05; **, P<0.01; ****, P<0.000 1.

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POR基因敲除、回补及过表达LO2细胞株的构建及作为AFB₁染毒模型的初步应用

Construction of POR gene knockout, complementation and overexpression LO2 cell lines and preliminary application as AFB₁ exposed model

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