WO2020006787A1 - Mettl3基因敲除的细胞系及其构建方法与干扰载体 - Google Patents

Mettl3基因敲除的细胞系及其构建方法与干扰载体 Download PDF

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WO2020006787A1
WO2020006787A1 PCT/CN2018/096554 CN2018096554W WO2020006787A1 WO 2020006787 A1 WO2020006787 A1 WO 2020006787A1 CN 2018096554 W CN2018096554 W CN 2018096554W WO 2020006787 A1 WO2020006787 A1 WO 2020006787A1
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mettl3
shrna
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宗鑫
汪以真
赵婧
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Zhejiang University
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Definitions

  • the invention belongs to the field of genetic engineering, and relates to a METTL3 gene knockout cell line, a construction method thereof and an interference vector.
  • M6A methylation modification is mainly located on the adenosine A sixth nitrogen atom of mRNA. It is a dynamically reversible RNA modification. As its methyltransferase, Mettl3 usually forms a complex with Mettl14 to perform the function of writing m6A. . It has been reported that changes in the level of RNA methylation regulated by Mettl3 are involved in the entire process of biological organisms from gene transcription to protein translation. For example, knocking out Mettl3 can significantly improve the proliferation and self-renewal of glioma stem cells or stem-like cells.
  • Mettl3 can mediate the translation of m6A-dependent cir-RNA (Yang et al., 2017); Mettl3 can regulate gene transcription by combining with the promoter region of DNA to act as a gene transcription switch Role (Barbieri et al., 2017); knocking out Mettlel3 can prevent the inflammatory response by preventing the shearing of the key factor of the inflammation-related pathway MyD88 (Feng et al., 2018; Jiang Yizhou et al., 2017); also have articles It is reported that Mettl3 can promote adipogenesis by promoting cell cycle during adipogenic differentiation (Batista et al., 2014). However, its role in fatty acid absorption and transport has not been reported.
  • the present invention provides a METTL3 gene knockout cell line, a construction method thereof, and an interference vector.
  • a method for constructing a METTL3 gene knockout cell line The m6A methyltransferase gene Mettl3 was used to obtain a pig intestinal epithelial cell line that was resistant to LPS stimulation and caused fatty acid malabsorption through a gene editing method.
  • the establishment method includes the following steps:
  • the Mettl3 sequence is shown in SEQ ID No. 1, and the shRNA sequence is shown in SEQ ID No. 2 and SEQ ID NO. 3;
  • step 4 Infect the intestinal epithelial cells with the Mettl3 shRNA lentivirus obtained in step 4), and use puromycin to screen to obtain intestinal epithelial cells whose gene editing knocks out the m6A methyltransferase gene Mettl3.
  • the method for constructing the Mettl3 shRNA lentiviral expression vector is as follows: the shRNA lentivirus expression vector is digested with BbsI, and the gel is recovered to obtain a linear vector; step 2) is used to obtain phosphorylated Mettl3 shRNA, using T4 ligase , Linked to the pRSI9-U6 lentiviral expression vector.
  • An intestinal epithelial cell line resistant to LPS stimulation resulting in fatty acid absorption disorders obtained according to the established method.
  • a gene interference vector targeting the pig-derived m6A methyltransferase gene Mettl3 A gene interference vector targeting the pig-derived m6A methyltransferase gene Mettl3,
  • the interference vector is obtained by designing shRNA against the m6A methyltransferase gene Mettl3 and inserting it into the shRNA lentiviral expression vector to knock out the expression of Mettl3 in intestinal epithelial cells.
  • the invention discloses a new function of the Mettl3 gene, participates in regulating fatty acid absorption and transport in intestinal epithelial cells, and provides a method for improving fatty acid absorption disorders caused by LPS stimulation by means of gene editing.
  • Mettl3 is able to regulate fatty acid absorption in intestinal epithelial cells for the first time. Analysis of transcription level and protein level showed that the expression of Mettl3 in intestinal epithelial cells undergoing gene editing was significantly reduced. Fatty acid absorption function verification experiments show that intestinal epithelial cells undergoing gene editing can tolerate fatty acid absorption disorders caused by LPS stimulation.
  • the invention not only lays a foundation for studying the role of Mettlel3 in the fatty acid absorption process, but also improves the fatty acid absorption disorder caused by LPS stimulation by using a gene editing method.
  • Figure 1 shows the agarose gel electrophoresis of Mettl3 shRNA lentiviral expression vector by PCR identification
  • Figure 2 is a schematic diagram of the pRSI9-U6 lentiviral vector
  • FIG. 3A is a Western-blot diagram of Mettl3 protein expression levels after Mettl3 shRNA lentivirus infects epithelial cells
  • FIG. 3B is the transcription level of intestinal epithelial cells Mettl3 after gene editing; the average value of the data represents 3 biological replicates, and the error bars represent standard errors (SE). Significance analysis * indicates p ⁇ 0.05, ** indicates p ⁇ 0.01.
  • FIG. 4 is a diagram of laser confocal detection of fatty acid absorption by gene-edited intestinal epithelial cells.
  • Figure 5 is a flow cytometry method for detecting fatty acid absorption of gene-edited intestinal epithelial cells.
  • the left part is a graph of fatty acid absorption in different treatment groups, and the right part is a quantitative statistics of the left graph; the average value of the data represents 3 biological replicates . Error bars represent standard error (SE). Significance analysis * indicates p ⁇ 0.05, ** indicates p ⁇ 0.01.
  • Fig. 6 shows the absorption of fatty acids in gene-edited intestinal epithelial cells by ultraviolet spectrometry; the average value of the data represents three biological replicates. Error bars represent standard error (SE). Significance analysis * indicates p ⁇ 0.05, ** indicates p ⁇ 0.01.
  • Fig. 7 shows the effect of the transfer of Mettl3 plasmid on the fatty acid absorption of intestinal epithelial cells in which the Mettl3 gene was knocked out by gene editing.
  • the Mettl3 shRNA in the present invention is an online shRNA design tool: http://www.broadinstitute.org/rnai/public/seq/search, and is designed according to the m6A methyltransferase gene Mettl3; wherein, the Mettl3 gene sequence As shown in SEQ ID NO.1, the shRNA sequence includes the upstream sequence P1 and the downstream sequence P2, as shown below:
  • P1 upstream sequence: ACCGG GAGATCCTAGAACTATTAAAT CTCGAG ATTTAATAGTTCTAG GATCTC TTTTTTG (SEQ ID NO. 2);
  • T4Polynucleotide Kinase was used to react at 37 ° C for 30 minutes, then at 95 ° C for 2 minutes, and then naturally cooled to room temperature to phosphorylate Mettl3 shRNA.
  • the shRNA lentivirus expression vector was digested with BbsI, and gel was recovered to obtain a linear vector. Phosphorylated Mettl3 shRNA was obtained, and T4 ligase was used to incubate at room temperature for 2 hours, and then ligated to the pRSI9-U6 lentivirus expression vector. , Escherichia coli DH5 ⁇ strain was transformed by heat shock method, and the plasmid was extracted for PCR identification. The primer sequence is shown below.
  • the reaction conditions were: pre-denaturation at 94 ° C for 3 min, denaturation at 94 ° C for 15 s, annealing at 56 ° C for 30 s, extension at 72 ° C for 2 min, and 25 reaction cycles. Positive clones were obtained. Group, indicating that the shRNA of Mettl3 was indeed inserted into the pRSI9-U6 lentiviral expression vector as shown in FIG. 2.
  • Intestinal epithelial cells knocked out of the Mettl3 gene by the gene editing method obtained in Example 3 were used, and normal cells (Scramble) were used as controls, and stimulated with 100 ⁇ g / ml lipopolysaccharide LPS for 0 hours, 3 hours, and 6 hours later; added Bodipy-labeled fatty acids were incubated at 37 ° C for 5 min, and the absorption of fatty acids was detected by confocal laser.
  • Example 5 Fatty acid absorption in intestinal epithelial cells by flow cytometry gene editing
  • Intestinal epithelial cells knocked out of the Mettl3 gene by the gene editing method obtained in Example 3 were used, and normal cells (Scramble) were used as controls, and stimulated with 100 ⁇ g / ml lipopolysaccharide LPS for 0 hours, 3 hours, and 6 hours later; added Bodipy labeled fatty acids were incubated at 37 ° C for 5 minutes, and the absorption of fatty acids was detected by flow cytometry (wavelength 488nm).
  • the results showed that intestinal epithelial cells with gene editing knocked out of Mettl3 gene were significantly more resistant to fatty acid malabsorption caused by LPS stimulation than normal cells. As shown in FIG.
  • LPS stimulation was performed on the intestinal epithelial cells (shMettl3) of the control group (Scramble) and Mettl3 gene editing for 0 hours, 3 hours, and 6 hours, respectively.
  • Flow cytometry was used to detect the absorption of fluorescently labeled fatty acids in the cells.
  • IPCE-J2 cells Normal and gene-edited IPCE-J2 cells were planted in gelatin-coated, 96-well, black, transparent bottom plates. After overnight culture, the serum was starved in FBS-free medium for at least 8 hours, and treated with 1 mg / ml LPS. Hours, 3 hours, 6 hours, and then briefly washed with phosphate buffered saline (PBS).
  • PBS phosphate buffered saline
  • BodiPy FA molecular probe ⁇ -D3823
  • BSA bovine serum albumin
  • LPS stimulation was performed on the intestinal epithelial cells (shMettl3) of the control group (Scramble) and Mettl3 gene editing for 0 hours, 3 hours, and 6 hours, respectively.
  • Ultraviolet spectrometry was used to detect the absorption of fluorescently labeled fatty acids in cells.
  • Example 7 Effect of the transfer of Mettl3 plasmid on the fatty acid absorption of intestinal epithelial cells with gene editing knock-out of Mettl3 gene
  • the intestinal epithelial cells knocked out of the Mettl3 gene by the gene editing method obtained in Example 3 were transfected into empty pcDNA3.1 plasmid and Mettl3 plasmid with Lipo2000. After 24 hours, they were stimulated with 100 ⁇ g / ml lipopolysaccharide LPS for 0 hours , 3 hours, 6 hours later; add Bodipy-labeled fatty acids, incubate at 37 ° C for 5 minutes, and measure the absorption of fatty acids by flow cytometry (wavelength 488nm).
  • the results show that compared with the empty vector group, the fatty acid absorption level of the transferred into the Mettlel3 plasmid group was significantly reduced under LPS stimulation, indicating that it is indeed Mettlel3 playing an important role in tolerating fatty acid absorption disorders.
  • the intestinal epithelial cells knocked out of the Mettl3 gene by gene editing were transferred into pcDNA3.1 empty plasmid and Mettl3 plasmid, and then subjected to LPS stimulation treatment for 0 hours, 3 hours, and 6 hours.
  • Laser confocal method was used to detect the absorption of fluorescently labeled fatty acids in cells.

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Abstract

提供了一种METTL3基因敲除的猪肠道上皮细胞系细胞系及其构建方法。还提供了一种针对猪源METTL3的基因干扰载体。

Description

METTL3基因敲除的细胞系及其构建方法与干扰载体 技术领域
本发明属于基因工程领域，涉及一种METTL3基因敲除的细胞系及其构建方法与干扰载体。
背景技术
我国现在普遍采用集约型模式进行养猪生产，生猪在每个生长环节都会受到病原微生物的感染，容易造成仔猪肠道(屏障)功能损伤，导致养分消化吸收障碍，并往往伴随着抵抗力下降和较严重的腹泻。在主要养分中，又以作为主要能量物质的脂肪酸的吸收利用影响较大。针对这一现象，目前主要采取抗病育种和长期高剂量添加药物两方面的措施。由于抗病育种世代周期长和普及面小等问题难以大范围推广，因此目前普遍采用的措施是长期高剂量添加饲用抗生素和氧化锌，然而其存在的问题也是显而易见的：极易引发细菌耐药性、肉产品残留、扰乱动物肠道微生态区系平衡、环境污染。因此寻求解决这一问题的新方法迫在眉睫。
m6A甲基化的修饰主要位于mRNA的腺苷A第六号氮原子上，是一种动态可逆的RNA修饰，Mettl3作为其甲基转移酶，通常和Mettl14形成复合物，执行写入m6A的功能。有文章报道，Mettl3所调控的RNA甲基化水平变化参与到生物机体从基因的转录到蛋白质的翻译的整个过程，如敲除Mettl3能够显著提高神经胶质瘤干细胞或类干细胞的增殖和自我更新(Cui et al.,2017)；Mettl3能够介导m6A依赖的cir-RNA的翻译(Yang et al.,2017)；Mettl3能够通过与DNA启动子区域相结合，调控基因转录，发挥着基因转录开关的作用(Barbieri et al.,2017)；敲除Mettl3能够通过阻止炎症相关通路关键因子MyD88的剪切，进而阻止炎症反应的发生(Feng et al.,2018；江一舟等，2017)；同时有文章报道Mettl3在成脂分化过程中能够通过促进细胞周期进而促进脂肪生成(Batista et al.,2014)。但是其在脂肪酸吸收转运过程中的作用，尚未报道。
发明内容
为了克服现有技术的不足，本发明提供了一种METTL3基因敲除的细胞系及其构建方法与干扰载体。
一种METTL3基因敲除的细胞系的构建方法，利用m6A甲基转移酶基因Mettl3在通过基因编辑方法获得耐受LPS刺激导致脂肪酸吸收障碍的猪肠道上皮细胞系。
所述的建立方法，包括以下步骤：
1)根据所述的m6A甲基转移酶基因Mettl3设计shRNA，
所述的Mettl3序列如SEQ ID NO.1所示，所述的shRNA序列如SEQ ID NO.2、SEQ ID NO.3所示；
2)针对m6A甲基转移酶基因Mettl3 shRNA进行磷酸化；
3)将磷酸化的Mettl3 shRNA连接到慢病毒表达载体获得Mettl3 shRNA慢病毒表达载体；
4)以293A细胞作为受体，将步骤3)获得的Mettl3 shRNA慢病毒表达载体进行慢病毒包被；
5)将步骤4)获得的Mettl3 shRNA慢病毒侵染肠道上皮细胞，利用嘌呤霉素进行筛选，获得基因编辑敲除m6A甲基转移酶基因Mettl3的肠道上皮细胞。
所述的Mettl3 shRNA慢病毒表达载体的构建方法具体如下：对shRNA慢病毒表达载体利用BbsI进行酶切，并进行胶回收获得线性载体；以步骤2)获得磷酸化的Mettl3 shRNA，采用T4连接酶，连接到pRSI9-U6慢病毒表达载体。
一种根据所述的建立方法得到的耐受LPS刺激导致脂肪酸吸收障碍的肠道上皮细胞系。
一种针对猪源m6A甲基转移酶基因Mettl3的基因干扰载体，
所述的干扰载体是将针对所述的m6A甲基转移酶基因Mettl3设计shRNA，插入到shRNA慢病毒表达载体所得，用于敲除肠道上皮细胞内Mettl3的表达。
本发明的有益效果为：
本发明公开了Mettl3基因的一种新功能，参与调控肠道上皮细胞脂肪酸吸收转运，并提供了通过基因编辑手段改善LPS刺激导致的脂肪酸吸收障碍的方法。Mettl3能够调控肠道上皮细胞脂肪酸吸收为首次报道。转录水平和蛋白水平分析表明进行基因编辑的肠道上皮细胞Mettl3表达量显著降低。脂肪酸吸收功能验证实验表明进行基因编辑的肠道上皮细胞能够耐受LPS刺激导致的脂肪酸吸收障碍。本发明不仅为研究Mettl3在脂肪酸吸收过程中的作用奠定基础，还利用基因编辑的方法改善了LPS刺激导致的脂肪酸吸收障碍。
附图说明
图1为PCR鉴定Mettl3 shRNA慢病毒表达载体琼脂糖凝胶电泳图(Agarose gel electrophoresis)；
图2为pRSI9-U6慢病毒载体的示意图；
图3A为Mettl3 shRNA慢病毒侵染上皮细胞之后Mettl3的蛋白表达水平Western-blot图；
图3B为基因编辑后的肠道上皮细胞Mettl3的转录水平；数据平均值代表3个生物学重复，误差线表示标准误差(SE)。显著性分析*表示p<0.05，**表示p<0.01。
图4为激光共聚焦检测基因编辑肠道上皮细胞的脂肪酸吸收情况图。
图5为流式细胞法检测基因编辑肠道上皮细胞的脂肪酸吸收情况，左部分是不同处理组脂肪酸的吸收情况图，右部分是对左图的量化统计；数据平均值代表3个生物学重复。误差线表示标准误差(SE)。显著性分析*表示p<0.05，**表示p<0.01。
图6为紫外分光法检测基因编辑肠道上皮细胞的脂肪酸吸收情况；数据平均值代表3个生物学重复。误差线表示标准误差(SE)。显著性分析*表示p<0.05，**表示p<0.01。
图7为转入Mettl3质粒对基因编辑敲除Mettl3基因的肠道上皮细胞脂肪酸吸收的影响。
具体实施方式
以下结合附图和实施例对本发明做进一步的阐述。
实施例1 Mettl3 shRNA慢病毒表达载体的获得
本发明中的Mettl3 shRNA是利用在线的shRNA设计工具：http://www.broadinstitute.org/rnai/public/seq/search，根据所述的m6A甲基转移酶基因Mettl3设计；其中，Mettl3基因序列如如SEQ ID NO.1所示，shRNA序列包括上游序列P1，下游序列P2，如下所示：
P1(上游序列)：ACCGG GAGATCCTAGAACTATTAAAT CTCGAG ATTTAATAGTTCTAG GATCTC TTTTTTG(SEQ ID NO.2)；
P2(下游序列):CGAACAAAAAA GAGATCCTAGAACTATTAAAT CTCGAG ATTTAATAGTTCTAG GATCTC C(SEQ ID NO.3)。
并利用T4Polynucleotide Kinase，37℃反应30分钟后，95℃反应2分钟，然后自然冷却至室温，对Mettl3 shRNA进行磷酸化。
对shRNA慢病毒表达载体利用BbsI进行酶切，并进行胶回收获得线性载体；以获得磷酸化的Mettl3 shRNA，采用T4连接酶，室温孵育2小时，将其连接到pRSI9-U6慢病毒表达载体上，通过热激法转化大肠杆菌DH5α菌株，提取质粒进行PCR鉴定，引物序列如下所示，
Forward:5`-GAGGGCCTATTTCCCATGATTCC-3`(SEQ ID NO.4)，
Reverse:5`-ACAGTCCGAAACCCCAAACGCACGAA-3`(SEQ ID NO.5)。
反应条件为反应条件94℃预变性3min，94℃变性15s，56℃退火30s，72℃延伸2min，25个反应循环，获得阳性克隆，如图1所示，所挑克隆PCR产物均大于阴性对照组，表明Mettl3的shRNA确实插入到如图2所示的pRSI9-U6慢病毒表达载体上。
实施例2 Mettl3 shRNA慢病毒的包被
将2μg实施例1中的方法获得的Mettl3 shRNA慢病毒表达质粒，利用脂质体Lipo2000，同时转入慢病毒元件0.25μg VsVg和1μg PsPax；24小时后换成1.5ml完全培养基；48小时后收集病毒，获得Mettl3 shRNA的慢病毒。
实施例3 基因编辑肠道上皮细胞系的获得
将500μl实施例2中的方法获得的Mettl3 shRNA慢病毒加入到肠道上皮细胞IPEC-J2中，24小时后换成完全培养基，48小时后加入4μg/ml嘌呤霉素进行筛选，至通过荧光定量PCR及Western-blot法验证获得基因编辑敲除Mettl3基因的肠道上皮细胞。如图3A-3B所示，与空载体组相比，Mettl3 shRNA慢病毒侵染组的肠道上皮细胞Mettl3的表达水平显著降低。
实施例4 激光共聚焦法分析基因编辑肠道上皮细胞的脂肪酸吸收情况
用实施例3中的方法获得的基因编辑敲除Mettl3基因的肠道上皮细胞，以正常细胞(Scramble)作为对照，分别用100μg/ml脂多糖LPS刺激0小时，3小时，6小时后；加入Bodipy标记的脂肪酸，37℃孵育5min，激光共聚焦检测脂肪酸的吸收情况。结果表明基因编辑敲除Mettl3基因的肠道上皮细胞对LPS刺激导致的脂肪酸吸收障碍的耐受显著高于正常细胞，如图4所示，分别针对对照组和Mettl3基因编辑后的肠道上皮细胞进行LPS刺激处理，结果表明，对照组在LPS刺激条件下，脂肪酸的吸收明显受阻，但是Mettl3基因编辑后的肠道上皮细胞则表面出明显的耐受。
实施例5 流式细胞分析法基因编辑肠道上皮细胞的脂肪酸吸收情况
用实施例3中的方法获得的基因编辑敲除Mettl3基因的肠道上皮细胞，以正常细胞(Scramble)作为对照，分别用100μg/ml脂多糖LPS刺激0小时，3小时，6小时后；加入Bodipy标记的脂肪酸，37℃孵育5min，流式细胞仪(波长488nm)检测脂肪酸的吸收情况。结果表明基因编辑敲除Mettl3基因的肠道上皮细胞对LPS刺激导致的脂肪酸吸收障碍的耐受显著高于正常细胞。如图5所示，分别针对对照组(Scramble)和Mettl3基因编辑后的肠道上皮细胞(shMettl3)进行LPS刺激处理0小时，3小时，6小时。流式细胞法检测细胞荧光标记脂肪酸的吸收情况。
实施例6 紫外分光法检测基因编辑肠道上皮细胞的脂肪酸吸收情况
分别将正常的和基因编辑的IPCE-J2细胞种植明胶包被、96孔、黑色、透明底板，过夜培 养后，用无FBS培养基对血清进行饥饿培养至少8小时，用1mg/ml LPS处理0小时、3小时、6小时，然后用磷酸盐缓冲盐水(PBS)进行短暂洗涤。同时在PBS中加入BodiPy FA(分子探针γ-D3823)和无脂肪酸牛血清白蛋白(BSA)(2:1摩尔比)在37℃水浴中预孵育10分钟；加入细胞后在37℃下孵育5分钟，用加入0.5％BSA的PBS洗涤2次，每次2min，为了抑制细胞外荧光，加入0.4％台盼蓝(每孔50uL)，并用紫外分光光度计立即测量细胞内荧光(激发488nm，发射515nm，cut-off 495nm)。如图6所示，分别针对对照组(Scramble)和Mettl3基因编辑后的肠道上皮细胞(shMettl3)进行LPS刺激处理0小时，3小时，6小时。紫外分光法检测细胞荧光标记脂肪酸的吸收情况。
实施例7 转入Mettl3质粒对基因编辑敲除Mettl3基因的肠道上皮细胞脂肪酸吸收的影响
用实施例3中的方法获得的基因编辑敲除Mettl3基因的肠道上皮细胞，用Lipo2000分别转入pcDNA3.1空质粒和Mettl3质粒，24小时后，分别用100μg/ml脂多糖LPS刺激0小时，3小时，6小时后；加入Bodipy标记的脂肪酸，37℃孵育5min，流式细胞仪(波长488nm)检测脂肪酸的吸收情况。结果表明相比于空载体组，转入Mettl3质粒组在LPS刺激下脂肪酸吸收水平显著降低，表明确实是Mettl3在耐受脂肪酸吸收障碍中发挥着重要作用。如图7所示，对基因编辑敲除Mettl3基因的肠道上皮细胞转入pcDNA3.1空质粒和Mettl3质粒后，进行LPS刺激处理0小时，3小时，6小时。激光共聚焦法检测细胞荧光标记脂肪酸的吸收情况。

Claims (5)

  1. 一种METTL3基因敲除的细胞系的构建方法，其特征在于，利用m6A甲基转移酶基因Mettl3在通过基因敲除方法获得耐受LPS刺激导致脂肪酸吸收障碍的猪肠道上皮细胞系。
  2. 根据权利要求1所述的建立方法，其特征在于，包括以下步骤：
    1)根据所述的m6A甲基转移酶基因Mettl3设计shRNA，
    所述的Mettl3序列如SEQ ID NO.1所示，所述的shRNA序列如SEQ ID NO.2、SEQ ID NO.3所示；
    2)针对m6A甲基转移酶基因Mettl3 shRNA进行磷酸化；
    3)将磷酸化的Mettl3 shRNA连接到慢病毒表达载体获得Mettl3 shRNA慢病毒表达载体；
    4)以293A细胞作为受体，将步骤3)获得的Mettl3 shRNA慢病毒表达载体进行慢病毒包被；
    5)将步骤4)获得的Mettl3 shRNA慢病毒侵染肠道上皮细胞，利用嘌呤霉素进行筛选，获得基因编辑敲除m6A甲基转移酶基因Mettl3的肠道上皮细胞。
  3. 根据权利要求1所述的建立方法，其特征在于，所述的Mettl3 shRNA慢病毒表达载体的构建方法具体如下：对shRNA慢病毒表达载体利用BbsI进行酶切，并进行胶回收获得线性载体；以步骤2)获得磷酸化的Mettl3 shRNA，采用T4连接酶，连接到pRSI9-U6慢病毒表达载体。
  4. 一种根据权利要求1所述的建立方法得到的耐受LPS刺激导致脂肪酸吸收障碍的猪肠道上皮细胞系。
  5. 一种针对猪源m6A甲基转移酶基因Mettl3的基因干扰载体，其特征在于，
    所述的干扰载体是将针对所述的m6A甲基转移酶基因Mettl3设计shRNA，插入到shRNA慢病毒表达载体所得，用于敲除肠道上皮细胞内Mettl3的表达。
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