阅读说明：本技术 Map3k8基因敲除pk-15细胞系的构建方法及其应用 (Construction method and application of PK-15 cell line knocked out by MAP3K8 gene ) 是由 郑海学 张克山 闫鸣昊 郝军红 �田宏 李丹 朱紫祥 茹毅 杨帆 张大俊 刘湘涛 于 2019-10-30 设计创作，主要内容包括：本发明属于生物技术领域,具体涉及MAP3K8基因敲除PK-15细胞系的构建方法及其应用。MAP3K8基因敲除的PK-15细胞系PK-15-KO-MAP3K8的保藏编号为CCTCC NO:C2019176。该细胞系的构建方法包括1：根据猪源MAP3K8基因序列分别设计两条sgRNA,将双链sg RNA与酶切后的lentiGuide-EGFP载体连接得到重组慢病毒表达质粒lentiGuide-EGFP-MAP3K8-sg RNA；2：将重组慢病毒表达质粒、病毒包装辅助质粒共转染PK-15细胞；3：收集病毒液,过滤、超速离心浓缩纯化病毒；4：用感染复数MOI=30的慢病毒感染PK-15细胞,然后用超速流式细胞分选系统进行单细胞分选；5：将分选得到的单克隆细胞进行扩大培养并测序鉴定。该细胞系能够促进FMDV和SVV增殖,提高病毒产量,可用于FMDV和SVV疫苗株的大规模细胞化培养和生产,并可为研究MAP3K8在病毒感染过程中的作用机制提供有力工具。(The invention belongs to the technical field of biology, and particularly relates to a construction method and application of a PK-15 cell line knocked out by a MAP3K8 gene. The preservation number of the PK-15 cell line PK-15-KO-MAP3K8 with the MAP3K8 gene knockout is CCTCC NO: C2019176. The construction method of the cell line comprises the following steps of 1: respectively designing two sgRNAs according to a swine MAP3K8 gene sequence, and connecting the double-stranded sg RNA with a digested lentiGuide-EGFP vector to obtain a recombinant lentivirus expression plasmid lentiGuide-EGFP-MAP3K8-sg RNA; 2: co-transfecting the recombinant lentivirus expression plasmid and the virus packaging helper plasmid to a PK-15 cell; 3: collecting virus liquid, filtering, ultracentrifuging, concentrating and purifying virus; 4: infecting PK-15 cells with lentiviruses with multiplicity of infection MOI =30, and then performing single cell sorting with an ultra-speed flow cytometric sorting system; 5: and carrying out amplification culture and sequencing identification on the sorted monoclonal cells. The cell line can promote the proliferation of FMDV and SVV, improve the virus yield, can be used for large-scale cell culture and production of FMDV and SVV vaccine strains, and can provide a powerful tool for researching the action mechanism of MAP3K8 in the virus infection process.)

1. The PK-15 cell line PK-15-KO-MAP3K8 with the MAP3K8 gene knocked out is characterized in that the preservation number of the PK-15 cell line PK-15-KO-MAP3K8 is CCTCC NO: C2019176.

2. The construction method of the PK-15 cell line PK-15-KO-MAP3K8 with the MAP3K8 gene knocked out is characterized by comprising the following steps:

step 1: primer design and plasmid construction: respectively designing two sgRNAs according to a swine MAP3K8 gene sequence, and connecting double-stranded sg RNA with a digested lentiGuide-EGFP vector to obtain a recombinant lentivirus expression plasmid lentiGuide-EGFP-MAP3K8-sg RNA;

step 2: plasmid transfection: co-transfecting lentiGuide-EGFP-MAP3K8-sg RNA lentivirus expression plasmid and virus packaging auxiliary plasmid into PK-15 cells;

and step 3: and (3) slow virus concentration: collecting virus liquid, filtering, ultracentrifuging, concentrating and purifying virus, and storing for later use;

and 4, step 4: lentivirus infection and flow sorting: infecting PK-15 cells with lentiviruses with multiplicity of infection MOI =30, and then performing single cell sorting with an ultra-speed flow cytometric sorting system;

and 5: culturing and sequencing identification of monoclonal cells: and carrying out amplification culture and sequencing on the single cell obtained by sorting to identify the single cell clone.

3. The method for constructing a MAP3K8 gene knock-out PK-15 cell line PK-15-KO-MAP3K8 as claimed in claim 2, wherein the sequence of sgRNA is as follows:

P-MAP3K8-sgRNA1 Forward：5’-CACCGTTCCATAATGTCTATTACAT-3’，

P-MAP3K8-sgRNA1Reverse：5’-AAACATGTAATAGACATTATGGAAC-3’；

P-MAP3K8-sgRNA2Forward：5’-CACCGAGATCCCAGATTCCTGGGGT-3’，

P-MAP3K8-sgRNA2 Reverse: 5'-AAACACCCCAGGAATCTGGGATCTC-3', respectively; the lentiGuide-EGFP vector in step 1 was digested with BsmBI I enzyme and ligated to sg RNA.

4. The PK-15 cell line PK-15-KO-MAP3K8 with the MAP3K8 gene knocked out is applied to the research of the action mechanism of virus infection.

5. The use of the MAP3K8 gene knock-out PK-15 cell line PK-15-KO-MAP3K8 of claim 4 for studying the mechanism of action of a viral infection, wherein said virus is FMDV or SVV.

6. Use of MAP3K8 gene knock-out PK-15 cell line PK-15-KO-MAP3K8 for isolating and culturing FMDV or SVV.

7. The PK-15 cell line PK-15-KO-MAP3K8 with the MAP3K8 gene knocked out is applied to large-scale cell culture and production of FMDV or SVV vaccine strains.

Technical Field

The invention belongs to the technical field of biology, and particularly relates to a construction method and application of a PK-15 cell line knocked out by a MAP3K8 gene.

Background

The porcine kidney epithelial cell PK-15 is derived from porcine kidney and is named as porcine kidney epithelial cell in Chinese. The cell is sensitive to various viruses, such as Porcine Circovirus (PCV), Porcine Parvovirus (PPV), Classical Swine Fever Virus (CSFV) and the like, and can be applied to the preparation of porcine circovirus vaccines, porcine parvovirus vaccines, classical swine fever virus vaccines and the like.

TPL2 is a serine/threonine kinase, also known as COT or MAP3K8, and TPL2 forms a complex with p105 and ABIN2 to remain inactive when unstimulated (Gantke, T., s.sriskharajah, and s.c. Ley, Regulation and function of TPL-2, an ikappa B kinase-regulated kinase. cell Res,2011.21(1): p.131-45.) when stimulated, it can activate multiple signal transduction pathways such as downstream ERK, JNK, p38 and NF-kb through multiple receptors such as TLR, TNFR and IL1R, and it can also stimulate multiple innate immune cells such as macrophages, keratinocytes and TNF- α to produce a number of cytokines such as IFN- γ, TNF- α (gante T, s.s, Sadowski 2, and l2, which are essential for the Regulation of tumor production of tumors of TPL 734 + T β T + T493 β + T β + T β T3K 493, T.

The CRISPR-Cas9 gene editing technology is a third generation genome editing technology that has been rapidly developed following ZFN and TALEN technologies. The technology comes from a CRISPR-Cas acquired immune system resisting phage invasion existing in bacteria and archaea, and is gradually developed through artificial modification (development and application of CRISPR/Cas9 technology of Sichuan university of agriculture [ N ] scientific report, 2019-08-20 (B02)). Bacteria, with the help of CRISPR and Cas9, can target and silence key parts of invader's genetic information via the guidance of small RNA molecules. The CRISPR-Cas9 genome editing technology is that a target gene sequence is specifically recognized by a gRNA, a Cas9 endonuclease is guided to cut double-stranded DNA at a targeted site, and then a non-homologous end joining repair mechanism (NHEJ) of a cell rejoins the genomic DNA at a break and introduces an insertion or deletion mutation. Compared with ZFN and TALEN technologies, the CRISPR-Cas9 system has higher gene editing efficiency, and the Cas9 system is more convenient to construct and use, has been applied to various species and is the most widely used gene editing technology at present.

Disclosure of Invention

The invention constructs a PK-15 cell line PK-15-KO-MAP3K8 for knocking out MAP3K8 gene by using CRISPR-Cas9 gene editing technology, and the cell line can promote the proliferation of Foot and Mouth Disease Virus (FMDV) and Sernica Valley Virus (SVV) and improve the virus yield. Can be used for large-scale cell culture and production of FMDV and SVV vaccine strains, and can provide a powerful tool for researching the action mechanism of MAP3K8 in the virus infection process.

In a first aspect, the preservation number of the PK-15 cell line PK-15-KO-MAP3K8 with the MAP3K8 gene knocked out is CCTCC NO: C2019176.

In a second aspect, the invention also provides a construction method of the PK-15 cell line PK-15-KO-MAP3K8 with the MAP3K8 gene knocked out, which specifically comprises the following steps:

step 1: primer design and plasmid construction: respectively designing two sgRNAs according to a swine MAP3K8 gene sequence, and connecting double-stranded sg RNA with a digested lentiGuide-EGFP vector to obtain a recombinant lentivirus expression plasmid lentiGuide-EGFP-MAP3K8-sg RNA;

specifically, double-stranded sg RNA was ligated to the digested lentiGuide-EGFP vector using T4 DNA ligase.

Step 2: plasmid transfection: co-transfecting lentiGuide-EGFP-MAP3K8-sg RNA lentivirus expression plasmid and virus packaging auxiliary plasmid into PK-15 cells;

specifically, the transfection procedure was performed according to the Lipofectamine 2000 transfection reagent instructions.

Before transfection, PK-15 cells were cultured in DMEM medium supplemented with 10% fetal bovine serum, 0.2mg/mL streptomycin, and 200IU/mL penicillin.

And step 3: and (3) slow virus concentration: collecting virus liquid, filtering, ultracentrifuging, concentrating and purifying virus, and storing for later use.

Lentivirus titer determination before lentivirus infection: PK-15 cells were infected after virus stock solution was diluted in a gradient, and virus titer was calculated.

And 4, step 4: lentivirus infection and flow sorting: PK-15 cells were infected with lentivirus with a multiplicity of infection MOI of 30 and single cell sorting was performed using an ultra-speed flow cytometric sorting system.

And 5: culturing and sequencing identification of monoclonal cells: and carrying out amplification culture and sequencing on the single cell obtained by sorting to identify the single cell clone.

Specifically, the expansion culture is as follows: and (3) inoculating the sorted monoclonal cells into a 96-well plate, carrying out passage to a 48-well plate after the monoclonal cells grow to full, and sequentially carrying out amplification culture to a 24-well plate, a 12-well plate, a 6-well plate and a T25 culture bottle.

Further, the sequence of the sgRNA is as follows:

P-MAP3K8-sgRNA1 Forward：5’-CACCGTTCCATAATGTCTATTACAT-3’，

P-MAP3K8-sgRNA1Reverse：5’-AAACATGTAATAGACATTATGGAAC-3’；

P-MAP3K8-sgRNA2Forward：5’-CACCGAGATCCCAGATTCCTGGGGT-3’，

P-MAP3K8-sgRNA2Reverse：5’-AAACACCCCAGGAATCTGGGATCTC-3’；

the lentiGuide-EGFP vector in step 1 was digested with BsmBI I enzyme and ligated to sg RNA.

In a third aspect, the invention provides application of a PK-15 cell line PK-15-KO-MAP3K8 with a MAP3K8 gene knocked out in the research of the action mechanism of virus infection.

Further, the virus is FMDV or SVV.

In a fourth aspect, the invention provides the use of a MAP3K8 gene knock-out PK-15 cell line PK-15-KO-MAP3K8 for the isolation and culture of FMDV or SVV.

In a fifth aspect, the invention provides application of a PK-15 cell line PK-15-KO-MAP3K8 with a MAP3K8 gene knockout gene in large-scale cell culture and production of FMDV or SVV vaccine strains.

The invention has the following beneficial effects:

1. the PK-15 cell line PK-15-KO-MAP3K8 for knocking out MAP3K8 gene is constructed by using a CRISPR-Cas9 system, and the establishment of the cell line provides a powerful tool for researching the action mechanism of MAP3K8 in the virus infection process.

2. The PK-15 cell line PK-15-KO-MAP3K8 with the MAP3K8 gene knocked out cannot correctly express the MAP3K8 protein due to frame shift mutation caused by insertion or deletion of certain fragments. Since the MAP3K8 protein has an antiviral effect, FMDV virus and SVV virus cannot proliferate in large amounts in wild-type PK-15 cells. And the PK-15 cell line PK-15-KO-MAP3K8 with the MAP3K8 gene knocked out cannot correctly express the MAP3K8 protein, so that FMDV virus or SVV virus can be propagated in the cell line to obtain larger high-titer virus, and the method can be used for large-scale cell culture and production of FMDV or SVV vaccine strains.

Drawings

FIG. 1 is a structural diagram of lentiGuide-EGFP-MAP3K8-sg RNA lentivirus expression vector constructed in the present invention. a is lentiGuide-EGFP-MAP3K8-sg RNA1, and b is lentiGuide-EGFP-MAP3K8-sg RNA 2.

FIG. 2 is a diagram of transfection of PK-15 cells with lentiGuide-EGFP-MAP3K8-sg RNA lentiviral expression vector under a fluorescence microscope (transmission light and fluorescence detection, X200).

FIG. 3 is a diagram showing the analysis of insertion and deletion mutation types of PK-15-KO-MAP3K8 cell lines #1, #19, #20 clone MAP3K 8. a is clone #1, b is clone #19, and c is clone # 20.

FIG. 4 is a graph of detecting the abundance of MAP3K8 protein in PK-15-WT-MAP3K8 and PK-15-KO-MAP3K8 cells by Western blotting.

FIG. 5 is a graph of absolute quantitation of FMDV in PK-15-WT-MAP3K8 and PK-15-KO-MAP3K8 cells in accordance with the present invention.

FIG. 6 is a graph showing the absolute quantitative determination of the viral copy number of SVV in PK-15-WT-MAP3K8 and PK-15-KO-MAP3K8 cells in accordance with the present invention.

FIG. 7 is a graph showing the relative quantitative determination of the transcript levels of FMDV replication in PK-15-WT-MAP3K8 and PK-15-KO-MAP3K8 cells in accordance with the present invention.

FIG. 8 is a graph showing the relative quantification of the transcript levels of SVV replication in PK-15-WT-MAP3K8 and PK-15-KO-MAP3K8 cells in accordance with the present invention.

FIG. 9 is a graph showing the abundance of proteins replicated by Western blotting to detect FMDV in PK-15-WT-MAP3K8 and PK-15-KO-MAP3K8 cells.

FIG. 10 is a graph showing the abundance of proteins replicated by SVV in PK-15-WT-MAP3K8 and PK-15-KO-MAP3K8 cells detected by Western blotting in the present invention.

FIG. 11 is a graph showing the fluorescence expression level and the corresponding optical density value of FMDV in PK-15-WT-MAP3K8 and PK-15-KO-MAP3K8 cells observed under a fluorescence microscope in accordance with the present invention.

FIG. 12 is a graph showing the fluorescence expression level and the corresponding optical density of SVV in PK-15-WT-MAP3K8 and PK-15-KO-MAP3K8 cells under a fluorescence microscope in accordance with the present invention.

In the figure, WT represents the control PK-15-WT-MAP3K8 cell line and KO represents the PK-15-KO-MAP3K8 cell line.

In the figure denotes p < 0.05; denotes p < 0.001.

Preservation information:

preservation time: 23 months 7 in 2019;

the name of the depository: china center for type culture Collection;

the preservation number is: CCTCC NO: C2019176;

the address of the depository: wuhan university in Wuhan, China;

and (3) classification and naming: porcine kidney epithelial cells PK-15-KO-MAP3K 8.

Detailed Description

The invention is described in further detail below with reference to the drawings and specific examples, which are provided for illustration only and are not intended to limit the scope of the invention. The test methods used in the following examples are all conventional methods unless otherwise specified; the materials, reagents and the like used are all commercially available reagents and materials unless otherwise specified.