阅读说明：本技术 Tpl2基因敲除hek293t细胞系及其构建方法和应用 (TPL 2gene knockout HEK293T cell line and construction method and application thereof ) 是由 郑海学 张克山 闫鸣昊 郝军红 申超超 朱紫祥 李丹 �田宏 茹毅 杨帆 曹伟军 于 2020-04-30 设计创作，主要内容包括：本发明涉及一种TPL2基因敲除的HEK293T细胞系及其构建方法和应用。本发明提供TPL2基因敲除HEK293T细胞系的构建方法,将人TPL2基因的第二外显子区域作为靶序列,具体的,将SEQ ID NO:1的TPL2等位基因1和等位基因2第二外显子的第390～459bp序列或第391～448bp序列作为靶序列。本发明提供TPL2基因敲除HEK293T细胞系HEK293T-KO-TPL2,其保藏编号为CCTCC NO:C2019328。本发明获得的敲除细胞系形态、增殖速度等方面均与对照细胞无明显差异,是较为理想的TPL2敲除细胞模型；改造后细胞系稳定,为探究TPL2蛋白抑制病毒复制的方式及病毒致病机理提供关键的生物材料,也可用于分离和培养SVA以及在SVA疫苗株的大规模细胞化培养和生产中进行应用。(The invention relates to a TPL 2gene knockout HEK293T cell line and a construction method and application thereof. The invention provides a construction method of a TPL 2gene knockout HEK293T cell line, which takes a second exon region of a human TPL 2gene as a target sequence, and concretely takes a 390 th to 459 th bp sequence or a 391 th to 448 th bp sequence of a TPL2 allele 1 and a second exon of an allele 2 of SEQ ID NO:1 as the target sequence. The invention provides a TPL 2gene knockout HEK293T cell line HEK293T-KO-TPL2 with a preservation number of CCTCC NO: C2019328. The morphology, proliferation speed and other aspects of the knockout cell line obtained by the invention have no obvious difference from those of a control cell, and the knockout cell line is an ideal TPL2 knockout cell model; the modified cell line is stable, provides a key biological material for researching the mode of inhibiting virus replication by TPL2 protein and the pathogenic mechanism of the virus, and can be used for separating and culturing SVA and applied to large-scale cell culture and production of SVA vaccine strains.)

A construction method of a TPL 2gene knockout HEK293T cell line is characterized in that a second exon region of a human TPL 2gene is used as a target sequence, and specifically, 390 th to 459 th bp sequences or 391 th to 448 th bp sequences of a TPL2 allele 1 and a second exon of an allele 2 shown in SEQ ID NO. 1 are used as target sequences.

2. The method for constructing the TPL 2gene knockout HEK293T cell line as claimed in claim 1, wherein an exogenous sequence is inserted into the target sequences of allele 1 and allele 2 or the target sequence is knocked out.

3. The construction method of the TPL 2gene knockout HEK293T cell line as claimed in claim 1, characterized in that an exogenous sequence is inserted between 390 th to 391bp of TPL2 allele 1 shown in SEQ ID NO. 1, and 449bp base "G" is knocked out, 58bp in total of 391 th to 448bp of allele 2 is knocked out;

or knocking out the 393bp 'C' base of the TPL2 allele 1 shown in SEQ ID NO. 1, knocking out 18bp altogether from 442-459 bp, and knocking out 58bp altogether from 391-448 bp of the allele 2.

4. The method for constructing the TPL 2gene knockout HEK293T cell line as claimed in claim 1, which comprises the following steps:

step 1: constructing two pairs of sgRNAs targeting a human TPL2 genome sequence and cloning the sgRNAs to the same CRISPR/Cas9 vector plasmid pX459 to obtain a pX-EZ-TPL2-sgRNA recombinant plasmid; the sgRNAs of the two pairs of targeted human TPL2 genome sequences are sgRNA1 and sgRNA2, and the sequences of the sgRNA1 and the sgRNA2 are respectively synthesized by sequences shown as SEQ ID NO. 2-3 and SEQ ID NO. 4-5;

step 2: transfecting HEK293T cells with the constructed pX-EZ-TPL2-sgRNA recombinant plasmid, and killing negative cells by drug screening;

and step 3: carrying out monoclonal sorting by a limiting dilution method;

and 4, step 4: and (4) carrying out expanded culture and verification on the sorted monoclonal cells.

5. The cell line constructed by the construction method of the TPL 2gene knockout HEK293T cell line as claimed in any one of claims 1 to 4.

6. The cell line of claim 5, wherein the cell line has a collection number of CCTCC NO: C2019328, HEK293T-KO-TPL 2.

7. Use of the cell line of claim 6 to study the manner in which TPL2 inhibits viral replication and the pathogenesis of a virus.

8. The use of the cell line of claim 6 to study the manner in which TPL2 inhibits viral replication and the pathogenesis of a virus, wherein said virus is SVA.

9. Use of the cell line of claim 6 to isolate and culture SVA.

10. The TPL 2gene knockout HEK293T cell line of claim 6, applied to large-scale cell culture and production of SVA vaccine strains.

Technical Field

The invention belongs to the technical field of biology, and particularly relates to a TPL 2gene knockout HEK293T cell line, and a construction method and application thereof.

Background

The HEK293 cell line is a human embryonic kidney epithelial cell transfected with the adenovirus E1A gene. The HEK293T cell is a high-trans derivative formed by transferring SV40T-antigen gene into HEK293 cell, can express SV40 large T antigen, and can replicate a plasmid containing SV40 replication starting point and promoter region. The cell can be used for gene expression and protein production of various types, and can also be used for producing high-titer retroviruses and other viruses, such as adenoviruses and other mammalian viruses.

TPL2 is a serine/threonine kinase, also known as COT or MAP3K8, that when unstimulated TPL2 forms a complex with p105 and ABIN2 to remain inactive (GANTKE T, SRISKANTHARAJAH S, LEY S C. Regulation and function of TPL-2, an IkappaB kinase-regulated MAP kinase [ J ]. Cell Res, 2011, 21(1): 131-45.). After various receptors such as TLR, TNFR, IL1R and the like are stimulated, the activation of various signal proteins such as downstream ERK, JNK, p38, NF-kappa B and the like can be regulated and controlled through signal transduction mediated by TPL 2; it also stimulates a variety of innate immune cells such as macrophages, dendritic cells, neutrophils to produce a large number of cytokines such as type I interferons, tumor necrosis factors, etc. (GANTKE T, SRISKANTHARAAJAH S, SADOWSKI M, et al. I.I.kappa.B kinase regulation of the TPL-2/ERK MAPK pathway [ J ]. Immunol Rev, 2012,246(1): 168-82.). TPL2 is also essential in regulating the differentiation of CD4+ T cells to generate different Th cell lineages (ZHU J, PAUL W E. CD 4T cells: tissues, functions, and functions [ J ] Blood,2008, 112(5): 1557-69.), is an important participant in innate immunity, inflammation and tumor, and plays an important role in both innate immunity and acquired immunity.

The CRISPR/Cas9 gene editing technology is a third generation genome editing technology that has evolved rapidly 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, then a non-homologous end joining repair mechanism (NHEJ) of a cell rejoins the genomic DNA at a break, and insertion or deletion mutations are introduced (CONG L, F Z. Genome engineering using CRISPR/Cas9system [ J ] Methods mol Bio, 2015, 1239: 197-217.). Three gene editing endonucleases, namely ZNF, TALEN and CRISPR/Cas9, are applied to clinic at present. Among them, the CRISPR/Cas9system is the most widely used gene editing technology in this field due to its advantages of high efficiency, rapidness, multiple functions, easy use, low cost, etc., and has been applied to various species (MEMI F, NTOKOU A, PAPANGELII. CRISPR/Cas9 gene-editing: Research technologies, clinical applications and clinical diagnostics [ J ]. SeminPerinato, 2018, 42(8): 487-500.).

The Chinese invention patent with the publication number of CN 110862968A discloses a PK-15 cell line PK-15-KO-MAP3K8 knocked out by MAP3K8 gene, a construction method and application thereof. 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. However, in later practice, the cell line is not stable enough in terms of cell morphology and proliferation rate compared with a wild cell line, and is not favorable for application in basic research.

Disclosure of Invention

The invention provides a TPL 2gene knockout HEK293T cell line HEK293T-KO-TPL2, which aims to solve the problem that the cell line of the TPL 2gene knockout cell line is poor in stability in aspects of cell morphology, proliferation speed and the like. The growth speed, the cell morphology and the propagation speed of the cell line HEK293T-KO-TPL2 are not different from those of a wild HEK293T cell line, so that the cell line is an ideal TPL2 knockout cell model and is a key biological material for researching a mode of inhibiting virus replication by TPL2 protein and accumulating a virus pathogenesis.

The invention specifically adopts the following technical scheme:

in a first aspect, the invention provides a construction method of a TPL 2gene knockout HEK293T cell line, wherein a second exon region of a human TPL 2gene is used as a target sequence, and specifically, 390-459 bp sequences or 391-448 bp sequences of second exons of a TPL2 allele 1 and an allele 2 shown in SEQ ID NO. 1 are used as target sequences.

Preferably, the target sequences of allele 1 and allele 2 are knocked-in exogenous sequences or knocked-out target sequences.

Preferably, an exogenous sequence is inserted between 390 th to 391bp of TPL2 allele 1 shown in SEQ ID NO. 1, the base G at 449bp is knocked out, and 58bp of 391 th to 448bp of allele 2 are knocked out;

or knocking out the 393bp 'C' base of the TPL2 allele 1 shown in SEQ ID NO. 1, knocking out 18bp altogether from 442-459 bp, and knocking out 58bp altogether from 391-448 bp of the allele 2.

More preferably, the insertion exogenous sequence of the TPL2 allele 1 between 390 th bp and 391 th bp is shown as SEQ ID NO. 6.

The construction method of the TPL 2gene knockout HEK293T cell line specifically comprises the following steps:

step 1: design of sgRNA oligo sequence: two pairs of sgRNAs specifically targeting human TPL2 genes are constructed according to the sequence of the human TPL2 gene: sgRNA1 and sgRNA 2;

specifically, the sgRNA targets the second exon region of the human TPL2 gene.

Further, the sgRNA1 was synthesized from the following sequence:

H-TPL2-sgRNA1Forward：5’-TCCTCGGGGCGCCTTTGGAA-3’；

H-TPL2-sgRNA1Reverse：5’-TTCCAAAGGCGCCCCGAGGA-3’；

the sgRNA2 was synthesized from the following sequence:

H-TPL2-sgRNA2Forward：5’-CCGATGTTCTCCTGATCCCC-3’；

H-TPL2-sgRNA2Reverse：5’-GGGGATCAGGAGAACATCGG-3’；

step 2: construction of pX-EZ-TPL2-sgRNA recombinant plasmid: cloning two sgRNAs of the constructed specific target knockout human TPL 2gene to the same CRISPR/Cas9 vector plasmid pX459 to obtain a recombinant plasmid pX-EZ-TPL 2-sgRNA;

specifically, two sgrnas were cloned onto the same CRISPR/Cas9 vector plasmid pX459 using an EZ-guideexh helper plasmid.

And step 3: plasmid transfection: respectively transfecting the pX-EZ-TPL2-sgRNA recombinant plasmid and the pX-EZ no-load plasmid into HEK293T cells;

specifically, the transfection process was performed according to the instructions of the HighGene transfection reagent.

Prior to transfection, HEK293T cells were cultured in DMEM medium supplemented with 10% fetal bovine serum and 1% penicillin streptomycin.

And 4, step 4: drug screening monoclonal cell lines: the CRISPR/Cas9system is utilized to achieve the purpose of gene silencing, negative cells are killed through drug screening, and then a positive monoclonal cell line is obtained through limited dilution sorting;

specifically, the screening drug is puromycin (puromycin) antibiotic.

And 5: identification of knockout cell lines: and (3) carrying out amplification culture on the sorted monoclonal cell line, identifying the knockout condition of a positive monoclonal cell line gene by genotyping PCR sequencing, and then further identifying the screened homozygous knockout cell line by Westernblotting to verify the knockout condition of the TPL2 protein in the cell line.

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 length, 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 genotyping PCR detection primer sequence is as follows:

H-TPL2 genetyping Forward：5’-GACCAGGCACCTGCATCTGTT-3’，

H-TPL2 genetyping Reverse：5’-TGAGGCAGTGCACCCTCAGA-3’。

in a second aspect, the invention provides a cell line constructed by the construction method of the TPL 2gene knockout HEK293T cell line.

Furthermore, the invention provides a TPL 2gene knockout HEK293T cell line HEK293T-KO-TPL2 with a preservation number of CCTCC NO: C2019328.

In a third aspect, the invention provides application of a TPL 2gene knockout HEK293T cell line in researching a mode that TPL2 inhibits virus replication and virus pathogenesis.

Further, the virus is SVA.

In a fourth aspect, the invention provides the use of a TPL2 knock-out HEK293T cell line for isolation and culture of SVAs.

In a fifth aspect, the invention provides application of the TPL 2gene knockout HEK293T cell line in large-scale cell culture and production of SVA vaccine strains.

The invention has the following beneficial effects:

1. the TPL 2gene knockout HEK293T cell line HEK293T-KO-TPL2 is constructed by using a CRISPR/Cas9system, the cell line is stable after being modified, the aspects of cell morphology, proliferation speed and the like are not obviously different from those of a control cell, the cell line is an ideal TPL2 knockout cell model, and a key biological material is provided for researching a mode of TPL2 protein inhibiting virus replication and a virus pathogenic mechanism.

2. The TPL 2gene knockout HEK293T cell line HEK293T-KO-TPL2 can not correctly express TPL2 protein due to the fact that certain fragments are deleted to change the open reading frame of TPL2 coding protein to cause frame shift mutation, and therefore the gene knockout purpose is achieved. As the TPL2 protein has an antiviral effect, the proliferation of SVA virus in wild-type HEK293T cells can be inhibited, and TPL 2gene knockout HEK293T cell line HEK293T-KO-TPL2 cannot correctly express TPL2 protein, so that the SVA virus can be favorably propagated in the cell line to obtain larger virus with high titer, and the feasibility strategy that the engineering cell line for vaccine production edited by the CRISPR/Cas9 gene editing technology is a feasible strategy for improving the virus yield is proved, and the method has important significance for large-scale cell culture and production of SVA vaccine strains in the future.

Drawings

FIG. 1 is a diagram showing the results of sequencing verification of pX459-sgRNA1 and EZ-sgRNA2 recombinant plasmids constructed in the examples of the present invention.

FIG. 2 is a diagram showing the PCR identification result of the recombinant plasmid pX-EZ-TPL2-sgRNA constructed in the example of the present invention, in which lanes 1 to 4 are sequentially Marker DL2000, recombinant plasmid pX-EZ-TPL2-sgRNA, blank lane, and recombinant plasmid pX-EZ-TPL 2-sgRNA.

FIG. 3 is a diagram showing the results of PCR identification of TPL2 knock-out cell line genome in the present example, wherein lane 1 is Marker DL2000, lane 2 is HEK293T-WT-TPL2 cell line, lane 3 is HEK293T-KO-TPL2-A2 cell line, lane 5 is HEK293T-KO-TPL2-B1 cell line, and the other lanes are non-positive clones.

FIG. 4 is a diagram showing the sequencing results of the genotype alignment of the TPL 2gene knockout cell line HEK293T-KO-TPL2-A2 and HEK293T-KO-TPL2-B1 in the example of the present invention.

FIG. 5 is a diagram for detecting the abundance of TPL2 protein in HEK293T-WT-TPL2 and HEK293T-KO-TPL2 cells by Western blotting in the embodiment of the invention.

FIG. 6 is a cell morphology of HEK293T-WT-TPL2 and HEK293T-KO-TPL2 cells under an inverted microscope in the examples of the present invention.

FIG. 7 is a graph showing the time taken for cells to form cell monolayers in HEK293T-WT-TPL2 and HEK293T-KO-TPL2 in examples of the present invention.

FIG. 8 is a graph showing the fluorescence expression level of SVA in HEK293T-WT-TPL2 and HEK293T-KO-TPL2 cells observed under a fluorescence microscope in the examples of the present invention.

FIG. 9 is a graph of absolute quantification of SVA replication in HEK293T-WT-TPL2 and HEK293T-KO-TPL2 cells in an example of the invention.

FIG. 10 is a graph showing the relative quantification of SVA viral transcript levels in HEK293T-WT-TPL2 and HEK293T-KO-TPL2 cells in accordance with an embodiment of the present invention.

FIG. 11 is a diagram showing the abundance of structural proteins of viruses replicated by SVA in HEK293T-WT-TPL2 and HEK293T-KO-TPL2 cells detected by Western blotting in the examples of the present invention.

FIG. 12 is a SVATCID of cell expansion of HEK293T-WT-TPL2 and HEK293T-KO-TPL2 in an example of the invention₅₀Titre profile.

In the drawings there is shown in detail,p<0.05 indicated statistically significant difference; **,p<0.01 indicates that the statistical difference is very significant.

Preservation information:

preservation time: 11/29/2019;

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

the preservation number is: CCTCC NO: C2019328;

the address of the depository: wuhan university in China;

and (3) classification and naming: human embryonic kidney cells HEK293T-KO-TPL 2.

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.