CN114539379A

CN114539379A - AQP1 mutant gene, protein, cell line and application thereof

Info

Publication number: CN114539379A
Application number: CN202210214020.3A
Authority: CN
Inventors: 唐娜; 沈志强; 管宇; 徐倩倩; 苗立中; 王金良; 苑文娴; 高庆芳; 张倩
Original assignee: Shandong Lvdu Bio Sicience & Technology Co ltd; Shandong Binzhou Animal Science & Veterinary Medicine Academy
Current assignee: Shandong Lvdu Bio Sicience & Technology Co ltd; Shandong Binzhou Animal Science & Veterinary Medicine Academy
Priority date: 2022-03-03
Filing date: 2022-03-03
Publication date: 2022-05-27
Anticipated expiration: 2042-03-03

Abstract

The invention discloses a mutant gene and a mutant protein of chicken AQP1, a mutant cell line of the gene and application thereof in cell suspension culture. The invention also discloses a preparation method of the AQP1 mutant cell line. According to the invention, the spacer sequence behind the first transmembrane region of the AQP1 protein of the chicken embryo fibroblast line DF-1 cell is knocked out, so that the obtained AQP1 mutant cell line is easy to suspend and domesticate, has stable genetic character, does not have tumorigenicity, is not influenced in virus multiplication capacity, is applied to the production of poultry virus vaccines, replaces a vaccine production process of solid-phase culture, and can greatly improve the production efficiency.

Description

AQP1 mutant gene, protein, cell line and application thereof

Technical Field

The invention belongs to the field of biological products, relates to the field of cell and virus vaccines, and particularly relates to AQP1 mutant genes, proteins, a cell line and application thereof.

Background

At present, most cells for producing vaccines adopt three modes of rotary bottle adherent culture, microcarrier adherent culture and suspension culture, the efficiency is increased gradually, and the production cost is decreased gradually. For large-scale industrial vaccine production, suspension culture is superior to microcarrier culture and spinner flask culture: from the perspective of production cost, the cell density is higher, the virus titer is higher, and the production cost of unit vaccine is lower; from the perspective of process control, suspension culture cells are cultured in a large bioreactor, fermentation parameters are easy to monitor and adjust, the automation degree is high, the batch-to-batch difference is small, and exogenous pollution is easy to control. Therefore, suspension culture will be the trend for cell culture methods to produce antiviral vaccines.

There are many strategies for adapting cells to suspension culture, such as using specially designed low serum or serum free media, using shake flasks of cells or by gene regulation, etc. However, the traditional domestication method is only effective for a part of cells with suspension growth potential, and is still inexperienced for most cells. At present, the types of suspension cells which can be applied to industrial vaccine production at home and abroad are still very rare, and only comprise BHK21, MDCK, PER.C6, AGE1, HEK293, EB66 and CAP. Animal virus vaccine production still uses a large amount of cells or cell lines with low production efficiency, such as primary cells of chicken embryo fibroblasts widely used in poultry virus culture, and continuous cell lines of chicken embryo fibroblasts, DF-1, and the like. In order to improve the production efficiency of poultry vaccines, a new suspension domestication method and a new technology are urgently needed to change the adherent growth characteristics of poultry cell lines such as DF-1 cell line and the like and realize large-scale culture.

The suspension cell line prepared by genetic engineering can solve the uncertainty and randomness of the traditional suspension domestication method in the aspects of cell yield, genetic background, domestication time and the like. Several studies have shown that altering certain anchorage-associated genes of cells can adapt cells to suspension culture. Inhibition of PTEN gene expression in 293T cells by RNA interference techniques in 2005 enabled cells to lose anchorage capacity, altering cell morphology (Miseomata S, et al 2005). In 2009, Chia Chu successfully achieved suspension culture of MDCK cells by overexpressing the ST6GALNAC5 gene in dog kidney cells MDCK (Chia Chu et al 2009). However, loss of anchorage-dependent capacity of cells does not indicate that the cells are capable of growth in suspension, and deletion of many anchorage-dependent genes does not result in suspensible cells. In addition, many adherent genes are closely related to cancer, and the PTEN gene is an anti-cancer gene, and the inhibition of the expression of the PTEN gene is likely to cause the canceration of cells. ST6GALNAC5 is also a cancer-associated gene, and suspension cells overexpressing this gene may be at risk for carcinogenesis (Huang D, et al 2015). Therefore, when developing a suspension cell line for genetic engineering, it is a problem to be solved urgently how to improve the suspension performance of cells by changing the functions of related proteins and on the premise of not generating the risk of tumorigenesis.

Aquaporins (AQPs) are a series of specific pore channels capable of efficiently and selectively transporting water molecules on cell membranes, and 13 different types of aquaporins (AQP0-AQP12) have been discovered, are expressed in host cells involved in liquid secretion and absorption, have the functions of transporting water, urine, glycerol and the like, and participate in various physiological mechanisms of organisms. The poultry cells can also express a plurality of AQPs, wherein AQP1 can be expressed in tissue cells of kidney, stomach, intestinal tract, ovary, oviduct and the like, and can also be obviously expressed in chick chorioallantoic membrane ectoderm, endoderm epithelial cells and vascular endothelial cells. No studies have been reported on the relationship between AQP1 protein structure and the adherent growth status of poultry cells.

Disclosure of Invention

The purpose of the invention is as follows: it is discovered accidentally during the course of poultry pathology research that the destruction of AQP1 protein structure can cause obvious change of the adherent growth state of poultry cells, and continuous passage does not produce tumorigenic effect. Therefore, in order to improve the suspensibility of chicken embryo fibroblast line DF-1 cells (from ATCC cell bank, number ATCCRL-12203), the invention knocks out partial sequence of AQP1 gene transmembrane region of the DF-1 cells through gene editing, changes the function of AQP1 protein, thereby improving the suspension growth capacity of the cells, accelerating suspension domestication and providing excellent cell lines for the large-scale production of vaccines for avian viral diseases. Therefore, the invention improves the cell suspension domestication efficiency without generating tumorigenic risk by knocking out or mutating AQP1 gene in chicken embryo fibroblast line DF-1 cell, and develops the avian cell line which can be cultured in high density.

The technical scheme is as follows: in order to solve the technical problems, the invention provides an AQP1 mutant gene, wherein the AQP1 mutant gene lacks nucleotides 92 to 214 in an AQP1 wild-type gene.

Wherein, the nucleotide sequence of the AQP1 mutant gene is shown as SEQ ID NO: 1 is shown.

SEQ ID NO: 1 the sequence is as follows:

ATGGCCAGTGAATTCAAAAAGAAAATGTTCTGGAGGGCGGTGGTGGCTGAGTTCCTCGCCATGATCCTCTTTATTTTTATCAGCATTGGCTGTTCTGCGGTGCCCACTTGAACCCGGCGGTGACCCTGGGCCTCCTGCTGAGCTGCCAGATCAGCATCTTCAAGGCGCTGATGTACATCCTCGCCCAGTGCCTGGGGGCAGTGGTGGCCACCGCCATCCTGTCCGGAGTCACCTCCTCTCTGCCCTACAACTCCCTGGGGCTCAATGCGCTTGCAAAAGGAATCAATGCAGGCCAAGGACTAGGAATTGAAATCATTGCTACTCTCCAGCTGGTTTTGTGCGTTCTTGCCACCACAGACCGGAGAAGGAATGATGTCTCAGGATCAGCACCTCTGGCCATTGGTCTCTCTGTTGCCTTGGGACATCTCCTTGCAATTGATTACACCGGTTGTGGAATTAACCCAGCCAGATCTTTTGGCTCAGCACTGATTGCCAACAACTTTGAAAATCATTGGATCTTCTGGGTTGGCCCAATCATTGGAGGAGCAGGTGCTGCCCTGATCTATGACTTCATCCTGGCTCCCAGAAGCAGTGACCTGACTGATCGCGTGAAGGTGTGGACCAGCGGCCAAGTAGAAGAGTATGATCTGGAAGGAGATGATATGAACTCCAGAGTTGAAATGAAGCCAAAATAA

the present disclosure also includes mutant proteins encoding said AQP1 mutant genes.

Wherein the amino acid sequence of the mutant protein is shown as SEQ ID NO: 2, respectively.

SEQ ID NO: 2, the following:

MASEFKKKMFWRAVVAEFLAMILFIFISIGFCGAHLNPAVTLGLLLSCQISIFKALMYILAQCLGAVVATAILSGVTSSLPYNSLGLNALAKGINAGQGLGIEIIATLQLVLCVLATTDRRRNDVSGSAPLAIGLSVALGHLLAIDYTGCGINPARSFGSALIANNFENHWIFWVGPIIGGAGAALIYDFILAPRSSDLTDRVKVWTSGQVEEYDLEGDDMNSRVEMKPK.

the present disclosure also includes expression cassettes, expression vectors, cell lines or recombinant strains comprising said AQP1 mutant gene.

The present disclosure also includes AQP1 mutant DF-1 cell lines containing the AQP1 mutant genes.

The invention also provides a preparation method of the AQP1 mutant DF-1 cell line, which comprises the following steps: obtaining an AQP1 gene sequence of a DF-1 cell by amplification sequencing, designing sgRNA after a protein coding initiation codon in a second exon, carrying out gene knockout on 92 th-214 th nucleotides of

transmembrane regions

1 and 2 of AQP1 genes of the DF-1 cell by applying a CRISPR/Cas9 gene editing technology, and obtaining an AQP1 mutant DF-1 cell which is named as DF-1/AQP1 through cloning, screening and purifying^-A cell.

Wherein the targeting sequence of the sgRNA is shown in SEQ ID NO: 3, respectively.

SEQ ID NO：3：ATCAGCATTGGCTCGGCTCT；

Wherein, the PCR primer sequence adopted in the clone screening is shown as SEQ ID NO: 4 and SEQ ID NO: 5, respectively.

SEQ ID NO：4：GGGCAGAGAAAGAGGAGAGAT；

SEQ ID NO：5：AGTAGAGGGGGACAGCAAAGT；

The invention also comprises the application of the mutant gene, the mutant protein, the expression cassette, the expression vector, the cell line or the recombinant strain in cell suspension culture or in preparation of infectious bursal disease virus vaccines.

Has the advantages that: compared with the prior art, the invention has the following advantages: according to the invention, the 1 st and 2 nd transmembrane region spacer sequences in the AQP1 gene of the DF-1 cell are knocked out by gene editing, the function of the AQP1 protein is changed, and the obtained AQ P1 mutant cell has better suspension property and stable genetic character, can be applied to poultry virus vaccine production, replaces the traditional cell vaccine production process, and greatly improves the production efficiency.

Drawings

FIG. 1 shows the result of PCR identification of DF-1 cells after transfection, where M is DNA marker, 1 is the cell culture after transfection, and 2-4 are untransfected cell control, blank control and culture solution control. The 437bp band in each lane is unedited amplification product, the 315bp band is edited mutation gene amplification product, and the others are non-specific amplification bands.

FIG. 2 shows DF-1/AQP1^-And prediction of the transmembrane site of AQP1 protein expressed by DF-1 cells. 2A is DF-1/AQP1^-Prediction map of transmembrane site of gene encoded by cell line AQP1, wherein 33-51aa are membrane internal regions; 2B is a prediction map of the transmembrane site of AQP1 encoding gene of parent DF-1 cell, wherein 36-54aa are extracellular regions.

FIG. 3 shows DF-1/AQP1^-The cells were visualized as pictures during passage. 3A is the full cells at the moment of passage; 3B is a cell which is not digested by pancreatin and is simply blown; 3C, cells which are blown, dispersed, inoculated into a new bottle and grown for 24 hours; 3D is cells that grew 48h after seeding.

FIG. 4 shows DF-1/AQP1^-And the growth curves of the DF-1 cells in shake flask culture.

FIG. 5 shows DF-1/AQP1^-And DF-1 cells in nude mice tumor formation test. 5A is DF-1/AQP1^-The cell group was nude mice, 5B was CEF cell group, and 5C was positive control group. Red arrows indicate injection sites or tumor formation sites.

Detailed Description

Embodiments of the present invention will be described in detail below with reference to examples, but those skilled in the art will appreciate that the following examples are only illustrative of the present invention and should not be construed as limiting the scope of the present invention. The examples, in which specific conditions are not specified, were carried out according to conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by manufacturers, and are all conventional products which can be purchased in the market.

Example 1DF-1/AQP1^-Construction of cells

1.1 sgRNA design and preparation

According to a chicken AQP1 gene sequence (ENSGALG00000005209) registered in an Ensembl database, a sequence of-20 to 437nt near an initiation codon in a No. 2 exon is selected, a guide RNA sequence is designed on line by using a design tool (genscript. com) and named as AQP1_ g1 (table 1), and the AQP1_ g1 is compared with human, monkey, cow and sheep genomes to analyze the off-target effect. In vitro transcription primers AQP1_ g1 were designed and synthesized, PCR amplification and sgRNA transcription were performed according to the instructions of a commercial one-step sgRNA in vitro transcription kit (Inovogen technology), and the concentration of sgRNA was adjusted to 400 ng/. mu.L.

TABLE 1 sgRNA and in vitro transcription primers used in the assay

1.2 transfection

DF-1 cells were derived from the ATCC cell bank (accession number ATCC CRL-12203) and cultured to be in logarithmic growth phase and passaged to 24-well plates to prepare cell monolayers that grew to 90% of the well bottoms. 2.5 mu L of the sgRNA transcribed in 1.1 and 2.5 mu L of the commercialized Cas9 protein (Genescript company, 5 mu M) are sequentially added into a first 1.5ml tube and mixed uniformly, and 20 mu L of Buffer CRISPR Buffer solution is added after the reaction at room temperature is carried out for 10 minutes to obtain the Cas9/gRNA compound. 25 μ L of transfection reagent (0.4 μ L Viromer +24.6 μ L Buffer CRISPR) was dispensed in a second 1.5ml tube according to the instructions of the commercial CRISPR RNP transfection kit (Viromer, origin) and mixed rapidly with the Cas9/gRNA complex in the first tube in equal volume, incubated 15 minutes at room temperature and then added to a 24 well plate and incubated for 72 hours.

1.3 cloning and screening of Gene-deleted cell lines

72 hours after transfection, transfected cells in 24-well plates were digested and resuspended into single cells and subcloned by 96-well plate limiting dilution. Wells from which single cell clones grew were selected and screened for AQP1 gene-deleted cell clones by the established PCR method (primers see table 2).

TABLE 2 deletion identification and sequencing primers used in the assay

Selecting and breeding DF-1/AQP1^-After washing the cells with PBS 3 times, the cells were resuspended in PBS at a density of 10000 cells/ml, 200. mu.L of the supernatant was collected, and nucleic acid was extracted using a DNA/RNA extraction kit. Using extracted cell RNA as a template, carrying out RT-PCR amplification by using designed primers AQP1F and AQP1R, and selecting cells with deletion bands for further culture.

Obtaining a clone with deletion bands of about 120bp generated by PCR (see attached figure 1) through a first round of cell subclone culture and PCR identification, selecting the clone for further subclone purification to obtain a cell clone strain which grows well and is identified as pure gene deletion by PCR, expanding passage and storing, and naming as DF-1/AQP1^-。

1.4 DF-1/AQP1^-Sequencing and identification of strain gene

Primers were designed based on the sequence of the chicken AQP1 gene (ENSGALG00000005209) registered in Ensemb1 database, and the same pair DF-1/AQP1 as described in 1.3^-The AQP1 protein encoding gene of the strain was amplified in full length and sequenced (primers AQP1wF and AQP1wR are shown in Table 3). The sequencing result is compared with the related sequence in the reference gene.

TABLE 3 deletion identification and sequencing primers used in the assay

The sequencing comparison shows that the sequence of the DNA is determined,DF-1/AQP1^-the total length of an AQP1 gene coding region of a cell is 693bp, compared with a parent strain 813bp, the CRISPR system generates double-strand break at an AQP1_ g1 target sequence near a PAM locus, a base deletion of more than 120bp is caused at a cut, and the base deletion is recombined through random repair to form a gene mutant. Compared with the parent cell, the mutant cell strain has a deletion of 123bp base sequences in the 92-214 nt region, and simultaneously has 3bp insertion bases which are randomly repaired (see a sequence table SEQ ID NO: 1). The amino acid sequence was deduced and aligned with the parent cell, and the AQP1 gene of the mutant cell encodes the 230aa protein, mutated between 31-72aa and 42aa (40 aa deleted) compared to the parent cell, covering the 1 st and 2 nd transmembrane spacer (fig. 2). DF-1/AQP1^-The AQP1 gene coding sequence and the amino acid sequence of the strain are shown in SEQ ID NO: 2.

in conclusion, in this example, the sequence of the AQP1 gene near N-terminal portion was knocked out from DF-1 cell, and a cell clone was obtained by screening, wherein the molecular feature of the cell is that the AQP1 protein lacks the 1 st and 2 nd transmembrane region spacer sequence, which is hereinafter referred to as DF-1/AQP1^-A cell.

Example 2 DF-1/AQP1^-Growth characteristics of cells

2.1 DF-1/AQP1^-Adherent culture and morphology observation of cells

DMEM medium (GIBCO) containing 5% newborn calf serum is prepared as cell growth liquid, and DF-1/AQP1 is dispersed by blowing with a pipette^-Cells were cultured in 1X 10 flasks per T25⁶And (4) carrying out concentration passage on each cell, carrying out passage on the parent strain in the same way, and observing the cell morphology and the growth state. Mutant clone DF-1/AQP1^-The cell morphology of the mutant strain cell is not obviously different from that of the parent cell, but the mutant strain cell is easy to mechanically disperse after being attached to the wall, and particularly, the mutant strain cell can be separated from the bottle wall by blowing and beating the mutant strain cell through a straw when the cell enters the late logarithmic growth stage and the plateau stage, and can be dispersed into single cells or 4-16 cell clusters. After the cells are divided into bottles for passage, the cells are well spread and can still grow adherent to the wall, and the cells are in a more uniform polygonal or fusiform structure (see figure 3).

2.2 DF-1/AQP1^-Cell suspension culture

Cells in logarithmic growth phase were as 5X 10⁵concentration of cells/mLThe medium was inoculated into a 125mL shake flask and 30mL DMEM medium containing 5% serum was added. 5% CO₂The mixture was incubated at 37 ℃ for 72 hours at 150rpm with a shaker. The cells were collected by centrifugation at 1,200rpm for 5min, resuspended in 30ml of medium and split into flasks at an initial cell concentration of 5X 10⁵cells/mL were subcultured. If the cell concentration exceeds 1X 10 after 72 hours⁶cells/mL, the initial seeding density needs to be reduced to 1X 10⁵cells/mL to 3X 10⁵cells/mL. If the cell concentration is less than 1X 10 after 72 hours of culture⁶cells/mL, then replace fresh medium to continue the culture. Meanwhile, the parent DF-1 cell without mutation is set as a control cell, and the shake flask culture is also carried out to compare with the treatment group. After continuous passage and observation and recording of the cell concentration every 72 hours, the parental DF-1 cell can not stably reach 1 x 10 in continuous suspension culture for 5 generations⁶cells/mL, and DF-1/AQP1 cells were cultured in suspension for 48 hours at passage 2, which reached 1X 10⁶cells/mL, cells grown substantially stably for 5 successive generations of culture (FIG. 4).

EXAMPLE 3 DF-1/AQP 1-cell tumorigenicity test

Selecting DF-1/AQP1 with good growth state and in logarithmic growth phase^-Cells were harvested when they grew to 80-90% full. Resuspend cells to 5X 10 with PBS or serum-free Medium⁷Individual cells/ml. While setting 5X 10⁷CEF of individual cells/ml as negative control, 1X 10⁷BHK-21 cells at individual cells/ml served as positive control. 5-8 weeks old nude mice were selected, weighing about 18-20g, and the neck and back were inoculated with 5 cells subcutaneously, each group, 0.2ml each. Before inoculation, the cell suspension is fully blown away to prevent the cells from agglomerating. The observation was continued for 35 days to 12 weeks after the inoculation, and the presence or absence of nodules or tumors was checked. After inoculation, the positive control nude mice have obvious lumps at the inoculated part, the diameter can reach 10mm, the spirit is in the morning and the food intake is declined. Neither the negative control group nor the test group had nodules or suspected tumor tissues, no abnormal expression of spirit and diet (FIG. 5), and no tumor cells were found by examination of the dissections and tissue sections 35 days and 12 weeks later.

Example 4 DF-1/AQP1^-Subculture stability of the cellular AQP1 mutation

Mixing DF-1/AQP1^-Continuously passaging cells for more than 20 generations, taking samples of the 20 th generation, amplifying a target fragment with about 693bp of the total length of an AQP1 coding region according to the primers and steps used in full-length sequencing in example 1, sending the target fragment to the biological engineering (Shanghai) company Limited for sequencing, comparing the sequencing result with an AQP1 coding sequence of early generation of a mutant cell line, wherein the sequence homology is 100 percent, no back mutation occurs, and DF-1/AQP1 has the advantages of no occurrence of any back mutation^-The inheritance of the strain is stable.

Example 5 DF-1/AQP1^-Preliminary application of infectious bursal disease virus vaccine antigen in cell culture

DF-1/AQP1 in logarithmic growth phase^-The content of Cissus matsugaensis is 5 × 10⁵cells/mL were inoculated into 125mL shake flasks and 30mL DMEM medium, 5% CO was added₂After culturing for 48 hours at 150rpm on a shaker, the infectious bursal disease virus BJQ902 strain (vaccine strain of Shandong Lvdu Biotech Co., Ltd.) was inoculated at 0.005 MOI. Suspension culture was continued for 72 hours during which the degree of glucose consumption was periodically measured and fed to 4g/L when the glucose concentration was less than 1 g/L. With TCID₅₀The assay detects infectious bursal disease virus content. Use of suspension culture of DF-1/AQP1^-The cell proliferation of infectious bursal disease virus can reach the virus titer of 10 within 36 to 48 hours after the virus inoculation^9.1TCID₅₀And/ml, compared with the same number of DF-1 parent cells cultured by the traditional adherent culture method, the unit yield can be improved by 3.98-19.95 times as shown in Table 4.

TABLE 4 comparison of viral proliferation (TCID) after inoculation with infectious bursal disease Virus₅₀/ml)

	Batch 1	Batch 2	Batch 3
				Suspension culture in Shake flasks	10^9.1	10^8.9	10^9.1
Traditional adherent culture	10^8.1	10^7.6	10^8.5

Sequence listing

<110> Shandong province Binzhou animal husbandry veterinary research institute, Shandong Green city Biotech Co., Ltd

<120> AQP1 mutant gene, protein, cell line and application thereof

<160> 5

<170> SIPOSequenceListing 1.0

<210> 1

<211> 695

<212> DNA

<213> Aquaporin 1(Aquaporin1)

<400> 1

atggccagtg aattcaaaaa gaaaatgttc tggagggcgg tggtggctga gttcctcgcc 60

atgatcctct ttatttttat cagcattggc tgttctgcgg tgcccacttg aacccggcgg 120

tgaccctggg cctcctgctg agctgccaga tcagcatctt caaggcgctg atgtacatcc 180

tcgcccagtg cctgggggca gtggtggcca ccgccatcct gtccggagtc acctcctctc 240

tgccctacaa ctccctgggg ctcaatgcgc ttgcaaaagg aatcaatgca ggccaaggac 300

taggaattga aatcattgct actctccagc tggttttgtg cgttcttgcc accacagacc 360

ggagaaggaa tgatgtctca ggatcagcac ctctggccat tggtctctct gttgccttgg 420

gacatctcct tgcaattgat tacaccggtt gtggaattaa cccagccaga tcttttggct 480

cagcactgat tgccaacaac tttgaaaatc attggatctt ctgggttggc ccaatcattg 540

gaggagcagg tgctgccctg atctatgact tcatcctggc tcccagaagc agtgacctga 600

ctgatcgcgt gaaggtgtgg accagcggcc aagtagaaga gtatgatctg gaaggagatg 660

atatgaactc cagagttgaa atgaagccaa aataa 695

<210> 2

<211> 230

<212> PRT

<213> Aquaporin 1(Aquaporin1)

<400> 2

Met Ala Ser Glu Phe Lys Lys Lys Met Phe Trp Arg Ala Val Val Ala

1 5 10 15

Glu Phe Leu Ala Met Ile Leu Phe Ile Phe Ile Ser Ile Gly Phe Cys

20 25 30

Gly Ala His Leu Asn Pro Ala Val Thr Leu Gly Leu Leu Leu Ser Cys

35 40 45

Gln Ile Ser Ile Phe Lys Ala Leu Met Tyr Ile Leu Ala Gln Cys Leu

50 55 60

Gly Ala Val Val Ala Thr Ala Ile Leu Ser Gly Val Thr Ser Ser Leu

65 70 75 80

Pro Tyr Asn Ser Leu Gly Leu Asn Ala Leu Ala Lys Gly Ile Asn Ala

85 90 95

Gly Gln Gly Leu Gly Ile Glu Ile Ile Ala Thr Leu Gln Leu Val Leu

100 105 110

Cys Val Leu Ala Thr Thr Asp Arg Arg Arg Asn Asp Val Ser Gly Ser

115 120 125

Ala Pro Leu Ala Ile Gly Leu Ser Val Ala Leu Gly His Leu Leu Ala

130 135 140

Ile Asp Tyr Thr Gly Cys Gly Ile Asn Pro Ala Arg Ser Phe Gly Ser

145 150 155 160

Ala Leu Ile Ala Asn Asn Phe Glu Asn His Trp Ile Phe Trp Val Gly

165 170 175

Pro Ile Ile Gly Gly Ala Gly Ala Ala Leu Ile Tyr Asp Phe Ile Leu

180 185 190

Ala Pro Arg Ser Ser Asp Leu Thr Asp Arg Val Lys Val Trp Thr Ser

195 200 205

Gly Gln Val Glu Glu Tyr Asp Leu Glu Gly Asp Asp Met Asn Ser Arg

210 215 220

Val Glu Met Lys Pro Lys

225 230

<210> 3

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 3

atcagcattg gctcggctct 20

<210> 4

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 4

gggcagagaa agaggagaga t 21

<210> 5

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 5

agtagagggg gacagcaaag t 21

Claims

An AQP1 mutant gene, characterized in that said AQP1 mutant gene lacks nucleotides 92 to 214 from the wild type gene of AQP 1.
2. The AQP1 mutant gene of claim 1, wherein the nucleotide sequence of said AQP1 mutant gene is as set forth in SEQ ID NO: 1 is shown.
3. A mutant protein encoding the AQP1 mutant gene of claim 1 or 2.
4. The mutein of claim 3, wherein the amino acid sequence of the mutein is set forth in SEQ ID NO: 2, respectively.
5. An expression cassette, expression vector, cell line or recombinant strain comprising the AQP1 mutant gene of claim 1 or 2.
An AQP1 mutant DF-1 cell line comprising the AQP1 mutant gene of claim 1 or 2.
7. The method of making an AQP1 mutant DF-1 cell line of claim 6, comprising the steps of: obtaining AQP1 gene sequence of DF-1 cell by amplification sequencing, designing sgRNA after protein coding initiation codon in the second exon, implementing gene knockout on 92 th-214 th nucleotides of 1 st and 2 th transmembrane regions of AQP1 gene of DF-1 cell by CRISPR/Cas9 gene editing technology, cloning, screening and purifying to obtain the productObtaining AQP1 mutant DF-1 cell, named as DF-1/AQP1^—A cell.
8. The method of claim 7, wherein the targeting sequence of the sgRNA is as set forth in SEQ ID NO: 3, respectively.
9. The method for preparing the AQP1 mutant DF-1 cell line of claim 7, wherein a PCR primer sequence adopted in the clone screening is as shown in SEQ ID NO: 4 and SEQ ID NO: 5, respectively.
10. Use of the mutant gene of claim 1 or 2, the mutant protein of claim 3 or 4, the expression cassette, the expression vector, the cell line or the recombinant strain of claim 5 in cell suspension culture or in the preparation of an infectious bursal disease virus vaccine.