CN114574526A

CN114574526A - Construction method of RPSA gene humanized mouse model

Info

Publication number: CN114574526A
Application number: CN202210005243.9A
Authority: CN
Inventors: 金晶; 张瑞瑞; 苏会敏; 赵静
Original assignee: Jiangsu Jicui Yaokang Biotechnology Co ltd
Current assignee: Jiangsu Jicui Yaokang Biotechnology Co ltd
Priority date: 2021-01-28
Filing date: 2022-01-04
Publication date: 2022-06-03
Anticipated expiration: 2042-01-04
Also published as: CN114574526B

Abstract

The invention provides a preparation method of an RPSA gene modified humanized mouse, and particularly relates to a method for replacing an Rpsa gene of a mouse by an RPSA gene of a pig on a fertilized egg of a C57BL/6 background mouse by using homologous recombination and CRISPR/Cas9 technology to construct a mouse model capable of expressing porcine RPSA.

Description

Construction method of RPSA gene humanized mouse model

Technical Field

The invention belongs to the technical field of biological medicines, and particularly relates to a construction method of an RPSA gene humanized mouse model.

Background

Classical Swine Fever (CSF), also known as rotten bowel disease (herein abbreviated as hog cholera), is a highly infectious and fatal viral disease caused by Classical Swine Fever Virus (CSFV), and has caused great economic loss in the swine industry all over the world. The animal health organization in the world determines the infectious diseases as A class infectious diseases, and China classifies the infectious diseases as a class infectious diseases, and the infectious diseases are one of the main epidemic diseases which endanger the development of the pig industry in China at present.

With respect to host cell surface receptors that bind CSF virus, laminin (RPSA) is one of the CSFV-infected host cell attachment receptors. RPSA (also known as LAMR1) is a highly sequence-conserved cell surface receptor that mediates high-affinity interactions between laminin and cells. Notably, overexpression of porcine-derived RPSA in various non-porcine kidney cell lines (BHK-21, CHO-K1, etc.) at the same time, and no invasion of CSFV was observed, and RPSA was presumed to contribute to viral attachment to the cell surface, and was an important, but not exclusive, binding receptor for CSFV infection.

In addition, in vitro experiments at home and abroad prove that RPSA can inhibit the replication of the Foot-and-mouth disease virus (FMDV) by inhibiting MAPK signal transduction in FMDV infected cells in the process of FMDV infection. Meanwhile, RPSA is a binding receptor for Dengue Virus (Dengue Virus) to enter hepatocytes.

At present, the effective means for preventing and treating classical swine fever is vaccination, although China has better effect on controlling and eliminating classical swine fever, the virus is not completely eliminated, once epidemic situation is found, killing and harmless treatment are carried out according to relevant regulations and regulations of China, and great economic pressure is brought to the pig industry of China. Under the background that CSFV infection epidemic situation is difficult to be completely cured quickly, the epidemic situation is controlled mainly by means of enhancing the biological safety guarantee precaution of breeding enterprises, Chinese swine fever epidemic situation prevention and control are normalized, toxic production is normalized, how to closely monitor the breeding environment is achieved, and great challenge is brought to the pig raising industry.

The research and development of vaccine drugs and screening experiments need to be evaluated on animal models, rodents serve as the most widely applied experimental animal models, are one of the best experimental animal models because of small body size, short growth and breeding period and easy operation, and no pig-derived mouse model prepared aiming at pig-derived RPSA genes exists at present.

The gene editing function of the CRISPR/Cas9 system is mainly applied to genome targeting of multiple species, modification (such as knockout, mutation and knock-in) operation is carried out on alleles, various modification models can be established quickly and efficiently to research gene functions or establish disease models, compared with a traditional gene editing mouse model manufactured based on an embryonic stem cell targeting technology, the gene editing mouse model is simple in design and convenient to operate, complex targeting vector in-vitro splicing is omitted, work such as screening and culturing of embryonic stem cells is carried out, and the gene editing mouse model has great application prospect no matter the manufacturing cost or period of the model. Although the CRISPR/Cas9 gene editing system has high cleavage efficiency and is generally applied to the establishment of a gene knockout model, the requirements of more complex gene modification are met, for example, when a gene of a longer fragment is targeted, the targeting efficiency is obviously reduced along with the increase of the length of a knock-in fragment, and if the sequence of a targeting region has a homologous structure in a mouse body, an obvious off-target effect occurs. In addition, when the CRISPR/Cas9 system containing the donor recombinant fragment is directly micromanipulated on fertilized eggs, the donor fragment can be recombined at a target site, and also can be randomly integrated into the genome of a mouse with high probability, the integrated site is random, high-copy knock-in can occur, and the mouse with random integration can have unexpected phenotypes such as exogenous gene ectopic expression, non-target site endogenous gene silencing or activation and the like under the influence of the integrated site and copy number, so that the experimental result is interfered. Therefore, the recombination efficiency of the CRISPR/Cas9 system is still required to be further searched and optimized, and how to reduce or avoid the random integration probability of the donor during targeting and improve the detection rate of the random integration undoubtedly brings a series of challenges to gene editors.

Disclosure of Invention

A method for constructing a mouse model capable of expressing porcine RPSA is carried out by replacing porcine RPSA with murine Rpsa gene on fertilized eggs of C57BL/6 background mice by using homologous recombination and CRISPR/Cas9 technology.

The specific technical scheme for realizing the invention is as follows:

a construction method of an RPSA gene humanized mouse model is characterized in that the RPSA gene of a pig is replaced by the RPSA gene of a mouse on a fertilized egg of a C57BL/6 background mouse through homologous recombination and CRISPR/Cas9 technology, so that the mouse model capable of expressing the porcine RPSA is constructed.

A construction method of an RPSA gene humanized mouse model comprises the following steps:

1) according to the structure and the function of the porcine RPSA, the coding regions of the porcine RPSA and the mouse Rpsa are selected, the difference sites are identified, and a porcine sequence for replacement is determined.

2) Determining a targeting site according to the sequence of the mouse RPSA gene determined in the step 1), and designing a homologous DNA donor containing the porcine RPSA gene.

3) Preparing Cas9 or an expression vector thereof based on CRISPR/Cas9 technology; sgRNA for murine sequences was designed in the pig-derived replacement region. Designing and synthesizing and recognizing a 5 'end target site and a 3' end target site, and constructing a sgRNA expression vector.

4) The Cas9/sgRNA system and the targeting vector are injected into mouse fertilized eggs and transplanted into a pseudopregnant female mouse, and after the mouse is born, a targeted mouse (a positive F0 generation mouse) is obtained through gene identification.

Preferably, the amino acid sequence of the RPSA humanized mouse used for replacement is shown as SEQ No.2 (three difference sites are A241T, A272T and A290T).

Preferably, the mouse RPSA gene sequences identify the targeting sites as exon6 and exon7 of the mouse RPSA gene.

Preferably, the 5 'and 3' sgRNA recognition sites are located within the mouse Rpsa gene intron3-4, and downstream of exon7, respectively.

Preferably, the sgRNA expression vector is constructed by cloning the sgRNA sequence into pUC57kan-T7-delG vector to construct pUC57-sgRNA plasmid.

Preferably, the sgRNA is prepared by transcription by: PCR is carried out by taking PrimerStar or PrimerStar Max system and sgRNA-F, sgRNA-R as primers and puc57-sgRNA plasmid with correct sequencing as a template, and a PCR product is purified to prepare a sgRNA transcription preparation template. The transcription of sgRNA was performed using T7-ShortScript in vitro transcription kit (AM 1354).

Preferably, the vector is injected using 0.5 day mouse zygotes for targeting.

Preferably, the obtained positive F0 mouse is bred with a wild type C57BL/6J mouse, and the born mouse is subjected to genotype identification to obtain the RPSA swine-derived mouse animal model capable of being stably inherited.

Preferably, the positive swine-origin mouse genotype obtained is identified. And identifying by PCR and sequencing the PCR product.

1) exon6 and exon7 of the mouse Rpsa gene were selected as targeting sites and designed to contain a homologous DNA donor of the porcine Rpsa gene.

2) Preparing Cas9 or an expression vector thereof; sgRNA for murine sequences was designed in the pig-derived replacement region. Designing and synthesizing and recognizing a 5 'end target site and a 3' end target site, and constructing a sgRNA expression vector. Two sgRNA recognition sites are respectively positioned in mouse Rpsa gene intron3-4 and downstream of exon 7.

3) The Cas9/sgRNA system and the targeting vector are injected into a mouse fertilized egg for 0.5 day, the fertilized egg is transplanted into a pseudopregnant female mouse, and after the mouse comes out, a target positive mouse (a positive F0 generation mouse) is obtained through gene identification.

Furthermore, the obtained positive F0 mouse can be bred with a wild type C57BL/6J mouse, and the born mouse is subjected to genotype identification to obtain the RPSA humanized mouse animal model capable of being stably inherited.

Further, the sequences of target sites of grnas on RPSA in step 2) are shown in the following table:

table 1 sgRNA sequences

sgRNA name	sgRNA sequence (5 '→ 3')	PAM
			Rpsa-5S1	GAGATAGTTTTGGTACGTGG	TGG
Rpsa-5S2	GTTGAGATAGTTTTGGTACG	TGG
			Rpsa-3S1	ATTACGGCCACAGCCTATTA	AGG
Rpsa-3S2	ATCTACTCATATTCCTTAAT	AGG

Each sgRNA sequence was cloned into a pUC57kan-T7-delG vector to construct a pUC57-sgRNA plasmid.

Further, the sgRNA transcription preparation method is to use PrimerStar or PrimerStar Max system, sgRNA-F, sgRNA-R as a primer, use the correctly sequenced puc57-sgRNA plasmid as a template to perform PCR, purify the PCR product, and prepare a sgRNA transcription preparation template. Transcription of sgRNA was performed using T7-ShortScript in vitro transcription kit (AM 1354).

Further, the genotype of the swine-derived mice is identified.

Further, the identification of the pig-derived mouse genotype comprises: for the obtained rat tail genomic DNA of the positive mouse, two pairs of primers are respectively designed and used for PCR identification after target hitting. The genotype identifying primers designed and used are shown in the following table.

TABLE 2 RPSA primers for genotyping pig-derived mice

Note: KI is an on-target genotype; WT was wild type.

The primers Rpsa-wt-tF1/Rpsa-wt-tR1 are respectively positioned outside the 5' homologous arm and inside intron6-7, and PCR products generated by the amplification of the primers are sequenced. Sequencing and analyzing the targeting region and the exon6 swine origin site, and if the sequence is consistent with a theoretical sequence, the target vector is effectively recombined in the 5' of the mouse genome; Rpsa-wt-tF2/Rpsa-wt-tR2 were located outside intron6-7 and 3' homology arms, respectively, and PCR products generated by amplification of the primers were sequenced, and the sequencing primers were as described in Table 3 below. And (3) sequencing and analyzing the targeting region and the exon7 swine origin site, wherein the sequence is consistent with a theoretical sequence, and the target vector is effectively recombined in the 3' of the mouse genome. Mice positive for PCR amplification and sequencing at both ends are positive mice.

TABLE 3 sequencing primers

Primer name	Primer sequences
			Rpsa-wt-tF3	CTGCCATTTGGGTGTGTGGAAT(SEQID NO.13)	5' full Length product sequencing
Rpsa-wt-seqR1	AACCCATGTGTTCACATGCTTA(SEQID NO.14)	3' full Length product sequencing

Further, QPCR detection was performed on mice positive for the targets.

Drawings

FIG. 1: constructing a targeting strategy of a mouse model capable of expressing porcine RPSA.

FIG. 2: 5-terminal and 3-terminal identification electrophorograms of pRPSA PCR, wherein a WT negative control is genomic DNA; TRANS 2K PLUS II band: 8000bp \5000bp \3000bp \2000bp \1000bp \750bp \500bp \250bp \100 bp.

FIG. 3: pRPSA PCR 5-terminal sequencing alignment map.

FIG. 4: and (3) sequencing and aligning the 272 th codon site at the 3 end of pRPSA PCR.

FIG. 5: sequencing and aligning map of 290 th codon site at 3 end of pRPSA PCR.

The CRISPR/Cas9 gene editing system has high cutting activity, designs and constructs sgRNA aiming at a target gene, and can guide Cas9 protein to cut efficiently at the target gene site, so as to manufacture a gene knockout model, if an exogenous sequence needs to be knocked in the target gene site, a doror vector for recombination needs to be constructed at the same time, the doror vector needs to comprise homologous arm structures and insertion fragments at two sides of an insertion site, and the doror vector constructed in vitro is injected into a fertilized egg together with the sgRNA and the Cas9 for recombination.

The mouse Rpsa gene is located on chromosome 9, a structure homologous with the mouse Rpsa exists in a plurality of chromosome sites such as chromosome 3, chromosome 17, chromosome 10, chromosome X, chromosome 4 and chromosome 14, a gRNA is designed in a region where point mutation exists, an off-target effect is inevitably brought, in order to avoid off-target brought by a homologous sequence, the targeted region is enlarged, gRNA sites are respectively designed at intro 3-4 and exon7 downstream of the Rpsa gene so as to ensure the specificity of homologous arms and a cutting site sequence, but the longer the inserted fragment is, the lower the recombination efficiency is, and in general, when the length of the inserted fragment is more than 1kb, the recombination difficulty is greatly improved. And designing gRNA at the site where the exogenous gene is inserted, and making donor, wherein the closer the gRNA is to the knock-in site, the higher the recombination efficiency is, the length of the donor gene sequence designed for avoiding off-target effect reaches 3424bp, and the positive mouse (F0 generation) is successfully verified and obtained by PCR and sequencing detection means of mouse offspring obtained after microinjection and transplantation.

The invention verifies the correct integration of the target site gene by a PCR and sequencing method, simultaneously carries out QPCR detection on the mouse with positive targets, detects the possibility of other site integration in the mouse genome, further verifies that the positive mouse is the correct target, and has no random insertion of other non-target sites of the positive mouse.

The mouse of the invention has functional genes of pigs, the knocked-in genes are derived from pig source RPSA genes and knocked into Rpsa gene sites of the mouse, and the self regulation and control elements of the mouse are reserved, so that the expression of the pig source RPSA is controlled by utilizing the self regulation and control mechanism of the mouse, and the Rpsa genes of the mouse are silenced, thereby avoiding drug cross reaction caused by the homology relation of species amino acids. The model has functional genes of the pig and has important guiding significance for researches on infection, invasion, immunity, drug development and the like of related viruses of the pig source.

The construction method for constructing the mouse model capable of expressing the porcine RPSA by replacing the Rpsa gene of the mouse with the porcine RPSA gene on the fertilized egg of the C57BL/6 background mouse by using the homologous recombination and CRISPR/Cas9 technology has high recombination efficiency, reduces or avoids the random integration probability of donor during targeting, has high detection rate of random integration, and can accurately and efficiently construct the RPSA gene humanized mouse model.

Detailed Description

The following examples illustrate specific steps of the present invention, but are not intended to limit the invention.

Terms used in the present invention generally have meanings commonly understood by those of ordinary skill in the art, unless otherwise specified.

The present invention is described in further detail below with reference to specific examples and with reference to the data. It should be understood that the following examples are intended only to illustrate the present invention and are not intended to limit the scope of the present invention in any way.

In the following examples, various procedures and methods not described in detail are conventional methods well known in the art.

The present invention is further illustrated by the following specific examples.

Example 1 determination of the regions for obtaining porcine-derived fragment substitutions and inserted porcine-derived sequences

Selecting three different sites of a porcine RPSA and a mouse Rpsa coding region according to the structure and the function of the porcine RPSA, wherein the three different sites of replacing a mouse Rpsa gene with a porcine sequence are A241T, A272T and A290T respectively, the rest regions all retain the murine structure, and the mouse wild type sequence is shown as SEQ No. 1. SEQ No. 1:

Msgaldvlqmkeedvlkflaagthlggtnldfqmeqyiykrksdgiyiinlkrtweklllaaraivaienpadvsvissrntgqravlkfaaatgatpiagrftpgtftnqiqaafreprllvvtdpradhqplteasyvnlptialcntdsplryvdiaipcnnkgahsvglmwwmlarevlrmrgtisrehpwevmpdlyfyrdpeeiekeeqaaaekavtkeefqgewtapapeftaaqpevadwsegvqvpsvpiqqfptedwsaqpatedwsaaptaqatewvgattews

the corresponding nucleotide sequence is SEQ ID NO. 3:

ATGTCCGGAGCCCTTGACGTCCTGCAGATGAAGGAGGAGGATGTCCTCAAATTCCTTGCTGCGGGAACCCACTTAGGTGGCACCAACCTTGACTTTCAGATGGAGCAGTACATCTACAAAAGGAAAAGTGACGGTATCTACATCATAAACCTGAAGAGGACCTGGGAGAAGCTGTTGCTCGCAGCTCGAGCTATTGTTGCCATCGAGAATCCTGCTGACGTCAGCGTCATCTCCTCCAGGAACACTGGCCAGCGAGCTGTGCTGAAGTTTGCTGCTGCCACAGGAGCCACTCCGATCGCTGGCCGCTTCACACCTGGGACCTTCACTAACCAGATCCAAGCAGCCTTCAGGGAGCCACGGCTTCTAGTGGTGACCGATCCCAGGGCTGACCATCAGCCACTCACAGAGGCCTCTTATGTCAACCTGCCCACCATTGCTCTGTGTAACACAGATTCTCCCCTGCGCTATGTGGACATTGCCATCCCATGCAACAACAAGGGAGCTCACTCAGTGGGTCTGATGTGGTGGATGCTGGCCAGGGAAGTACTCCGCATGCGAGGTACTATCTCCCGTGAGCACCCCTGGGAGGTCATGCCTGATCTTTACTTCTACAGAGACCCAGAGGAGATTGAGAAGGAGGAGCAGGCTGCTGCTGAGAAGGCTGTGACCAAGGAGGAATTCCAGGGTGAATGGACCGCACCAGCTCCTGAGTTCACTGCTGCTCAGCCTGAGGTGGCCGACTGGTCTGAGGGTGTGCAGGTTCCCTCTGTGCCCATCCAGCAGTTCCCCACGGAAGACTGGAGTGCACAGCCAGCCACTGAGGATTGGTCAGCAGCTCCCACAGCGCAGGCCACTGAGTGGGTTGGAGCCACCACTGAGTGGTCCTGA

example 2 determination of porcine-derived RPSA protein sequences

Three different sites of the porcine RPSA gene are replaced by corresponding regions of B6 murine Rpsa by homologous recombination, the sequences of B6 mice are reserved in the rest sequences, and the amino acid sequence of the RPSA humanized mouse which successfully targets is shown as SEQ No. 2.

SEQ No.2：

Msgaldvlqmkeedvlkfluagthlggntldfqmeqyikrksrksdgiyinlkrtwerlaoalarvaientnpavsvisvrtgqrtfatprtgftnqqqaaffretprlvtdprhppltetyvpltlylplipplndiopltrylpurwrawmwlwlmlargrwrretsrhpwevmopdpurdplyfplyfpirefpiredfyevejeqjefaqweqweqpifwefpltwratterwgtttetfpwefpedjefpedaafpedtafpatafpataqpataqatwegtvgttts corresponding nucleotide sequence is SEQ ID No. 4:

ATGTCCGGAGCCCTTGACGTCCTGCAGATGAAGGAGGAGGATGTCCTCAAATTCCTTGCTGCGGGAACCCACTTAGGTGGCACCAACCTTGACTTTCAGATGGAGCAGTACATCTACAAAAGGAAAAGTGACGGTATCTACATCATAAACCTGAAGAGGACCTGGGAGAAGCTGTTGCTCGCAGCTCGAGCTATTGTTGCCATCGAGAATCCTGCTGACGTCAGCGTCATCTCCTCCAGGAACACTGGCCAGCGAGCTGTGCTGAAGTTTGCTGCTGCCACAGGAGCCACTCCGATCGCTGGCCGCTTCACACCTGGGACCTTCACTAACCAGATCCAAGCAGCCTTCAGGGAGCCACGGCTTCTAGTGGTGACCGATCCCAGGGCTGACCATCAGCCACTCACAGAGGCCTCTTATGTCAACCTGCCCACCATTGCTCTGTGTAACACAGATTCTCCCCTGCGCTATGTGGACATTGCCATCCCATGCAACAACAAGGGAGCTCACTCAGTGGGTCTGATGTGGTGGATGCTGGCCAGGGAAGTACTCCGCATGCGAGGTACTATCTCCCGTGAGCACCCCTGGGAGGTCATGCCTGATCTTTACTTCTACAGAGACCCAGAGGAGATTGAGAAGGAGGAGCAGGCTGCTGCTGAGAAGGCTGTGACCAAGGAGGAATTCCAGGGTGAATGGACCGCACCAGCTCCTGAGTTCACTGCTACTCAGCCTGAGGTGGCCGACTGGTCTGAGGGTGTGCAGGTTCCCTCTGTGCCCATCCAGCAGTTCCCCACGGAAGACTGGAGTGCACAGCCAACCACTGAGGATTGGTCAGCAGCTCCCACAGCGCAGGCCACTGAGTGGGTTGGAACAACCACTGAGTGGTCCTGA

example 3 Positive mice obtained by vector injection transplantation

1) Selecting exon6 and exon7 of mouse Rpsa gene as targeting sites, designing homologous DNA donor containing pig source RPSA gene and identifying scheme;

2) preparing Cas9 or an expression vector thereof based on CRISPR/Cas9 technology; sgRNA for murine sequences was designed in the pig-derived replacement region. Designing and synthesizing and recognizing a 5 'end target site and a 3' end target site, and constructing a sgRNA expression vector. The recognition sites of the two sgRNAs are respectively positioned in intron3-4 of the mouse Rpsa gene and downstream of exon7, and the target site sequence of each sgRNA on the Rpsa is shown in Table 1. Each sgRNA sequence was cloned into a pUC57kan-T7-delG vector to construct a pUC57-sgRNA plasmid.

Table 1 sgRNA sequences

The sgRNA transcription preparation method comprises the following steps: PCR is carried out by taking PrimerStar or PrimerStar Max system and sgRNA-F, sgRNA-R as primers and puc57-sgRNA plasmid with correct sequencing as a template, and a PCR product is purified to prepare a sgRNA transcription preparation template. Transcription of sgRNA was performed using T7-ShortScript in vitro transcription kit (AM 1354).

3) The Cas9/sgRNA system and the targeting vector are injected into a fertilized egg of a mouse for 0.5 day, the fertilized egg is transplanted into a pseudopregnant female mouse, and after the mouse is born, the gene identification is carried out on the screened targeted mouse, namely the positive F0 mouse.

4) And breeding the positive F0 mouse and a wild C57BL/6J mouse, and identifying the genotype of the born mouse to obtain the RPSA humanized mouse animal model capable of being stably inherited.

Example 4 genotyping of porcine-derived mice.

For the obtained rat tail genomic DNA of the positive mouse, performing PCR identification after targeting by using two pairs of primers respectively (the primers are detailed in a table 2), wherein the primers Rpsa-wt-tF1/Rpsa-wt-tR1 are respectively positioned outside a 5 'homology arm and in intron6-7, sequencing a PCR product generated by amplification of the primers (the sequencing primers are detailed in a table 3), and analyzing a targeting region and an exon6 swine origin site by sequencing, wherein if the sequence is consistent with a theoretical sequence, the target vector is effectively recombined in the mouse genome 5'; Rpsa-wt-tF2/Rpsa-wt-tR2 are respectively positioned outside intron6-7 and 3 'homology arms, PCR products generated by the primer amplification are sequenced (the sequencing primer is shown in table 3 in detail), and the target region and the exon7 swine origin site are analyzed through sequencing, so that the sequence is consistent with a theoretical sequence, and the target vector is effectively recombined in the 3' of the mouse genome. Mice positive for PCR amplification and sequencing at both ends are positive mice.

TABLE 2 RPSA primers for genotyping pig-derived mice

Note: KI is an on-target genotype; WT was wild type.

TABLE 3 sequencing primers

Primer name	Primer sequences
			Rpsa-wt-tF3	CTGCCATTTGGGTGTGTGGAAT	5' full Length product sequencing
Rpsa-wt-seqR1	AACCCATGTGTTCACATGCTTA	3' full Length product sequencing

The PCR reaction system and reaction conditions are shown in the following table:

TABLE 4 PCR reaction System

Reagent (Vazyme P112-03)	Volume (μ l)	Specification of
			2×Taq Master Mix，Dye Plus	12.5	\
ddH2O	9.5	\
			Primer A(10pmol/μl)	1	10pmol/μl
Primer B(10pmol/μl)	1	10pmol/μl
			Template(≈100ng/μl)	1	≈100ng/μl

TABLE 5 PCR reaction conditions

Example 5QPCR

PCR identification results of pRPSA mouse model:

the results of 5-terminal and 3-terminal PCR amplification are shown in FIG. 2, and FIG. 2 shows the 5-terminal and 3-terminal identification electrophoretograms of pRPSA PCR. WT negative control was genomic DNA; TRANS 2K PLUS II band: 8000bp \5000bp \3000bp \2000bp \1000bp \750bp \500bp \250bp \100 bp.

The sequencing alignment map of the pRPSA PCR 5 end is shown in figure 3, an arrow indicates an exon6 porcine source site mutation site, and a241 th codon is mutated from GCT to ACT;

the sequencing comparison map of 272 th codon site at the 3 terminal of pRPSA PCR is shown in attached figures 4 and 5, wherein the shading indicates the mutation site of exon7 swine origin site, the 272 th codon is mutated from GCC to ACC, and the 290 th codon is mutated from GCC to ACA;

as a result: successful acquisition of B6 background resulted in 3 positive mice. And (3) identifying an electrophoretogram of pRPSA rat tail DNA, identifying that the target bands are positive 5 'and 3', and sequencing comparison shows that the mouse is a positive mouse with correct target.

Cas9/sgRNA system and targeting vector are injected into mouse fertilized eggs of 0.5 day, 3 times of microinjection and transplantation are carried out altogether, 9 and 17F 0 mice are born by three times of transplantation respectively, and the data (MGI) shows that homozygote mice with Rpsa gene knocked out have embryonic-stage death phenotype, so the birth rate is obviously lower by the data shown by three times of transplantation. After further PCR and sequencing detection, 1, 0 and 2 positive mice were obtained from the F0 mouse, and the total positive rate was 8.6%.

The transplant, birth and positive rate profiles are shown in Table 6 below.

TABLE 6 transplant, birth and Positive rates

The results show that the gRNA designed near the point mutation effectively avoids off-target effect, and the gRNA with enlarged target area is involved, so that the specificity of homologous arms and cutting site sequences can be ensured, and meanwhile, the high recombination efficiency can be realized. Meanwhile, the condition that orthotopic forward recombination exists in the positive mouse is eliminated through verification, and the positive mouse is guaranteed to be a correct targeting without random insertion of other non-target sites.

Sequence listing

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aacactggcc agcgagctgt gctgaagttt gctgctgcca caggagccac tccgatcgct 300

ggccgcttca cacctgggac cttcactaac cagatccaag cagccttcag ggagccacgg 360

cttctagtgg tgaccgatcc cagggctgac catcagccac tcacagaggc ctcttatgtc 420

aacctgccca ccattgctct gtgtaacaca gattctcccc tgcgctatgt ggacattgcc 480

atcccatgca acaacaaggg agctcactca gtgggtctga tgtggtggat gctggccagg 540

gaagtactcc gcatgcgagg tactatctcc cgtgagcacc cctgggaggt catgcctgat 600

ctttacttct acagagaccc agaggagatt gagaaggagg agcaggctgc tgctgagaag 660

gctgtgacca aggaggaatt ccagggtgaa tggaccgcac cagctcctga gttcactgct 720

gctcagcctg aggtggccga ctggtctgag ggtgtgcagg ttccctctgt gcccatccag 780

cagttcccca cggaagactg gagtgcacag ccagccactg aggattggtc agcagctccc 840

acagcgcagg ccactgagtg ggttggagcc accactgagt ggtcctga 888

<210> 4

<211> 888

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 4

atgtccggag cccttgacgt cctgcagatg aaggaggagg atgtcctcaa attccttgct 60

gcgggaaccc acttaggtgg caccaacctt gactttcaga tggagcagta catctacaaa 120

aggaaaagtg acggtatcta catcataaac ctgaagagga cctgggagaa gctgttgctc 180

gcagctcgag ctattgttgc catcgagaat cctgctgacg tcagcgtcat ctcctccagg 240

aacactggcc agcgagctgt gctgaagttt gctgctgcca caggagccac tccgatcgct 300

ggccgcttca cacctgggac cttcactaac cagatccaag cagccttcag ggagccacgg 360

cttctagtgg tgaccgatcc cagggctgac catcagccac tcacagaggc ctcttatgtc 420

aacctgccca ccattgctct gtgtaacaca gattctcccc tgcgctatgt ggacattgcc 480

atcccatgca acaacaaggg agctcactca gtgggtctga tgtggtggat gctggccagg 540

gaagtactcc gcatgcgagg tactatctcc cgtgagcacc cctgggaggt catgcctgat 600

ctttacttct acagagaccc agaggagatt gagaaggagg agcaggctgc tgctgagaag 660

gctgtgacca aggaggaatt ccagggtgaa tggaccgcac cagctcctga gttcactgct 720

actcagcctg aggtggccga ctggtctgag ggtgtgcagg ttccctctgt gcccatccag 780

cagttcccca cggaagactg gagtgcacag ccaaccactg aggattggtc agcagctccc 840

acagcgcagg ccactgagtg ggttggaaca accactgagt ggtcctga 888

<210> 5

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 5

gagatagttt tggtacgtgg 20

<210> 6

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 6

gttgagatag ttttggtacg 20

<210> 7

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 7

attacggcca cagcctatta 20

<210> 8

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 8

atctactcat attccttaat 20

<210> 9

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 9

aaagtgacgg tcagttgcca cct 23

<210> 10

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 10

actgtaaaag ctccctggca gc 22

<210> 11

<211> 26

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 11

ggatacactg tgtcttgaca ctgctc 26

<210> 12

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 12

cacttggaga ctctgggaaa gct 23

<210> 13

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 13

ctgccatttg ggtgtgtgga at 22

<210> 14

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 14

aacccatgtg ttcacatgct ta 22

Claims

1. A construction method of an RPSA gene swine mouse model is characterized in that a mouse model capable of expressing porcine RPSA is constructed by replacing the mouse RPSA gene with the porcine RPSA gene on a mouse fertilized egg through homologous recombination and CRISPR/Cas9 technology.

2. A construction method of an RPSA gene humanized mouse model comprises the following steps:

1) according to the structure and the function of the porcine RPSA, selecting a porcine RPSA and mouse Rpsa coding region, identifying a difference site and determining a porcine sequence for replacement;

2) determining a targeting site according to the mouse RPSA gene sequence determined in the step 1), and designing a homologous DNA donor containing a porcine RPSA gene;

3) preparing Cas9 or an expression vector thereof based on CRISPR/Cas9 technology; designing sgRNA aiming at a murine sequence in a pig-derived replacement region; designing and synthesizing and recognizing a 5 'end target site and a 3' end target site, and constructing an sgRNA expression vector;

4) cas9/sgRNA system and carrier are injected into mouse fertilized eggs and planted into a pseudopregnant female mouse, and after the mouse is born, the gene identification is carried out to obtain a target mouse, namely a positive F0 generation mouse.

3. The method for constructing a mouse model of porcine humanized RPSA genes according to claim 1 or 2, wherein the amino acid sequence of the mouse model of porcine humanized RPSA for replacement is shown in SEQ No. 2.

4. The method of constructing a porcine model of an RPSA gene according to any of claims 1-3, wherein the mouse RPSA gene sequence is determined to target the exon6 and exon7 of the mouse Rpsa gene.

5. The method for constructing a porcine humanized mouse model of RPSA gene according to any one of claims 2 to 4, wherein the sgRNA recognition sites at 5 'end and 3' end are located in intron3-4 and downstream of exon7 of mouse Rpsa gene, respectively.

6. The method of constructing a porcine mouse model of RPSA genes according to any of claims 2-5, wherein the sgRNA expression vector is constructed by cloning the sgRNA sequence into a pUC57kan-T7-delG vector to construct a pUC57-sgRNA plasmid.

7. The method of constructing a porcine humanized mouse model of the RPSA gene of any one of claims 2-6, wherein transcription of the sgRNA is performed using T7-ShortScript in vitro transcription kit.

8. The method of constructing a porcine humanized mouse model of RPSA genes according to any one of claims 1 to 7, wherein fertilized eggs of a mouse are used for 0.5 days.

9. The method of constructing a porcine mouse model of RPSA genes according to any of claims 2-8, wherein the obtained positive F0 generation mice are bred with wild type C57BL/6J mice, and the born mice are genotyped to obtain a porcine mouse model of RPSA genes which can be stably inherited.

10. A porcine mouse model of the RPSA gene obtained by the method of any one of claims 1 to 9.

11. The model of claim 10, wherein the porcine-derived RPSA amino acid sequence is set forth in SEQ ID No. 2.

12. Use of the mouse model obtained by the construction method of claims 1-19 in vaccine drug development.

13. Use of the mouse model obtained by the construction method of claims 1-9 in vaccine drug development in drug screening.

14. Use according to claim 12 or 13 for screening or evaluating drugs and diagnostic products related to swine fever or foot and mouth disease.

15. The sgRNA group for constructing the RPSA gene pig-derived mouse model is characterized in that the nucleotide sequence is as follows:

SEQ ID NO.5：GAGATAGTTTTGGTACGTGG；

SEQ ID NO.6：GTTGAGATAGTTTTGGTACG；

SEQ ID NO.7：ATTACGGCCACAGCCTATTA；

SEQ ID NO.8：ATCTACTCATATTCCTTAAT。

16. a primer for identifying the genotype of an RPSA swine-derived mouse, which is characterized by comprising the following nucleotide sequences:

SEQ ID NO.9：AAAGTGACGGTCAGTTGCCACCT

SEQ ID NO.10：ACTGTAAAAGCTCCCTGGCAGC

SEQ ID NO.11：GGATACACTGTGTCTTGACACTGCTC

SEQ ID NO.12：CACTTGGAGACTCTGGGAAAGCT。