Pig CD163 protein mutant and application thereof

Abstract

The invention provides a pig CD163 protein mutant and application thereof, the amino acid sequence of the current pig CD163 protein as shown in SEQ ID NO: 1 is selected as the basis, glutamine amino acid at the 488 site is mutated into arginine, the amino acid sequence of the mutated pig CD163 protein is as shown in SEQ ID NO: 2, II-type PRRSV infection can be effectively inhibited after mutation, and the pig CD163 protein can be used for inhibiting infection of a porcine reproductive and respiratory syndrome virus (PRRSV). And a theoretical basis is provided for subsequent acquisition of swinery which can effectively inhibit PRRSV infection and does not affect piglet growth performance and CD163 physiological functions.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and in particular relates to a pig CD163 protein mutant and an application thereof. Background Art

[0002] Porcine reproductive and respiratory syndrome (PRRS) is a highly contagious swine disease caused by porcine reproductive and respiratory syndrome virus (PRRSV). The reported PRRSV is divided into two types: European PRRSV-1 and American PRRSV-2, with a homology of 60%. The clinical symptoms are mainly reduced fertilization rate of breeding sows, reproductive disorders such as abortion, stillbirth and mummified fetuses in pregnant sows, and respiratory symptoms in pigs of different ages. PRRS is widely spread around the world and has become one of the most important infectious diseases threatening the global pig industry, causing huge economic losses to the pig industry. PRRSV virus particles are enveloped, single-stranded positive-strand RNA viruses. The virus has the characteristics of high mutation, immunosuppression and persistent infection, making the disease difficult to prevent and control. Therefore, PRRSV has always been the focus of research in the veterinary field.

[0003] Current studies have found that PRRSV infects host cells through the essential receptor CD163. CD163 has been shown to be an essential host factor for PRRSV infection, and its SRCR5 domain mediates PRRSV infection of the host by interacting with the viral GP2a and GP4 proteins. CD163 is an important member of the scavenger receptor family and consists of an extracellular region, a transmembrane region, and an intracellular region. Its extracellular region contains nine cysteine-rich domains and two proline-serine-threonine-rich motifs. Among them, the SRCR5 domain of the CD163 extracellular region plays a key role in the process of PRRSV infection of host cells.

[0004] A single base editor is a gene editing system composed of a fusion expression of Cas9 protein and deaminase (mainly adenine deaminase and cytosine deaminase). The system can accurately and irreversibly achieve the transition from one base pair to another without introducing double-strand breaks in DNA and exogenous repair templates. Adenine base editors are fusion proteins composed of nCas9 (D10A) and artificially modified adenine deaminase, etc. Under the guidance of sgRNA, they can specifically recognize and bind to the target sequence to achieve the conversion from A to G. Due to the high efficiency of base editing and simple operation of the base editing system, various versions of ABEs (mainly ABE7.10, ABEmax and ABE8e, etc.) have been widely used. Pigs with deletion or replacement of the CD163 SRCR5 domain have been shown to be effective in resisting PRRSV-2 infection. However, the growth performance of pigs with large fragment modifications is inevitably worrying. Since CD163 has the same biological function in vivo, and the only species infected by PRRSV is pigs, there are key amino acids in pig CD163 that affect the proliferation of PRRSV. By mutating the functional amino acid sites of CD163, the effects of CD163 gene modification on the normal physiological function of CD163 and the growth performance of piglets can be minimized, providing a new direction for anti-PRRS breeding. According to literature reports, PRRSV has strict host specificity (pigs) and cell tropism (pig primary alveolar macrophages, PAMs). In addition, artificially induced African green monkey kidney cells MA-104 and their derived Marc145 and CL2621 cells are also susceptible to PRRSV and show persistent infection in epidemiology. Since CD163 is an essential host factor for PRRSV, researchers artificially transferred pCD163 CDS into various cell lines, including 3D4 / 21, PK-15 and CHO-K1 cells, so that these cell lines acquired PRRSV susceptibility, laying the foundation for the study of the pathogenic mechanism and treatment of PRRSV. Summary of the invention

[0005] The purpose of the present invention is to provide a porcine CD163 protein mutant, which selects the wild-type porcine CD163 protein as a basis, mutates the 488th glutamine (Q) amino acid to arginine (R), and the other amino acid residues remain unchanged. The amino acid sequence of the wild-type porcine CD163 protein is shown in SEQ ID NO: 1, and the 488th glutamine is located in the SRCR5 domain of the CD163 extracellular region. The mutated porcine CD163 protein amino acid sequence is shown in SEQ ID NO: 2, which can effectively inhibit type II PRRSV infection, and provides a theoretical basis for the subsequent acquisition of a pig herd that can effectively inhibit PRRSV infection without affecting the growth performance of piglets and the physiological function of CD163.

[0006] Another object of the present invention is to provide a nucleotide sequence of a kind of pig CD163 protein mutant encoding, compared with wild-type pig CD163 gene, the 61st position of exon 7 is A, while the 61st position of exon 7 is G.

[0007] The sequence of exon 7 of the wild-type porcine CD163 gene in the present invention is shown in SEQ ID NO: 3, the sequence of exon 7 of the edited porcine CD163 gene is shown in SEQ ID NO: 4, the amino terminal sequence after mutation of exon 7 is shown in SEQ ID NO: 5, and the amino acid sequence before mutation of exon 7 is shown in SEQ ID NO: 6.

[0008] Another object of the present invention is to provide the described pig CD163 protein mutant and the application of the nucleotide sequence as described and the material that makes the pig CD163 protein realize the described mutation: A. Preparation of products for the prevention or treatment of porcine reproductive and respiratory syndrome; B. Or preparing products that improve the resistance of organisms or organism cells to porcine reproductive and respiratory syndrome virus; C. or preparing cells resistant to porcine reproductive and respiratory syndrome virus; D. or preparing pigs resistant to porcine reproductive and respiratory syndrome virus; E. or breeding of pigs resistant to porcine reproductive and respiratory syndrome virus; F. Or construct a porcine reproductive and respiratory syndrome virus-resistant cell model; G. Or construct a porcine reproductive and respiratory syndrome virus-resistant pig model.

[0009] The organism is a mammal.

[0010] The mammal is a pig.

[0011] The substances that enable the porcine CD163 protein to achieve the mutation are ABEs and a guide sequence that matches ABEs.

[0012] The guide sequence guides ABEs to achieve the following single base editing: cooperate with ABEs to mutate the 61st A in the 7th exon of the wild-type CD163 gene to G, and the nucleotide sequence of the wild-type CD163 gene is shown in SEQ ID NO: 8.

[0013] The guiding sequence is shown in SEQ ID NO:7.

[0014] Another object of the present invention is to provide a method for constructing a non-diagnostic and therapeutic CD163 protein mutant cell, comprising the steps of mutating the 488th position Q of the wild-type CD163 protein in pig kidney cells to R, while keeping other amino acid residues unchanged to obtain the CD163 protein mutant cell.

[0015] Another object of the present invention is to provide a method for constructing a CD163 protein mutation gene-edited pig for non-diagnostic and therapeutic purposes, which are the following two methods: The first method includes the following steps: The 488th position Q of CD163 protein in isolated pig kidney was mutated to R, while other amino acid residues remained unchanged to obtain CD163 protein mutant cells; The CD163 protein mutant cells are used as nuclear transplant donor cells and are transplanted into sows through somatic cell nuclear transplantation, and the offspring produced are CD163 protein mutant gene-edited pigs; The second method includes the following steps: The substance causing the above mutation in CD163 protein is introduced into pig zygotic embryos to obtain CD163 gene-modified embryos, and then the CD163 gene-modified embryos are transplanted into the mother's body for pregnancy to obtain CD163 protein mutation gene-edited pigs.

[0016] The CD163 protein mutant gene-edited pig mentioned above refers to a pig whose CD163 protein encoding gene on its genome is a gene encoding the CD163 mutant protein.

[0017] In the above, the present invention does not limit whether CD163 before mutation also contains other mutation sites, so the precursor of the CD163 protein mutant provided by the present invention can be wild-type CD163, or has been based on wild-type CD163, mutated CD163 mutants at other sites. The precursor of CD163 mutant is the CD163 protein considered according to the general definition of this area.

[0018] In an alternative embodiment, the wild-type CD163 protein amino acid sequence is shown in SEQ ID NO: 1 or comprises an amino acid sequence having at least 80% identity to SEQ ID NO: 1, for example, can be but is not limited to comprising an amino acid sequence having at least 80%, 85%, 90%, 95% or 98% identity to SEQ ID NO: 1.

[0019] The beneficial effects are as follows: The present invention discovered a site that can effectively inhibit type II PRRSV infection, namely, an amino acid site Q in the SRCR5 domain of the extracellular region of porcine CD163 was mutated to R, and PK-15-CD163 cells with Q488R mutation were successfully prepared using ABEs technology. The cells can effectively inhibit the replication of HP-PRRSV but maintain normal expression of CD163 protein.

[0020] It provides a theoretical basis for obtaining a pig herd that can effectively inhibit PRRSV infection without affecting the growth performance of piglets and the physiological activity function of CD163, avoids the impact of CD163 gene modification on the normal physiological activity function of CD163 and the growth performance of piglets, and provides a new idea for the prevention and control research of PRRSV.

[0021] The method for preparing gene-edited pigs using CD163 mutated cells has the advantages of easy operation and strong universality. The prepared amino acid mutation gene-edited pigs have good PRRSV resistance while retaining normal expression of CD163 protein. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 Sequence alignment of CD163 SRCR5 domains of various species; Figure 2 A is a schematic diagram of sgRNA targeting Q488R amino acid mutation, B is a sequencing peak diagram of recombinant vector construction, and C is a sequencing peak diagram for evaluating the sgRNA point mutation efficiency / mutation efficiency and cutting efficiency of Q488R mutation; Figure 3 Sequencing peak profile constructed for evaluation of PK-15-CD163-Q488R cell line; Figure 4 This is the result of qPCR detection of PK-15-CD163-Q488R cells infected with HP-PRRSV-JXA1; Figure 5 This is a graph showing the results of FACS detection of PK-15-CD163-Q488R cells infected with HP-PRRSV-GD; Figure 6 This is the result of fluorescence observation of PK-15-CD163-Q488R cells infected with HP-PRRSV-JXA1-GFP; Figure 7 This is the result of qPCR detection of the mechanism by which PK-15-CD163-Q488R cells inhibit the proliferation of HP-PRRSV-JXA1. DETAILED DESCRIPTION

[0023] The technical scheme of the present invention will be clearly and completely described below in conjunction with the embodiments. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments in the present invention, all other embodiments obtained by ordinary technicians in the field without making creative work are within the scope of protection of the present invention. Unless otherwise specified, the professional and scientific terms used in this article have the same meanings as those familiar to those skilled in the art. In addition, any method or material similar or equal to the recorded content can also be applied to the present invention.

[0024] In addition, if no specific technical operation steps or conditions are specified in the examples, they are all carried out according to the general techniques or conditions described in the literature in the field or according to the product instructions. If no manufacturer is specified for the reagents or instruments used, they are all conventional products that can be obtained through commercial purchase.

[0025] A porcine CD163 protein mutant that can effectively inhibit type II PRRSV infection is provided in the present embodiment. Compared with the wild-type porcine CD163 protein, the mutation site of the porcine CD163 protein mutant is that the 488th amino acid is mutated from glutamine amino acid to arginine, and the other amino acid residues remain unchanged.

[0026] In this embodiment, ABEs and a guide sequence compatible with ABEs are used to mutate the 61st A in the 7th exon sequence of the wild-type pig CD163 gene to G. The nucleotide sequence of the 7th exon of the wild-type pig CD163 gene is shown in SEQ ID NO: 3, the nucleotide sequence of the 7th exon of the edited pig CD163 gene is shown in SEQ ID NO: 4, the amino terminal sequence of the 7th exon of the edited pig CD163 gene is shown in SEQ ID NO: 5, and the wild-type amino acid sequence is shown in SEQ ID NO: 6.

[0027] Example 1. Comparison of the amino acid sequences of CD163 from various species and selection of the Q488 site.

[0028] The amino acid sequences of CD163 of pigs, African green monkeys, humans, chimpanzees, macaques, rats, house mice, cattle, horses, rabbits, dogs, and house cats were searched in the NCBI database. By comparing the SRCR5 domains of CD163 of various species, it was found that except for Q (Gln) at position 488 in pigs, all others were K (Lys). This site is located in the CD163 ligand binding pocket related to PRRSV binding. Therefore, this site was selected for mutation to determine the effect of the mutation on PRRSV infection at the cellular level. The sequence comparison results of the SRCR5 domains of CD163 proteins of various species can be found in Figure 1 .

[0029] Example 2: Construction of sgRNA-74707 expression vector and verification of point mutation efficiency.

[0030] A sgRNA sequence was designed and synthesized to mutate the glutamine amino acid at position 488 of the porcine CD163 protein to arginine (CD163Q488R), and ABEs were used to achieve the conversion from A to G to obtain the porcine CD163 gene mutant.

[0031] The two single-stranded sgRNA DNA sequences were annealed to form an oligonucleotide chain of sgRNA targeting porcine CD163Q488R mutation; the oligonucleotide chain was then linked to the 74707 plasmid vector to obtain sgRNA-74707, and the successful construction of the expression vector was verified by Sanger sequencing. The sgRNA targeting the Q488R amino acid mutation can be found in Figure 2 A, the sequencing peak diagram of the recombinant vector construction can be found in Figure 2 B.

[0032] The sgRNA sequence is: 5-AAGTACAACATGGAGACACG-3. (SEQ ID NO: 7) 30 μg of sgRNA-74707 expression vector and NG-ABE8e plasmid were introduced into PK-15-CD163 cells by electroporation transfection. After 72 hours of transfection, 1 / 3 of the cells were collected to extract the cell genome. PCR reaction was performed using specific mutation identification primers. After sequencing the PCR products, the efficiency of point mutation was evaluated by analyzing the sequencing peak graph. The sequencing peak graph for evaluating the sgRNA cutting efficiency of the Q488R mutation can be found in Figure 2 C.

[0033] Example 3: Acquisition of PK-15-CD163-Q488R mutant cells.

[0034] The remaining cells from the above electrotransfection were evenly plated in a 100 mm cell culture dish at 600 cells / dish by limiting dilution, and the cell culture medium was replaced every 2 to 3 days. After 8 to 10 days, when the cell clones have grown, the cell clones are marked under a microscope, and these marked clones are picked into a 24-well cell culture plate for further culture. After 2 to 3 days, when the cells in the 24-well plate have grown to 80% confluence, 1 / 4 of the cells are taken and genomic DNA is extracted using NP40 lysis buffer, which is used as a template for PCR identification. After electrophoresis sequencing, the sequencing peak graph is analyzed to verify the porcine CD163 Q488R mutation event. The sequencing peak graph used to evaluate the construction of the PK-15-CD163-Q488R cell line can be found in Figure 3 .

[0035] Example 4: qPCR detection of PK-15-CD163-Q488R cells infected with HP-PRRSV-JXA1.

[0036] The PK-15-CD163-Q488R cells and PK-15-CD163 cells constructed above were inoculated at a certain density in equal 12-well cell culture plates. After the cells adhered, the cells were incubated with a virus suspension at MOI = 0.1 for 3 hours. After the virus suspension was aspirated, 1 mL of RPMI culture medium containing 5% fetal bovine serum was added. The cells were collected at 72 hpi, and 1 mL of TRNzol was added to extract total cell RNA. Reverse transcription was performed using 1 μg RNA as a template, and qPCR was performed using cDNA as a template to detect the PRRSV copy number. The anti-PRRSV effect of PK-15-CD163-Q488R cells was determined by comparing the quantitative results with those of the control group PK-15-CD163 cells. The results of qPCR detection of PK-15-CD163-Q488R cells infected with HP-PRRSV-JXA1 can be found in Figure 4 .

[0037] Example 5: FACS detection of PK-15-CD163-Q488R cells infected with HP-PRRSV-GD.

[0038] PK-15-CD163-Q488R cells and PK-15-CD163 cells were seeded at a certain density in a 100 mm cell culture dish. The cells were incubated with HP-PRRSV-GD at MOI=0.1 for 3 hours, and the cells were harvested at 48 hpi and fixed with tissue fixative. FACS was performed using a PRRSV-N protein-specific antibody. The anti-PRRSV ability of PK-15-CD163-Q488R cells was further confirmed by comparison with the control group PK-15-CD163 cells. The results of FACS detection of PK-15-CD163-Q488R cells infected with HP-PRRSV-GD can be found in Figure 5 .

[0039] Example 6: Fluorescence observation of PK-15-CD163-Q488R cells infected with HP-PRRSV-JXA1-GFP.

[0040] PK-15-CD163-Q488R cells and PK-15-CD163 cells were seeded in 12-well plates in equal amounts. The cells were incubated with HP-PRRSV-JXA1-GFP at MOI = 0.1 for 3 hours, and the cell nuclei were stained with Hochest at 48 hpi. The fluorescence intensity of PRRSV-GFP in the sample was observed by fluorescence. By comparing with the control group PK-15-CD163 cells, it was visually observed that PK-15-CD163-Q488R cells had the best resistance to PRRSV. The results of fluorescence observation of PK-15-CD163-Q488R cells infected with HP-PRRSV-JXA1-GFP can be found in Figure 6 .

[0041] Example 7: qPCR detection of the mechanism by which PK-15-CD163-Q488R cells inhibit the proliferation of HP-PRRSV-JXA1.

[0042] PK-15-CD163-Q488R cells and PK-15-CD163 cells were inoculated in 60 mm cell culture dishes in equal amounts. HP-PRRSV-JXA1 was inoculated with an MOI of 1 to ensure that there was one virus particle on the surface of each cell. After the above adsorption and invasion experiments, the total RNA of the adsorbed and invaded cells was extracted, and the relative expression level of PRRSV mRNA was detected by reverse transcription PCR and qPCR to reflect the adsorption and invasion of virus particles. PK-15-CD163-Q488R cells inhibited the proliferation of HP-PRRSV-JXA1 by qPCR. The results are shown in Figure 7 .

[0043] In summary, the present invention specifically obtained positive Q488R mutation PK-15-CD163 cells infected with PRRSV-2 in vitro through ABEs bases, and found that the cells can effectively inhibit the replication of the highly pathogenic HP-PRRSV-JXA1 strain by inhibiting its adsorption and invasion.

[0044] The porcine CD163 protein mutant provided by the present invention, the nucleotide sequence and the substance that enables the porcine CD163 protein to achieve the mutation can be used to prepare products for preventing or treating porcine reproductive and respiratory syndrome; or to prepare products for improving the resistance of organisms or organism cells to porcine reproductive and respiratory syndrome virus; or to prepare cells resistant to porcine reproductive and respiratory syndrome virus; or to prepare porcine reproductive and respiratory syndrome virus-resistant pigs; or to breed porcine reproductive and respiratory syndrome virus-resistant pigs; or to construct a porcine reproductive and respiratory syndrome virus-resistant cell model; or to construct a porcine reproductive and respiratory syndrome virus-resistant pig model. .

[0045] In the present invention, sgRNA expression vector and NG-ABE8e are transfected into an in vitro porcine fibroblast cell line to obtain PK-15-CD163-Q488R cells, which are then transplanted into sows through somatic cell nuclear transplantation, and the offspring produced are mutant pigs.

[0046] The present invention uses the CD163 protein mutant cells as nuclear transplant donor cells into sows through somatic cell nuclear transplantation, and the produced offspring are CD163 protein mutant gene-edited pigs.

[0047] The present invention introduces a substance that causes the above-mentioned mutation in CD163 protein into pig zygotic embryos to obtain CD163 gene-modified embryos, and then transplants the CD163 gene-modified embryos into the mother's body for pregnancy to obtain CD163 protein mutation gene-edited pigs, and uses the gene-edited pigs for breeding to obtain pigs resistant to porcine reproductive and respiratory syndrome virus.

[0048] Although the embodiments of the present invention have been shown and described above, it is to be understood that the above embodiments are exemplary and are not to be construed as limitations of the present invention. A person skilled in the art may change, modify, replace and vary the above embodiments within the scope of the present invention.

[0049] Pig CD163 nucleotide sequence: (where the a base at position 63323077 is changed to g)

Claims

1. A porcine CD163 protein mutant, characterized in that: Compared with the wild-type porcine CD163 protein, the porcine CD163 protein mutant has a mutation site in which the 488th amino acid is mutated from glutamine amino acid to arginine, and other amino acid residues remain unchanged. The porcine CD163 protein mutant inhibits PRRSV infection.

2. a nucleotide sequence encoding the pig CD163 protein mutant as claimed in claim 1.

3. the application of the pig CD163 protein mutant as claimed in claim 1 and the nucleotide sequence as claimed in claim 2 and the material that makes the pig CD163 protein realize the mutation as claimed in claim 1, it is characterized in that: Preparation of products for preventing or treating porcine reproductive and respiratory syndrome; or preparation of products for improving the resistance of the body or body cells to porcine reproductive and respiratory syndrome virus; or preparation of cells resistant to porcine reproductive and respiratory syndrome virus; or preparation of pigs resistant to porcine reproductive and respiratory syndrome virus; or breeding of pigs resistant to porcine reproductive and respiratory syndrome virus; or construction of a porcine reproductive and respiratory syndrome virus-resistant cell model; or construction of a porcine reproductive and respiratory syndrome virus-resistant pig model.

4. The use according to claim 3, characterized in that: The organism is a mammal.

5. The use according to claim 4, characterized in that: The mammal is a pig.

6. The use according to claim 3, characterized in that: The material that enables the porcine CD163 protein to achieve the mutation as claimed in claim 1 is ABEs and a guide sequence that matches ABEs.

7. The use according to claim 6, characterized in that: The guide sequence guides ABEs to achieve the following single base editing: The 61st A in the 7th exon of the wild-type CD163 gene was mutated to G in cooperation with ABEs. The nucleotide sequence of the wild-type CD163 gene is shown in SEQ ID NO:

8.

8. The use according to claim 6, characterized in that: The guiding sequence is shown in SEQ ID NO:

7.

9. A method for constructing CD163 protein mutant cells for non-diagnostic and non-therapeutic purposes, comprising the following steps: mutating the 488th position Q of the wild-type CD163 protein in pig kidney cells to R, while keeping other amino acid residues unchanged to obtain CD163 protein mutant cells.

10. A method for constructing a CD163 protein mutation gene-edited pig for non-diagnostic and non-therapeutic purposes, which is the following method 1 or method 2: The method 1 comprises the following steps: The 488th position Q of CD163 protein in isolated pig kidney was mutated to R, while other amino acid residues remained unchanged to obtain CD163 protein mutant cells; The CD163 protein mutant cells are used as nuclear transplant donor cells and are transplanted into sows through somatic cell nuclear transplantation, and the offspring produced are CD163 protein mutant gene-edited pigs; The method 2 comprises the following steps: The substance causing the above mutation in CD163 protein is introduced into pig zygotic embryos to obtain CD163 gene-modified embryos, and then the CD163 gene-modified embryos are transplanted into the mother's body for pregnancy to obtain CD163 protein mutation gene-edited pigs.

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