Gene editing cell and method for producing extremely high molecular weight hyaluronic acid by using cell

Abstract

The invention provides a gene editing cell and a method for producing extremely high molecular weight hyaluronic acid by using the cell. The preparation method comprises the following steps: by taking PUC57 as a skeleton carrier, constructing a recombinant plasmid containing an HAS2 gene, and transfecting a mole NMRHas2 gene, a chicken CHICKHAS2 gene, an astronomy mouse SNMHAS2 gene, a dakalimeris mouse DMRHAS2 gene or a mole mouse BMRHAS2 gene into chicken fibroblasts through lipidosome to construct a stably transfected cell line, so that hyaluronic acid with extremely high molecular weight can be stably obtained. The method provided by the invention overcomes the defects of non-uniform molecular weight distribution of HA product sources, low molecular weight and single method in the prior art, and has important significance for developing medicines for treating osteoarthritis and novel medical equipment products.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biotechnology, in particular to a genetically edited cell and a method for producing very high molecular weight hyaluronic acid using the cell. BACKGROUND

[0002] Hyaluronic acid (HA) is a type of acid mucopolysaccharide distributed in the extracellular matrix (ECM) of vertebrate organs and blood and soft connective tissues. Natural hyaluronic acid has a simple chemical composition. HA is a chain-like high molecular polymer formed by the repeated alternation of glucuronic acid and N-acetylglucosamine through β-1, 3 and β-1, 4 glycosidic bonds. High molecular weight hyaluronic acid (HMM-HA) has anti-inflammatory, anti-hyperalgesia, anti-proliferation and anti-stress effects. In particular, it has been found in related studies that very high molecular weight hyaluronic acid (vHMM-HA) has been shown to have stronger antioxidant stress protection effects than human high molecular weight hyaluronic acid (HMM-HA). In addition, the very high molecular weight hyaluronic acid (vHMM-HA) found has also been shown to greatly contribute to animals' resistance to cancer and other age-related diseases.

[0003] High molecular weight HA has good applications in the medical field due to its physical and chemical and biological effect characteristics. Osteoarthritis (OA) is the most common form of arthritis and is a chronic disease characterized by slow degradation of cartilage, pain and increasing degree of disability. The disease has a negative impact on many aspects of the patient's life, including function and social activities, body image and emotional health. Intra-articular treatment with hyaluronic acid (HA) has recently been more widely accepted in the treatment of osteoarthritis pain. Hyaluronic acid (HA) determines the viscoelasticity of synovial fluid. Compared with healthy joints, the synovial fluid in osteoarthritis joints contains lower concentrations and smaller molecular weights of hyaluronic acid. Therefore, the goal of intra-articular hyaluronic acid treatment is to help replace the synovial fluid that has lost viscoelasticity. Several clinical trials have demonstrated the effectiveness and tolerability of intra-articular injection of hyaluronic acid in the treatment of knee osteoarthritis-related pain. In addition, HA has the effect of preventing tissue adhesion and promoting wound healing after surgery. Anti-adhesion is achieved by using the space blocking effect of HA, and studies have shown that the larger the molecular weight (Mr) of HA, the larger the space occupied by the molecule, and the stronger the physical blocking and lubricating effects. Similarly, in ophthalmic surgery, high molecular weight hyaluronic acid (generally selected with a molecular weight of 3 x 106) is used to prevent postoperative adhesion of the cornea and iris, and to prevent the adhesion of the cornea and iris to the lens. In addition, HA has the effect of preventing the adhesion of the cornea and iris to the lens. In addition, HA has the effect of preventing the adhesion of the cornea and iris to the lens. 6The viscoelasticity of the above HA) is good, the flowability is weak, and the molecular barrier is good, which can support the anterior chamber as a gel, provide a clear view for the operation, facilitate the operation, and reduce the damage to the corneal epithelial cells. At the same time, the viscoelastic agent can press the bleeding point, play a molecular barrier role, and inhibit the movement, proliferation, differentiation and phagocytosis of cells, reduce the postoperative inflammatory reaction, and the low molecular product cannot. However, due to the limitation of the size of the molecular weight of the current natural hyaluronic acid and the easy degradation characteristics, it is often necessary to modify the hyaluronic acid through cross-linking technology to achieve a higher molecular weight to better play its biomedical effect, so the breakthrough from high molecular weight hyaluronic acid to extremely high molecular weight hyaluronic acid has important significance for its application in the field of biomedicine.

[0004] The current method for obtaining HA is mainly divided into animal tissue extraction method, chemical synthesis method and microbial fermentation method. Among them, the biological method for obtaining HA has good performance and optimized process, and the inherent properties of hyaluronic acid (HA) must be maintained during the process, so the above method is standardized. However, the HA obtained by the above method cannot meet the development and use of higher quality medical products due to the limitation of the size of the molecular weight. At the same time, all the above methods have the problems of uneven distribution of molecular weight size, low molecular weight, complex composition, risk of cross contamination, etc. At present, it has been reported that the extremely high molecular weight HA obtained is more than 6 MDa (referring to CN 118345118 A), but this method is single and not multiple, and finding a new method to achieve better results can effectively promote the research and industrial application in this field. SUMMARY

[0005] In view of the shortcomings of the prior art described above, the purpose of the present application is to provide a kind of gene editing cell and the method for producing extremely high molecular weight hyaluronic acid by using cell, to solve the molecular weight size distribution of HA product source in prior art is uneven, the molecular weight is low, the method is single and the shortcomings of the prior art.

[0006] To achieve the above-mentioned purpose and other related purposes, the present application provides a kind of gene editing cell, the cell is the chicken fibroblast integrated with HAS2 gene, the HAS2 gene is any one or several of the mole NMRHas2 gene, chicken CHICKHAS2 gene, star mole SNMHAS2 gene, damalan mole DMRHAS2 gene or mole BMRHAS2 gene.

[0007] Further, the HAS2 gene is located in a plasmid.

[0008] Further, the plasmid uses PUC57 as a backbone vector and adds protein tracing and positive cell screening elements. Those skilled in the art can use the commonly used plasmids in the prior art.

[0009] Further, the plasmid is PUC57-CAG-HAS2-mCherry-Puro. The CAG is an exogenous gene high-efficiency strong promoter, mCherry is a red fluorescent protein element, and Puro is a puromycin resistance gene.

[0010] Another aspect of the present application provides the use of the above-mentioned gene edited cell for preparing the ultra-high molecular weight hyaluronic acid.

[0011] Further, the ultra-high molecular weight hyaluronic acid has a molecular weight greater than 6 MDa. In some embodiments, the molecular weight is greater than 8 MDa.

[0012] Another aspect of the present application provides a method for preparing hyaluronic acid, specifically comprising the following steps:

[0013] 1) constructing a plasmid containing the HAS2 gene;

[0014] 2) transfecting the plasmid into chicken fibroblasts by liposomes, and screening positive cells;

[0015] 3) culturing the cell line and collecting the supernatant of the culture medium;

[0016] 4) treating the supernatant with alcohol precipitation method, and freeze-drying the precipitate to obtain hyaluronic acid powder.

[0017] Further, the step 2) of screening positive cells specifically uses puromycin to screen positive cells.

[0018] Further, the step 2) of screening positive cells further comprises adding a tracer element to generate fluorescent protein in the plasmid.

[0019] Further, the fluorescent protein is mCherry.

[0020] Another aspect of the present application prepares an ultra-high molecular weight hyaluronic acid. The hyaluronic acid secreted by the chicken fibroblasts overexpressing the chicken HAS2 gene has a molecular weight greater than 6 MDa, and the hyaluronic acid overexpressing the NMRHas2 gene of naked mole rat, the SNMHAS2 gene of star-nosed mole, the DMRHAS2 gene of damaran mole or the BMRHAS2 gene of mole rat has a molecular weight greater than 8 MDa.

[0021] As described above, the method for producing ultra-high molecular weight hyaluronic acid using cells according to the present application has the following beneficial effects:

[0022] The chicken embryo fibroblasts are used to prepare hyaluronic acid with a molecular weight greater than 8 MDa. BRIEF DESCRIPTION OF DRAWINGS

[0023] Figure 1The chicken fibroblast carrier map capable of secreting high molecular weight hyaluronic acid provided by the application is constructed, wherein the HAS2 can be naked mole rat NMRHas2 gene, chicken CHICKHAS2 gene, star-nosed mole SNMHAS2 gene, damalin mouse DMRHAS2 gene or mole-shaped mouse BMRHAS2 gene;

[0024] Figure 2 The agarose gel electrophoresis map of the naked mole rat NMRHas2 gene, chicken CHICKHAS2 gene, star-nosed mole SNMHAS2 gene, damalin mouse DMRHAS2 gene or mole-shaped mouse BMRHAS2 gene provided by the application is provided;

[0025] Figure 3 The double enzyme digestion identification map of different species PUC57-CAG-HAS2-mCherry-Puro provided by the application is provided;

[0026] Figure 4 The microimaging map of the chicken fibroblast expressing the naked mole rat NMRHas2 gene, chicken CHICKHAS2 gene, star-nosed mole SNMHAS2 gene, damalin mouse DMRHAS2 gene or mole-shaped mouse BMRHAS2 gene provided by the application is provided;

[0027] Figure 5 The RT-PCR technology for detecting the expression of various HAS2 genes in the gene-edited chicken fibroblast provided by the application is provided;

[0028] Figure 6 The Western blot technology for detecting the expression of HAS2 protein in the gene-edited chicken fibroblast provided by the application is provided;

[0029] Figure 7 The immunofluorescence technology for detecting the expression of HAS2 protein and hyaluronic acid binding protein (HABP) in the gene-edited chicken fibroblast provided by the application is provided;

[0030] Figure 8 The gel electrophoresis technology for detecting the secreted high molecular weight hyaluronic acid of the gene-edited chicken fibroblast provided by the application is provided. DETAILED DESCRIPTION

[0031] In order to solve the defects of uneven molecular weight size distribution, low molecular weight and single method of the existing HA product source, we provide a new method for producing high molecular weight HA, which provides new raw materials for promoting the development of medical products of HA and promotes the application and development of medical HA.

[0032] Construct a vector that can produce high molecular weight hyaluronic acid, which includes a CAG promoter, and any one of naked mole rat NMRHAS2 gene, chicken CHICKHAS2 gene, star-nosed mole SNMHAS2 gene, damalin rat DMRHAS2 gene, mole rat BMRHAS2 gene, in addition, in order to monitor the expression of the exogenous HAS2 gene, we use red fluorescent protein to trace it, and add the puromycin resistance gene Puro for screening of positive cells.

[0033] Wherein the gene sequences of naked mole rat NMRHAS2, star-nosed mole SNMHAS2, damalin rat DMRHAS2, mole rat BMRHAS2 are all synthesized in vitro, and the chicken HAS2 gene is cloned from chicken embryo fibroblasts. The CAG sequence and other elements include IRES, mCherry, 2A sequence and Puro are all synthesized, and the above elements are cloned into the PUC57 backbone vector to obtain five recombinant plasmid vectors containing naked mole rat NMRHas2 gene, chicken CHICKHAS2 gene, star-nosed mole SNMHAS2 gene, damalin rat DMRHAS2 gene, mole rat BMRHAS2 gene.

[0034] The above overexpression plasmid is transfected into chicken fibroblasts using liposomes, and chicken fibroblasts expressing naked mole rat NMRHas2 gene, chicken CHICKHAS2 gene, star-nosed mole SNMHAS2 gene, damalin rat DMRHAS2 gene, mole rat BMRHAS2 gene are screened to obtain five gene editing cell lines.

[0035] The above gene editing cell lines are cultured for 3 days, and the supernatant of the above gene editing cell lines is collected. The above medium is subjected to ethanol precipitation method, 15ml of anhydrous ethanol is added to 10ml of cell culture supernatant to obtain a precipitate, and the collected precipitate is freeze-dried to obtain hyaluronic acid powder.

[0036] The hyaluronic acid secreted by the chicken fibroblast overexpressing the chicken HAS2 gene has a molecular weight greater than 6 MDa by gel electrophoresis technology; the hyaluronic acid secreted by the chicken fibroblast overexpressing the NMRHas2 gene of naked mole rat, the SNMHAS2 gene of star-nosed mole, the DMRHAS2 gene of damaran mole and the BMRHAS2 gene of mole rat has a molecular weight greater than 8 MDa. Such high molecular weight hyaluronic acid has been confirmed to have high anti-inflammatory ability, can reduce the release of inflammatory factors and protect cell viability (Takasugi M, Firsanov D, Tombline G, Ning H, Ablaeva J, Seluanov A, Gorbunova V. Naked mole-rat very-high-molecular-mass hyaluronan exhibits superior cytoprotective properties. Nat Commun. 2020 May 12; 11 (1): 2376.). The present application provides technical support for the development of clinical anti-inflammatory drugs.

[0037] Unless otherwise defined, technical or scientific terms used herein should be understood as commonly understood by one of ordinary skill in the art to which the present application pertains.

[0038] The advantages and effects of the present application can be easily understood by those skilled in the art from the disclosure of the present specification. The present application can also be implemented or applied by other different specific embodiments, and the details in the specification can be modified or changed based on different views and applications without departing from the spirit of the present application.

[0039] Before further describing the specific embodiments of the present application, it should be understood that the scope of protection of the present application is not limited to the following specific embodiments; it should also be understood that the terms used in the embodiments of the present application are used to describe the specific embodiments, but not to limit the scope of protection of the present application; in the specification and claims of the present application, unless otherwise explicitly stated, the singular forms "one", "a" and "this" include the plural forms.

[0040] When the embodiments give numerical ranges, it is understood that unless the present invention indicates otherwise, every numerical range's two endpoints, and any number between the two endpoints, can be selected. Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as commonly understood by one of ordinary skill in the art. Except in the examples, or where otherwise explicitly indicated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Unless otherwise specified, any method, device, material or reagent of the present invention can be used in accordance with the present invention, with any method, device, material or reagent of the present invention being similar or equivalent to those described in the embodiments of the present invention.

[0041] Unless otherwise indicated, the experimental methods, detection methods, preparation methods disclosed in the present invention all use conventional techniques in the fields of molecular biology, biochemistry, chromatin structure and analysis, analytical chemistry, cell culture, recombinant DNA technology and related fields.

[0042] Example 1 Construction of Recombinant Plasmid

[0043] As shown in Figure 1 , the backbone vector is PUC57, and CAG sequence (the promoter has a higher starting effect compared to other promoters such as CMV, EF1-α, etc.) is synthesized in Shanghai General Biosynthesis, and the CDs sequences of naked mole rat NMRHAS2 gene (genbank ID: 101724130), star-nosed mole SNMHAS2 gene (genbank ID: 101625921), damalin rat DMRHAS2 gene (genbank ID: 104870025), mole-shaped rat BMRHAS2 gene (genbank ID: 103738152), and chicken CHICKHAS2 gene (genbank ID: 395594) are synthesized. The cDNA sequence of CHICKHAS2 gene is cloned from chicken embryo fibroblasts. The HAS2 sequences of the above species are all 1659 bp in length.

[0044] Sequence 1: CAG sequence (SEQ ID NO. 1)

[0045]

[0046] The method for extracting the chicken CHICKHAS2 gene is as follows: 5x10 5 The cell amount of the chicken embryo fibroblast is extracted by using the Trizol method. Then, the reverse transcription kit of Novozyme is used to carry out the reverse transcription library, 500 ng of total RNA is used for reverse transcription, and the transcription product is stored at -20℃ for standby. The chicken embryo fibroblast is isolated and cultured from a 12-day chicken embryo, the culture medium is high-sugar DMEM, 10% FBS, and the culture is carried out in a 37℃, 5% CO2 incubator.

[0047] The amplification primers of the chicken CHICKHAS2 gene are as follows: Forward: 5'-ATGTATTGTGAGAGGTTTAT-3' (SEQ ID NO. 2), Reverse: 5'-TCATACATCTAGCACCATGT-3' (SEQ ID NO. 3); the cDNA of the obtained chicken CHICKHAS2 gene is used as a template to carry out the conventional PCR amplification. The PCR system is as follows: premix Extaq 25 μl, 50 pmol / L of each of the upper and lower primers, 0.1 μg of the cDNA template, and water is added to 50 μl. The reaction program is as follows: pre-denaturation at 94℃ for 3 min, denaturation at 94℃ for 30 s, annealing at 60℃ for 30 s, extension at 72℃ for 30 s, 34 cycles, extension at 72℃ for 10 min, and 16℃. Then, 1% agarose gel electrophoresis is carried out for identification. Then, the product is recovered from the gel for sequencing, the sequencing result of Shengong is consistent with the expected sequence, and is used for subsequent experiments.

[0048] As shown in Figure 2 , the naked mole rat NMRHas2 gene, the star-nosed mole snmHAS2 gene, the damaran mole dmrHAS2 gene, the mole-shaped mouse bmrHAS2 gene, and the chicken CHICKHAS2 gene are subjected to gel electrophoresis identification, and then they are respectively cloned and linked with CAG, mCherry, Puro and other elements, as shown in Figure 1 , the recombinant HAS2 plasmid is finally constructed, and is subjected to agarose gel electrophoresis identification after enzyme digestion as shown in Figure 3 , the size is consistent with the expectation, and it is subjected to sequencing identification, the sequencing result of Shengong is 100% homologous to the expected sequence, and is stored at -20℃ for subsequent experiments.

[0049] Example 2 Construction of HAS2-overexpressing cells

[0050] The successfully cloned recombinant HAS2 plasmids were respectively transfected into chicken fibroblasts by liposome, and the single clone screening was performed by limiting dilution method to establish five kinds of gene edited chicken fibroblast cell lines respectively expressing naked mole rat NMRHas2 gene, chicken CHICKHAS2 gene, star-nosed mole SNMHAS2 gene, damaran mole DMRHAS2 gene and mole-shaped mouse BMRHAS2 gene.

[0051] As shown in Figure 4 , the expression of mCherry fluorescence was observed, indicating the successful expression of the exogenous gene HAS2. In addition, the above gene edited cell lines were collected and subjected to genome extraction, and the above genes were subjected to PCR amplification identification, as shown in Figure 2 , the exogenous genes were amplified and subjected to sequencing detection, which was consistent with the expected sequence.

[0052] The gene edited chicken fibroblasts overexpressing naked mole rat NMRHas2 gene, chicken CHICKHAS2 gene, star-nosed mole SNMHAS2 gene, damaran mole DMRHAS2 gene and mole-shaped mouse BMRHAS2 gene were cultured to logarithmic growth phase, and the cells were collected. The trizol method was used to extract RNA, and the Norgen reverse transcription kit was used for reverse transcription to cDNA. The transcription level of HAS2 gene in the gene edited cells was detected by qPCR method, as shown in Figure 5 , the transcription level of HAS2 gene in all gene edited cells was increased compared with the control group. In addition, the above transgenic cells were collected to extract protein, and the WB method was used to detect the expression level of HAS2 protein, as shown in Figure 6 , the results showed that the expression of HAS2 protein in the gene edited cells was significantly improved compared with the control group. The above results showed that the exogenous naked mole rat NMRHas2 gene, chicken CHICKHAS2 gene, star-nosed mole SNMHAS2 gene, damaran mole DMRHAS2 gene and mole-shaped mouse BMRHAS2 gene were successfully overexpressed in chicken fibroblasts.

[0053] Hyaluronic acid binding protein (HABP) can bind to intracellular HA, which can indirectly analyze the content change of intracellular HA. We performed HABP immunofluorescence staining on the gene edited chicken fibroblasts overexpressing naked mole rat NMRHas2 gene, chicken CHICKHAS2 gene, star-nosed mole SNMHAS2 gene, damaran mole DMRHAS2 gene and mole-shaped mouse BMRHAS2 gene, and found that the protein expression of HABP was analyzed, as shown in Figure 7As shown, compared with the control group, the expression level of HABP protein in gene-edited cells was significantly upregulated, indicating a significant increase in intracellular HA content. These results demonstrate that exogenous overexpression of the naked mole rat NMRHas2 gene, chicken CHICKHAS2 gene, star-nosed rat SNMHAS2 gene, damaran rat DMRHAS2 gene, and mole rat BMRHAS2 gene can synthesize large amounts of HA in chicken fibroblasts.

[0054] Example 3: Identification of the molecular weight of HA

[0055] Gene-edited chicken fibroblasts overexpressing the NMRHas2 gene from naked mole rats, the CHICKHAS2 gene from chickens, the SNMHAS2 gene from star-nosed rats, the DMRHAS2 gene from damalan rats, and the BMRHAS2 gene from mole rats were cultured, and the supernatant was collected after 72 hours of culture. The cells and cell debris were first removed by centrifugation at 300g for 10 minutes. Then, the supernatant was precipitated using anhydrous ethanol by adding 15ml of anhydrous ethanol to 10ml of cell culture supernatant. The collected precipitate was lyophilized to obtain hyaluronic acid powder, which was then dissolved in 1×TE buffer to obtain a hyaluronic acid solution.

[0056] The molecular weight of hyaluronic acid was identified by gel electrophoresis. The specific steps included: preparing a 0.4% gold gel; adding 5 μL of 4 mol / L sucrose buffer to 25 μL of sample, mixing and spotting the mixture into the gel wells, and spotting 10 μL of hyaluronic acid marker into the gel wells; running the gel overnight at 4℃ and 75V; gel staining: placing the electrophoresed gel in 0.005% (w / v) Stains-All and staining overnight in the dark; destaining: placing the gel in deionized water for 48 h in the dark, and then placing it under white light at room temperature for 4 h; and taking images under white light.

[0057] like Figure 8 As shown, the cell culture medium of chicken fibroblasts overexpressing the HAS2 gene for 3 days revealed that the HAS2 gene-modified cells secreted extremely high molecular weight hyaluronic acid with a molecular weight greater than 6 MDa; while chicken fibroblasts overexpressing the naked mole rat NMRHas2 gene, star-nosed rat SNMHAS2 gene, damaran rat DMRHAS2 gene, and mole rat BMRHAS2 gene secreted hyaluronic acid with a molecular weight greater than 8 MDa.

[0058] The foregoing examples are to be construed as merely illustrative of the presently disclosed embodiments, and do not exhaust the scope of the present application. Furthermore, various modifications to both the examples listed herein and the methods and compositions of the application will be apparent to those skilled in the art, and this application is intended to encompass such modifications within the scope and spirit of the application. Although the application has been described in conjunction with specific preferred embodiments thereof, it will be understood that the application is not limited to these embodiments. In fact, various modifications to the described embodiments will be apparent to those skilled in the art, and the general principles defined herein can be applied to other embodiments. Therefore, to the extent that modifications of the described embodiments incorporating some but not other features of the application are within the spirit and scope of the application, they should be and are intended to be included within the scope of the application.

Claims

1. A gene-editing cell, characterized in that, The cells are chicken fibroblasts with an integrated exogenous HAS2 gene, and the HAS2 gene is any one or more of the following: mole rat NMRHas2 gene, chicken CHICKHAS2 gene, star-nosed rat SNMHAS2 gene, damaran rat DMRHAS2 gene, or mole rat BMRHAS2 gene.

2. The gene-edited cell according to claim 1, characterized in that, The HAS2 gene is located in a plasmid.

3. The gene-edited cell according to claim 2, characterized in that, The plasmid uses PUC57 as its backbone vector and contains tracer and positive cell selection elements.

4. The gene-edited cell according to claim 3, characterized in that, The plasmid is PUC57-CAG-HAS2-mCherry-Puro.

5. Use of the gene-edited cells as described in any one of claims 1-4 for the preparation of extremely high molecular weight hyaluronic acid.

6. The use according to claim 5, characterized in that, The extremely high molecular weight hyaluronic acid has a molecular weight greater than 6 MDa.

7. A method for preparing hyaluronic acid, characterized in that, Specifically, the following steps are included: 1) Construct a plasmid containing the HAS2 gene; 2) The plasmid was transfected into chicken fibroblasts via liposomes, and positive cells were screened out; 3) Cell line culture, collect the supernatant from the culture medium; 4) The supernatant is treated by alcohol precipitation, and the precipitate is freeze-dried to obtain hyaluronic acid powder.

8. The method for preparing hyaluronic acid according to claim 7, characterized in that, In step 2), positive cells are specifically obtained through puromycin screening.

9. A method for preparing hyaluronic acid according to claim 7, characterized in that, The screening of positive cells in step 2) also includes adding a tracer element to the plasmid to generate fluorescent protein.

10. A method for preparing hyaluronic acid according to claim 9, characterized in that, The fluorescent protein is mCherry.

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