Construction method and application of overexpression growth inhibition and DNA damage induction gene 45 gamma adenovirus
By constructing and applying the overexpressing GADD45γ adenovirus, the treatment problem of rheumatoid arthritis was solved, and the disease progression and joint damage were slowed down in the CIA mouse model was achieved, providing a new target for the treatment of rheumatoid arthritis.
Patent Information
- Application Number
- CN202411481080.7
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-10-23
- Publication Date
- 2025-02-21
AI Technical Summary
The prior art has not yet fully identified the cause and pathogenesis of rheumatoid arthritis, and there is a lack of rapid and effective prevention and treatment methods.
By constructing the adenovirus of overexpression growth inhibition and DNA damage-induced gene 45γ (GADD45γ), the recombinant shuttle plasmid of the GADD45γ gene was used to transform E. coli cells and adenovirus backbone plasmid to generate the recombinant GADD45γ adenovirus plasmid, and obtained through 293 cell packaging Infecting viable adenovirus particles, the recombinant GADD45γ adenovirus was finally purified.
This method slows down the disease process in the CIA mouse model and avoids joint destruction. By inhibiting inflammatory cytokines related to rheumatoid arthritis, it delays the occurrence and development of the disease, and prevents and diagnoses rheumatoid arthritis. and treatments provide new targets.
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Figure CN119491020A_ABST
Abstract
Description
Technical Field
[0001] The invention relates to the field of cell biotechnology, in particular to a construction method and application of an adenovirus for overexpressing a growth inhibition and DNA damage induction gene 45γ. Background Art
[0002] Rheumatoid arthritis (RA) is an autoimmune disease characterized by chronic inflammation in the synovium of joints, which can cause joint swelling and pain, leading to cartilage destruction, narrowing of the inter-articular space, joint deformity in the late stage, and finally varying degrees of disability. It is more common in middle-aged women. The main pathological characteristics are inflammatory reactions in the synovium of joints, including synovial hyperplasia, infiltration of a large number of inflammatory cells in the interstitial layer, pannus formation, cartilage and bone destruction, microangiogenesis, and synovial cells expressing multiple activation antigens. It is a disease with a high disability rate, which seriously affects the daily life and work of patients, and also imposes a huge economic burden on the patients' families and society. However, the etiology and pathogenesis of RA have not yet been fully clarified, and no rapid and effective prevention and treatment methods have been developed.
[0003] GADD45 is a member of the family of growth inhibition and DNA damage-induced genes, and plays an important role in radiation-induced apoptosis, cell cycle arrest, DNA damage repair, and autoimmune diseases. The GADD45 family has three members: GADD45α (GADD45), GADD45β (MyD118), and GADD45γ (CR6). These molecules are very conservative in evolution. The Gadd45 gene family has a high degree of homology (amino acid level is about 55% to 58%) and is strongly acidic, with an isoelectric point of 4.0 to 4.2. At present, there are no reports on whether GADD45γ is related to the pathogenesis of RA at home and abroad. Summary of the invention
[0004] In order to overcome the above-mentioned shortcomings of the prior art, the present invention provides a construction method and application of an adenovirus overexpressing the growth inhibitory and DNA damage inducing gene 45γ.
[0005] The technical solution adopted by the present invention to solve the technical problem is: a method for constructing an adenovirus overexpressing the growth inhibition and DNA damage inducing gene 45γ, characterized in that it comprises the following steps:
[0006] 1) Construction of recombinant shuttle plasmid containing GADD45γ gene;
[0007] The GADD45γ gene was cloned into the multiple cloning site of the pShuttle-CMV plasmid by restriction digestion and ligation using the EcoR V and XhoI restriction sites to construct a recombinant shuttle plasmid containing the GADD45γ gene;
[0008] 2) Construction of recombinant GADD45γ adenovirus plasmid;
[0009] The recombinant shuttle plasmid containing the GADD45γ gene and the adenovirus backbone plasmid were co-transformed into Escherichia coli cells to generate the recombinant GADD45γ adenovirus plasmid through homologous recombination;
[0010] 3) Construction of recombinant GADD45γ adenovirus;
[0011] The recombinant GADD45γ adenovirus plasmid was transfected into 293 cells, and adenovirus particles with infectious activity were obtained by packaging in 293 cells, and amplified in 293 cells. After the packaging cells were collected and lysed, the recombinant GADD45γ adenovirus was purified.
[0012] As a further improvement of the present invention: the construction of the recombinant GADD45γ adenovirus plasmid in step 2) comprises the following steps:
[0013] (1) Preparing electrocompetent Escherichia coli;
[0014] (2) co-transforming E. coli cells with the recombinant shuttle plasmid containing the GADD45γ gene and the adenovirus backbone plasmid;
[0015] (3) extracting the recombinant GADD45γ adenovirus plasmid by alkaline lysis method and performing restriction digestion identification; performing single restriction digestion with PacI and identifying the recombinant GADD45γ adenovirus plasmid of the positive clone by 0.8% agarose gel electrophoresis;
[0016] (4) Take 1-5 μl of the positive recombinant GADD45γ adenovirus plasmid and transform it into DH5α Escherichia coli cells, amplify the bacteria, and purify the plasmid.
[0017] As a further improvement of the present invention: the preparation of electroporation competent Escherichia coli in step (1) comprises the following steps:
[0018] From a fresh agar plate, pick a single E. coli and inoculate it in LB medium, and shake it at 37°C overnight; inoculate 25 ml of the above culture in 500 ml of LB medium, and place it in a 37°C shaker until OD600 reaches 0.4; place the culture in an ice bath for 30 minutes to cool it down sufficiently; transfer the bacterial solution to a pre-cooled centrifuge tube, centrifuge it at 2500r / min for 15 minutes at 4°C, discard the supernatant, and recover the cells; wash the cells with 10 ml of pre-cooled 10% glycerol, centrifuge at low temperature, and repeat twice; add an appropriate amount of pre-cooled 10% glycerol to resuspend the cells, dilute the suspension 100 times, measure OD600, and dilute to a concentration of 2-3×1010 cells / ml (1.0OD600 is about 2.5×1010 cells / ml); package and store at -80°C for use.
[0019] As a further improvement of the present invention: the step (2) of co-transforming Escherichia coli cells with the recombinant shuttle plasmid containing the GADD45γ gene and the adenovirus backbone plasmid comprises the following steps:
[0020] Add 1-5 μl (about 1 μg) of the recombinant shuttle plasmid containing the GADD45γ gene and 1 μl (about 100 ng / μl) of the viral backbone plasmid pAdEasy-1 to the EP tube containing 40 μl of BJ5183 electroporation competent cells, mix well, and cool on ice; add the mixture to the electroporation cup and perform electric shock (1250 V / mm, 5 ms); after the electric shock, take out the sample, add 1 ml of LB culture medium, and shake at low speed at 37°C for 40 min; take the electroporated transformed cell fluid and spread it on several kanamycin-resistant plates (25-50 mg / ml), and culture at 37°C for 16-20 h; the next day, pick the colonies grown on the plates, inoculate them into 3 ml of LB culture medium, and culture at 37°C for 10-15 h.
[0021] As a further improvement of the present invention: the construction of the recombinant GADD45γ adenovirus in step 3) comprises:
[0022] The recombinant GADD45γ adenovirus plasmid was transfected into 293 cells, and adenovirus particles with infectious activity were obtained by packaging in 293 cells and amplified in 293 cells. When the cells became round and half of the cells appeared to float, all the cells were collected and centrifuged at 500g, the supernatant was discarded, sterilized PBS was added, the cells were resuspended, and the cells were repeatedly frozen and thawed 4 times; centrifuged at 7000g for 5 minutes at 4°C; CsCl continuous gradient centrifugation purification, 4.4g CsCl was weighed, 8ml of virus lysis supernatant was added to a 50ml centrifuge tube, the volume was about 10ml after mixing, and the tube was transferred to a 12ml ultracentrifuge tube, covered with 2ml mineral oil, balanced, centrifuged at 10°C, 32000rpm for 18-24h, and the virus band centrifuged was extracted with a syringe; dialysate was prepared, sterilized, dialyzed at 4°C, CsCl was removed after the dialysate was replaced 3 times, and the tube was stored at -80°C to obtain the purified recombinant GADD45γ adenovirus.
[0023] As a further improvement of the present invention: it also includes: 4) identification of the recombinant GADD45γ adenovirus;
[0024] The transfection efficiency of the recombinant GADD45γ adenovirus was identified by real-time fluorescence quantitative PCR. Total RNA was extracted from transfected 293A cells and reverse transcribed into cDNA, followed by real-time fluorescence quantitative PCR to identify the expression of the GADD45γ gene.
[0025] A use of the recombinant GADD45γ adenovirus in preparing a drug for treating rheumatoid arthritis.
[0026] Compared with the prior art, the present invention has the following beneficial effects:
[0027] The present invention successfully induced arthritis in DBA mice by homemade bovine type II collagen, detected the changes of GADD45γ in the disease process of CIA mice, and used collagen-induced CIA mice as the object to explore the correlation between GADD45γ and IL-1β, IL-6 and TNF-α, revealing the important role of GADD45γ signal in the occurrence and development of rheumatoid joints. Overexpression of GADD45γ adenovirus slowed down the disease process of CIA mice and avoided joint destruction of CIA mice, thereby playing a role in delaying the occurrence and development of CIA. It provides new methods and new theories for the prevention and diagnosis of RA in clinical practice, and provides new targets for the treatment of rheumatoid arthritis and autoimmune diseases. BRIEF DESCRIPTION OF THE DRAWINGS
[0028] Figure 1 The results of enzyme digestion identification.
[0029] Figure 2 Adenovirus overexpressing GADD45γ expressed green fluorescent protein in 293A cells.
[0030] Figure 3 Empty adenovirus vector expressing green fluorescent protein in 293A cells.
[0031] Figure 4 Adenovirus overexpressing GADD45γ was used in 293A cells.
[0032] Figure 5 This is the electrophoresis result of bovine cartilage sample after pepsin digestion.
[0033] Figure 6 This is the electrophoresis result of the precipitate obtained by centrifugation of dialyzed bovine type II collagen.
[0034] Figure 7 Comparison results of extracted bovine type II collagen and standard.
[0035] Figure 8 These are the knee joint sections of mice in the blank control group.
[0036] Fig. 9 This is a section of the knee joint of mice in the CIA-Mock experimental group.
[0037] Fig.10 These are knee joint sections of mice in the CIA-GADD45γ experimental group.
[0038] Fig.11 This is a section of the knee joint of mice in the CIA-Mock experimental group. DETAILED DESCRIPTION
[0039] In this document, relational terms such as first and second, etc. are used only to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Moreover, the terms "include", "comprises" or any other variants thereof are intended to cover non-exclusive inclusion, so that a process, method, article or device including a series of elements includes not only those elements, but also other elements not explicitly listed, or also includes elements inherent to such process, method, article or device. The present invention is further described in conjunction with the accompanying drawings and implementation examples:
[0040] A method for constructing an adenovirus that overexpresses the growth inhibitory and DNA damage-inducing gene 45γ, characterized in that it comprises the following steps:
[0041] 1) Construction of recombinant shuttle plasmid containing GADD45γ gene;
[0042] The GADD45γ gene was cloned into the multiple cloning site of the pShuttle-CMV plasmid to construct a recombinant shuttle plasmid containing the GADD45γ gene;
[0043] 2) Construction of recombinant GADD45γ adenovirus plasmid;
[0044] The recombinant shuttle plasmid containing the GADD45γ gene and the adenovirus backbone plasmid were co-transformed into Escherichia coli cells to generate the recombinant GADD45γ adenovirus plasmid through homologous recombination;
[0045] 3) Construction of recombinant GADD45γ adenovirus;
[0046] The recombinant GADD45γ adenovirus plasmid was transfected into 293 cells, and adenovirus particles with infectious activity were obtained by packaging in 293 cells and amplified in 293 cells. When the cells became round and half of the cells appeared to float, all cells were collected and centrifuged at 500g, the supernatant was discarded, sterilized PBS was added, the cells were resuspended, and the cells were repeatedly frozen and thawed 4 times; 4°C, 7000g centrifugation for 5min; CsCl continuous gradient centrifugation purification, 4.4g CsCl was weighed, 8ml of virus lysis supernatant was added to a 50ml centrifuge tube, the volume was about 10ml after mixing, and the tube was transferred to a 12ml ultracentrifuge tube, covered with 2ml mineral oil, balanced, 10°C, 32000rpm centrifugation for 18-24h, and the virus band centrifuged was extracted with a syringe; dialysate was prepared, sterilized, dialyzed at 4°C, CsCl was removed after changing the dialysate 3 times, and the tube was stored at -80°C to obtain the purified recombinant GADD45γ adenovirus;
[0047] 4) Identification of recombinant GADD45γ adenovirus;
[0048] The transfection efficiency of the recombinant GADD45γ adenovirus was identified by real-time fluorescence quantitative PCR. Total RNA was extracted from transfected 293A cells and reverse transcribed into cDNA, followed by real-time fluorescence quantitative PCR to identify the expression of the GADD45γ gene.
[0049] Furthermore, the construction of the recombinant GADD45γ adenovirus plasmid in step 2) comprises the following steps:
[0050] (1) Preparing electrocompetent Escherichia coli;
[0051] From the fresh agar plate, pick a single E. coli and inoculate it in LB medium, and place it in a 37°C shaker overnight; inoculate 25 ml of the above culture in 500 ml of LB medium, and place it in a 37°C shaker until OD600 reaches 0.4; place the culture in an ice bath for 30 minutes to cool it down sufficiently; transfer the bacterial solution to a pre-cooled centrifuge tube, centrifuge it at 2500r / min for 15 minutes at 4°C, discard the supernatant, and recover the cells; wash the cells with 10 ml of pre-cooled 10% glycerol, centrifuge at low temperature, and repeat twice; add an appropriate amount of pre-cooled 10% glycerol to resuspend the cells, dilute the suspension 100 times, measure OD600, and dilute to a concentration of 2-3×1010 cells / ml (1.0OD600 is about 2.5×1010 cells / ml); package and store at -80°C for use;
[0052] (2) co-transforming E. coli cells with the recombinant shuttle plasmid containing the GADD45γ gene and the adenovirus backbone plasmid;
[0053] Add 1-5μl (about 1μg) of the recombinant shuttle plasmid containing the GADD45γ gene and 1μl (about 100ng / μl) of the viral backbone plasmid pAdEasy-1 to the EP tube containing 40μl BJ5183 electroporation competent cells, mix well, and cool on ice; add the mixture to the electroporation cup and perform electric shock (1250V / mm, 5ms); after the electric shock, take out the sample, add 1ml of LB culture medium, and shake at a low speed at 37℃ for 40min; take the electroporation transformation cell liquid and apply it to several kanamycin resistance plates (25-50mg / ml), and culture at 37℃ for 16-20h; the next day, pick the colonies grown on the plate, inoculate them into 3ml of LB culture medium, and culture at 37℃ for 10-15h;
[0054] (3) extracting the recombinant GADD45γ adenovirus plasmid by alkaline lysis method and performing restriction digestion identification; performing single restriction digestion with PacI and identifying the recombinant GADD45γ adenovirus plasmid of the positive clone by 0.8% agarose gel electrophoresis;
[0055] (4) Take 1-5 μl of the positive recombinant GADD45γ adenovirus plasmid and transform it into DH5α Escherichia coli cells, amplify the bacteria, and purify the plasmid.
[0056] A use of the recombinant GADD45γ adenovirus in preparing a drug for treating rheumatoid arthritis.
[0057] Implementation Cases:
[0058] Experimental animals: 75 DBA mice, 8-12 weeks old, all male, weighing 20-35 g, housed in an SPF animal room;
[0059] The DBA mice were divided into three groups as shown in Table 1 below:
[0060]
[0061]
[0062] Table 1 Experimental animal groups and treatment methods
[0063] 1. Enzyme digestion and identification of recombinant adenovirus plasmid
[0064] The GADD45γ gene fragment was inserted into the pShuttle plasmid multiple cloning site by restriction digestion and ligation using EcoR V and XhoI restriction sites, and then identified by restriction digestion;
[0065] The results of enzyme digestion identification are shown in the attached Figure 1 As shown, where: M is a protein molecular weight marker, and lanes L1-L5 are recombinant expression fragments.
[0066] 2. Packaging and Identification of Recombinant Adenovirus
[0067] The plasmid containing the recombinant GADD45γ adenovirus was transfected into the packaging cell 293A cells. After 24 hours, green fluorescence could be observed under a fluorescence microscope. After 3-5 days of transfection, typical cytopathic effect (CPE) appeared in 293A cells. Figure 2 and Figure 3 The titer of Ad-GADD45γ was 1X10~10pfu / ml, and the titer of empty vector adenovirus was 5X10~9pfu / ml.
[0068] 3. Extraction and Identification of Bovine Type II Collagen
[0069] 3.1 SDS-PAGE electrophoresis of bovine cartilage after pepsin digestion
[0070] Take 20ul, 15ul, and 10ul of the crude protein extract and load them for SDS-PAGE electrophoresis. After the electrophoresis, the gel is stained and destained, and then photographed and analyzed using a gel imaging system. Figure 5 As shown in the figure, the molecular weight of bovine type II collagen is about 130 kD. M is a protein molecular weight marker; 1, 2, and 3 are crude extracts of bovine type II collagen.
[0071] 3.2 SDS-PAGE electrophoresis after dialysis
[0072] The precipitate obtained after dialysis was loaded with 10ul and 20ul samples for SDS-PAGE gel electrophoresis. After the electrophoresis, the gel was stained and destained, and then photographed and analyzed using a gel imaging system. Figure 6As shown in the figure, the molecular weight of bovine type II collagen is about 130 kD. M is a protein molecular weight marker; 1 and 2 are precipitates precipitated in 50 mM Tris-HCl buffer.
[0073] 3.3 SDS-PAGE electrophoresis detection of extracted bovine type II collagen and standard
[0074] Take 10ul of the dialyzed sample, 10ul of the ion exchange sample, and 25ul of the bovine type II collagen solution from Sigma for SDS-PAGE gel electrophoresis. After the electrophoresis, take a photo of the stained and destained gel using a gel imaging system and analyze the photo. Figure 7 As shown in the figure, the molecular weight of bovine type II collagen is about 130 kD, which is consistent with the size of the standard product (bovine type II collagen from Sigma). Among them, M is the protein molecular weight marker; 1 is the sample after dialysis; 2 is the sample after ion exchange; 3 is the standard product of Sigma.
[0075] 4. Establishment and evaluation of animal model of collagen-induced rheumatoid arthritis
[0076] Bovine type II collagen was used to induce the establishment of rheumatoid arthritis CIA in DBA mice, and the effect of GADD45γ on the occurrence and development of arthritis in CIA mice was observed. The mice were immunized subcutaneously with 100ul of bovine type II collagen containing Freund's complete adjuvant, and 21 days later, they were immunized subcutaneously with 100ul of bovine type II collagen containing Freund's complete adjuvant. On the 35th day, the mice were processed for analysis of related experiments.
[0077] The CIA model mice began to develop the disease on the 21st day and disappeared on the 42nd day, with an incidence rate of 100%, while the control group did not develop the disease. The joint clinical scores, paw swelling thickness and incidence rate of the CIA model group mice were higher than those of the control group mice, and this lasted until the 42nd day. Compared with the control group, the right hind limb joints and paws of the CIA mice were significantly red and swollen, and they could not bear weight and had difficulty walking. In the late stage of the disease, the joints gradually became deformed. In order to observe the pathological changes in the joints of the CIA model mice, the knee joints of the CIA group and the control group mice were fixed, decalcified, and HE stained. It was found that compared with the joint sections of the control group mice, the knee joints of the CIA group mice had obvious synovial hyperplasia, and a large number of inflammatory cells infiltrated the synovial tissue, the synovium became thicker and disordered, the number of cell layers increased, and the synovial pannus invaded the articular cartilage and bone to cause joint damage. The control group mice had normal joint spaces, regular articular surfaces and synovium, and neatly arranged cells ( Figure 8 and Fig. 9). Enzyme-linked immunosorbent assay was used to detect the levels of IgG, IgG1, and IgG2a in the serum of mice in the CIA group and the control group. It was found that the protein expression levels of IgG and IgG2a increased, while the expression level of IgG1 decreased significantly, both of which were significant. Enzyme-linked immunosorbent assay was used to detect collagen-specific IgG antibodies in the serum of mice in the CIA group and the control group. It was found that the collagen-specific IgG antibodies in the serum of CIA model mice were significantly higher than those in the control group. The experimental results showed that the CIA arthritis model of DBA mice was successfully induced using bovine type II collagen. The arthritis symptoms of this CIA model were similar to those of clinical RA, with severe destruction of bone and articular cartilage, and a large number of inflammatory cells infiltrating lubricating tissues.
[0078] 5. Changes of GADD45γ during the course of CIA disease
[0079] To detect the expression of GADD45γ in the disease progression of collagen-induced arthritis mice, real-time fluorescence quantitative PCR was used to detect the level of GADD45γ in CIA model mice. It was found that GADD45γ was expressed in macrophages, synovial tissue and peripheral blood monocytes during the course of CIA disease, and there was a significant increase in synovial tissue and macrophages at the peak of the disease.
[0080] To verify the effect of GADD45γ on the disease of CIA model mice, an adenovirus overexpressing GADD45γ was constructed and injected into the joint cavity of CIA animal model mice to observe the effect of GADD45γ on the occurrence and development of CIA mouse disease. First, 100ul of emulsified bovine II collagen containing Freund's complete adjuvant was injected subcutaneously into the tail of mice for a single immunization. On the 19th day, the adenovirus overexpressing GADD45γ was injected into the joint cavity of mice. On the 21st day, 100ul of bovine II collagen containing Freund's complete adjuvant was injected subcutaneously into the tail of mice for a secondary immunization. On the 25th day, the adenovirus overexpressing GADD45γ was injected into the joint cavity for a boost. On the 40th day, the mice were processed for analysis of related experiments. According to the results, GADD45γ significantly attenuated the splenomegaly caused by CIA compared with the CIA control group mice. The DBA-GADD45γ-ex and DBA-Mock experimental group mice were scored using the arthritis scoring method (0-3). 0 points: no redness or swelling in the joints; 1 point: mild swelling and erythema in the joints; 2 points: obvious swelling in the joints; 3 points: severe swelling in the joints and unable to bear weight; the total score of each mouse's limbs was averaged and the average clinical score of the joints was obtained. Compared with the CIA control group mice, the joint clinical score of the experimental group injected with GADD45γ overexpression adenovirus was reduced, and there was a significant difference on the 40th day after immunization and lasted until the 60th day.
[0081] To observe the effect of GADD45γ on the joint pathology of CIA mice, the knee joints of CIA mice and mice injected with GADD45γ overexpression adenovirus were fixed, decalcified, and stained with HE. It was found that compared with the obvious synovial hyperplasia, typical pannus formation, and severe destruction of articular cartilage and bone in CIA mice, the synovial tissue of the knee joints of the mice in the GADD45γ overexpression group had only slight hyperplasia, normal joint space, regular articular surface and synovium, and neatly arranged cells ( Fig.10 and Fig.11 ), and the swelling of the paws of CIA mice injected with GADD45γ overexpression adenovirus was significantly less than that of the CIA control group. The results of ELISA detection of mouse serum proinflammatory cytokine protein levels showed that GADD45γ overexpression adenovirus had a significant inhibitory effect on proinflammatory cytokines IL-1β, IL-6, and TNF-α; real-time fluorescence quantitative PCR results showed that GADD45γ overexpression adenovirus had different degrees of inhibitory effects on collagen-induced arthritis (CIA)-related proinflammatory cytokines IL-1β, IL-6, and TNF-α in synovium, macrophages, and peripheral blood lymphocytes. The experimental results show that overexpression of GADD45γ adenovirus slowed down the disease progression of CIA mice, avoided joint destruction in CIA mice, and inhibited inflammation by inhibiting inflammatory cytokines associated with rheumatoid arthritis.
[0082] The present invention uses bovine type II collagen to induce DBA mice to establish a CIA animal model, detects the changes of GADD45γ in the disease process of CIA mice, and the results show that GADD45γ is involved in the pathogenesis of CIA. In order to study the correlation between GADD45γ and proinflammatory cytokines IL-1β and IL-6TNF-α related to rheumatoid arthritis, a GADD45γ overexpression adenovirus is constructed and a CIA animal model is induced. The inhibitory effect of GADD45γ on the expression of these proinflammatory cytokines in peripheral blood lymphocytes, synovial tissues and macrophages in the CIA disease animal model is explored. In order to verify the effect of our homemade bovine type II collagen in inducing RA, we successfully established a CIA animal model by injecting an emulsion of DBA mouse collagen and adjuvant. The experimental results show that a CIA animal model similar to RA can be induced by homemade bovine type II collagen, and a more shortcut method for studying rheumatoid arthritis animal models has been found. On this basis, the changes of GADD45γ in the course of CIA disease were detected, and it was found that GADD45γ was highly expressed in synovial tissue and macrophages during the onset of CIA mice. Therefore, an adenovirus overexpressing GADD45γ was constructed and injected into the joint cavity of CIA model mice. Real-time fluorescence quantitative PCR histochemical detection found that GADD45γ overexpression inhibited the expression of proinflammatory cytokines IL-1β, IL-6, and TNF-α associated with rheumatoid arthritis in synovial tissue, peripheral blood monocytes, and macrophages. When the knee joints of the injected GADD45γ overexpression adenovirus and the CIA control group were detected by histochemical detection, it was found that compared with the obvious synovial hyperplasia of CIA mice, typical pannus formation, and severe destruction of articular cartilage and bone, the synovial tissue of the knee joints of the GADD45γ overexpression group of mice had only slight hyperplasia, normal joint space, regular articular surface and synovium, and neatly arranged cells.
[0083] The experimental results showed that adenovirus overexpressing GADD45γ slowed down the disease progression of CIA mice and avoided joint destruction in CIA mice, suggesting that GADD45γ may delay the occurrence and development of CIA by inhibiting the expression of proinflammatory cytokines IL-1β, IL-6, and TNF-α associated with rheumatoid arthritis.
[0084] In summary, the present invention successfully induced arthritis in DBA mice by homemade bovine type II collagen, and on this basis detected the changes of GADD45γ in the disease progression of CIA mice. At the same time, we used collagen-induced CIA mice as the object, and explored the correlation between GADD45γ and IL-1β, IL-6 and TNF-α, revealing the important role of GADD45γ signaling in the occurrence and development of rheumatoid joints, providing new methods and new theories for the prevention and diagnosis of RA in clinical practice, and providing new targets for the treatment of rheumatoid arthritis and autoimmune diseases.
[0085] The above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit the same. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that the technical solutions described in the aforementioned embodiments may still be modified, or some or all of the technical features thereof may be replaced by equivalents. Such modifications or replacements do not deviate the essence of the corresponding technical solutions from the scope of the technical solutions of the embodiments of the present invention, and should all be included in the scope of the claims and specification of the present invention.
Claims
1. A method for constructing an adenovirus overexpressing the growth inhibitory and DNA damage inducing gene 45γ, characterized in that: The following steps are involved: 1) Construction of recombinant shuttle plasmid containing GADD45γ gene; The GADD45γ gene was cloned into the multiple cloning site of the pShuttle-CMV plasmid by restriction digestion and ligation using the EcoR V and XhoI restriction sites to construct a recombinant shuttle plasmid containing the GADD45γ gene; 2) Construction of recombinant GADD45γ adenovirus plasmid; The recombinant shuttle plasmid containing the GADD45γ gene and the adenovirus backbone plasmid were co-transformed into Escherichia coli cells to generate the recombinant GADD45γ adenovirus plasmid through homologous recombination; 3) Construction of recombinant GADD45γ adenovirus; The recombinant GADD45γ adenovirus plasmid was transfected into 293 cells, and adenovirus particles with infectious activity were obtained by packaging in 293 cells, and amplified in 293 cells. After the packaging cells were collected and lysed, the recombinant GADD45γ adenovirus was purified.
2. The method for constructing an adenovirus overexpressing the growth inhibitory and DNA damage-inducing gene 45γ according to claim 1, characterized in that: The construction of the recombinant GADD45γ adenovirus plasmid in step 2) comprises the following steps: (1) Preparing electrocompetent Escherichia coli; (2) co-transforming E. coli cells with the recombinant shuttle plasmid containing the GADD45γ gene and the adenovirus backbone plasmid; (3) extracting the recombinant GADD45γ adenovirus plasmid by alkaline lysis method and performing restriction digestion identification; performing single restriction digestion with PacI and identifying the recombinant GADD45γ adenovirus plasmid of the positive clone by 0.8% agarose gel electrophoresis; (4) Take 1-5 μl of the positive recombinant GADD45γ adenovirus plasmid and transform it into DH5α Escherichia coli cells, amplify the bacteria, and purify the plasmid.
3. The method for constructing an adenovirus overexpressing the growth inhibitory and DNA damage-inducing gene 45γ according to claim 2, characterized in that: The step (1) of preparing electrocompetent Escherichia coli comprises the following steps: From a fresh agar plate, pick a single E. coli and inoculate it in LB medium, and shake it at 37°C overnight; inoculate 25 ml of the above culture in 500 ml of LB medium, and place it in a 37°C shaker until OD600 reaches 0.4; place the culture in an ice bath for 30 minutes to cool it down sufficiently; transfer the bacterial solution to a pre-cooled centrifuge tube, centrifuge it at 2500r / min for 15 minutes at 4°C, discard the supernatant, and recover the cells; wash the cells with 10 ml of pre-cooled 10% glycerol, centrifuge at low temperature, and repeat twice; add an appropriate amount of pre-cooled 10% glycerol to resuspend the cells, dilute the suspension 100 times, measure OD600, and dilute to a concentration of 2-3×1010 cells / ml (1.0OD600 is about 2.5×1010 cells / ml); package and store at -80°C for use.
4. The method for constructing an adenovirus overexpressing the growth inhibitory and DNA damage-inducing gene 45γ according to claim 3, characterized in that: The step (2) of co-transforming Escherichia coli cells with the recombinant shuttle plasmid containing the GADD45γ gene and the adenovirus backbone plasmid comprises the following steps: Add 1-5 μl (about 1 μg) of the recombinant shuttle plasmid containing the GADD45γ gene and 1 μl (about 100 ng / μl) of the viral backbone plasmid pAdEasy-1 to the EP tube containing 40 μl of BJ5183 electroporation competent cells, mix well, and cool on ice; add the mixture to the electroporation cup and perform electric shock (1250 V / mm, 5 ms); after the electric shock, take out the sample, add 1 ml of LB culture medium, and shake at low speed at 37°C for 40 min; take the electroporated transformed cell solution and spread it on several kanamycin-resistant plates (25-50 mg / ml), and culture at 37°C for 16-20 h; the next day, pick the colonies grown on the plates, inoculate them into 3 ml of LB culture medium, and culture at 37°C for 10-15 h.
5. The method for constructing an adenovirus overexpressing the growth inhibitory and DNA damage-inducing gene 45γ according to claim 4, characterized in that: The construction of the recombinant GADD45γ adenovirus in step 3) comprises: The recombinant GADD45γ adenovirus plasmid was transfected into 293 cells, and adenovirus particles with infectious activity were obtained by packaging in 293 cells and amplified in 293 cells. When the cells became round and half of the cells appeared to float, all the cells were collected and centrifuged at 500g, the supernatant was discarded, sterilized PBS was added, the cells were resuspended, and the cells were repeatedly frozen and thawed 4 times; centrifuged at 7000g for 5 minutes at 4°C; CsCl continuous gradient centrifugation purification, 4.4g CsCl was weighed, 8ml of virus lysis supernatant was added to a 50ml centrifuge tube, the volume was about 10ml after mixing, and the tube was transferred to a 12ml ultracentrifuge tube, covered with 2ml mineral oil, balanced, centrifuged at 10°C, 32000rpm for 18-24h, and the virus band centrifuged was extracted with a syringe; dialysate was prepared, sterilized, dialyzed at 4°C, CsCl was removed after the dialysate was replaced 3 times, and the tube was stored at -80°C to obtain the purified recombinant GADD45γ adenovirus.
6. The method for constructing an adenovirus overexpressing the growth inhibitory and DNA damage inducing gene 45γ according to claim 5, characterized in that: Also includes: 4) Identification of recombinant GADD45γ adenovirus; The transfection efficiency of recombinant GADD45γ adenovirus was identified by real-time fluorescence quantitative PCR; Total RNA was extracted from transfected 293A cells and reverse transcribed into cDNA, which was then used for real-time fluorescence quantitative PCR to identify the expression of GADD45γ gene.
7. Use of the recombinant GADD45γ adenovirus according to any one of claims 1 to 6 in the preparation of a drug for treating rheumatoid arthritis.