Application of SAA1 as target spot in preparation of medicine for preventing and treating kidney diseases

Abstract

The invention discloses application of SAA1 as a target spot in preparation of a medicine for preventing and treating kidney diseases, and belongs to the field of gene functions, application and biological medicine. In-vivo and in-vitro experiments prove that in an ischemia / reperfusion or ureteral obstruction induced mouse kidney disease model, the knockout of the SAA1 gene can relieve renal interstitial vascular endothelial cell injury, recover a renal capillary network, improve vascular permeability, reduce collagen deposition, inhibit renal fibrosis and effectively delay the progress of kidney diseases; furthermore, by adopting the SAA1 recombinant protein vaccine to immunize mice, the organism can be induced to generate a protective neutralizing antibody, and the SAA1 recombinant protein vaccine has a remarkable protective effect on kidney diseases induced by ischemia / reperfusion and ureteral obstruction. Therefore, the SAA1 is an intervention target for preventing and treating the kidney diseases, the SAA1 recombinant protein vaccine is an effective means for preventing and treating the kidney diseases, and active immunization by adopting the vaccine is an effective strategy for preventing and treating the kidney diseases.

Description

Technical Field

[0001] The present invention belongs to the field of gene function and application and biomedicine, and specifically relates to the application of SAA1 as a target in the preparation of drugs for the prevention and treatment of kidney diseases. Background Art

[0002] Kidney disease is a global public health issue and a common and serious disease in my country's medical field. According to statistics, the number of chronic kidney disease patients progressing to end-stage renal disease is increasing at a rate of 5-6% per year, placing a heavy burden on families and society. How to effectively prevent the occurrence of kidney disease, delay the progression of the disease, reduce the incidence of end-stage renal disease, effectively improve the quality of life of patients, and save social medical resources are huge difficulties and challenges facing the current medical community.

[0003] In response to this urgent medical problem, previous studies have reached a consensus that the pathological basis and common pathway leading to the progression of kidney disease to end-stage renal disease is mainly tubulointerstitial fibrosis, which is usually referred to as renal fibrosis. The degree of renal fibrosis is closely related to the prognosis of chronic kidney disease and is also a decisive factor leading to loss of renal function. The evolution of renal fibrosis mainly includes damage and shedding of tubular epithelial cells, dysfunction of vascular endothelial cells, infiltration of inflammatory cells, proliferation and activation of fibroblasts, etc. Histologically, typical pathological features such as tubular atrophy and collapse, decreased density of capillary network, and excessive deposition of extracellular matrix appear, which eventually lead to permanent loss of renal structure and function.

[0004] At present, there is still a lack of effective clinical prevention and treatment measures for patients with kidney disease. Given that the loss of peritubular capillary networks leading to renal tissue ischemia and hypoxia is one of the important reasons for accelerating the progression of kidney disease, reducing endothelial cell damage, restoring capillary network density, and improving tissue blood circulation are effective means to prevent and delay the occurrence and development of kidney disease, and are also a hot topic of research in recent years.

[0005] Serum amyloid A1 (SAA1) is an acute phase response protein. Under physiological conditions, its content in the body is extremely low. It is mainly secreted into the blood by liver cells and participates in cholesterol transport and lipid metabolism. Once the body suffers trauma, infection or stress, SAA1 expression rises rapidly. Clinically, it has been used together with C-reactive protein as an auxiliary indicator for early diagnosis, risk assessment, efficacy observation and prognosis evaluation of infectious diseases. Recent studies have also found that under pathological conditions, the rapidly elevated SAA1 in the body can recognize a variety of membrane surface receptors and transmit various instructions to the cells, such as binding to Toll-like receptor 2, promoting granulocyte proliferation and inducing the production of IL-12, IL-23, and G-CSF; binding to TLR-4 to activate downstream ERK1 / 2 and p38 MAPKs signals, inducing macrophages to produce NO; binding to scavenger receptors to mediate the uptake of cholesterol by macrophages; binding to the receptor for terminal glycosylation products RAGE to promote the release of serum high mobility group protein and induce inflammatory response. In summary, SAA1 regulates many cells through receptors, mediates inflammatory responses, lipid metabolism, and substance transport, and participates in infection, wound repair, and the occurrence of metabolic diseases and tumors. However, existing technologies have not yet studied how SAA1 causes kidney disease and whether it can be used as a drug target. Summary of the invention

[0006] The primary purpose of the present invention is to provide the use of SAA1 as a target in the preparation of drugs for the prevention and treatment of kidney diseases.

[0007] Another object of the present invention is to provide a SAA1 recombinant protein vaccine for preventing and treating kidney diseases.

[0008] Another object of the present invention is to provide the use of the above-mentioned SAA1 recombinant protein vaccine for preventing and treating kidney diseases.

[0009] To achieve the above object, the present invention provides the following solutions:

[0010] The application of SAA1 as a target in the preparation of drugs for the prevention and treatment of kidney diseases is based on the present invention's first discovery that SAA1 is an important kidney damage molecule, which binds to receptors on the surface of vascular endothelial cell membranes to induce vascular endothelial cell damage, leading to ischemia and hypoxia in kidney tissue and aggravating the progression of kidney disease; genetic knockout of SAA1 or blocking its recognition with receptors can reduce renal interstitial vascular endothelial cell damage, restore renal capillary network density and reduce vascular permeability, reduce extracellular matrix deposition, effectively delay the progression of renal fibrosis and improve renal function.

[0011] The kidney diseases include acute kidney injury and chronic kidney disease.

[0012] The application of drugs that can inhibit SAA1 expression or block SAA1 from recognizing receptors in the prevention and treatment of kidney diseases. The present invention prepares a recombinant protein vaccine for the prevention and treatment target SAA1, which can produce high-titer anti-SAA1 neutralizing antibodies in vivo, block its binding to cell membrane surface receptors, antagonize the pathological damage of SAA1, effectively prevent and delay the occurrence and development of kidney diseases, and improve renal vascular rarefaction and interstitial fibrosis.

[0013] A recombinant protein vaccine targeting SAA1 includes a fusion protein hSAA1-LTB of human SAA1, a linker and an immune adjuvant molecule, Escherichia coli heat-labile enterotoxin B subunit (LTB).

[0014] The recombinant protein vaccine targeting SAA1 also includes an adjuvant.

[0015] In the recombinant protein vaccine targeting SAA1, the fusion protein hSAA1-LTB solution with a concentration of 5 to 100 μg / mL and the emulsified adjuvant are mixed in a mass ratio of 40 to 60:40 to 60; more preferably, they are mixed in a mass ratio of 50:50.

[0016] The concentration of the fusion protein hSAA1-LTB solution is preferably 5-15 μg / mL; more preferably 10 μg / mL.

[0017] The adjuvants include emulsified adjuvants and aluminum gel adjuvants.

[0018] The emulsified adjuvant is preferably an ISA adjuvant; more preferably at least one of ISA 201VG adjuvant, ISA 71VG adjuvant and ISA15AVG.

[0019] The amino acid sequence of the fusion protein hSAA1-LTB is shown in SEQ ID NO.2.

[0020] The DNA sequence of the fusion protein hSAA1-LTB is optimized as shown in SEQ ID NO.1, and the sequence is more suitable for the prokaryotic expression system.

[0021] The fusion protein hSAA1-LTB can be obtained by chemical synthesis or expressed by genetic engineering.

[0022] The use of the above-mentioned recombinant protein vaccine targeting SAA1 in the preparation of drugs for the prevention and treatment of kidney diseases.

[0023] The kidney disease and its accompanying renal vascular rarefaction and interstitial fibrosis are caused by pathological factors such as renal ischemia / reperfusion or ureteral obstruction.

[0024] The present invention discloses the following technical effects:

[0025] (1) The present invention proves through cell experiments that SAA1 can induce damage, inflammation, apoptosis and endothelial-mesenchymal transformation of vascular endothelial cells, and inhibit the migration and angiogenesis ability of vascular endothelial cells.

[0026] (2) The present invention has demonstrated through animal experiments that in vivo knockout of SAA1 can significantly reduce renal interstitial vascular endothelial cell damage in a mouse kidney disease model, restore renal capillary network density and reduce vascular permeability, reduce extracellular matrix deposition and improve renal fibrosis, exert a kidney-protective effect and delay the progression of kidney disease.

[0027] (3) The present invention designs and prepares a SAA1 recombinant protein vaccine based on the molecular characteristics of SAA1 (a small molecule secretory protein with multiple receptors). Animal experiments have confirmed that the vaccine can induce the production of SAA1 neutralizing antibodies in mice and effectively antagonize the damaging effects of SAA1, protect the capillary network loss caused by various pathological factors such as ischemia / reperfusion and ureteral obstruction, reduce renal tissue fibrosis, reduce vascular endothelial cell damage and restore capillary network density, delay the progression of kidney disease, and improve renal function.

[0028] In summary, as an important vascular injury molecule, SAA1 can be used as a new target for the prevention and treatment of kidney diseases, and can be used to prepare new drugs for the prevention and treatment of kidney diseases, reducing tissue fibrosis, and improving tissue blood supply. Furthermore, the SAA1 recombinant protein vaccine we prepared has the above-mentioned protective effects and no obvious toxic and side effects, and can become a new and effective means of preventing and treating kidney diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 The graphs show the results of SAA1 inducing vascular endothelial cell injury, inflammation, apoptosis and endothelial-mesenchymal transition in vitro; A is a photograph of the expression changes of molecules related to endothelial injury, inflammation, apoptosis and endothelial-mesenchymal transition detected by protein immunoblotting (Western Blot); B to H are the statistical results of the changes in the molecular protein levels in A.

[0030] Figure 2 The graphs show the results of SAA1 inhibiting the migration and angiogenesis of vascular endothelial cells; wherein A is a photograph of the effect of SAA1 on the migration ability of vascular endothelial cells detected by invasion experiment, and C is the statistical result graph of A; B is a photograph of the effect of SAA1 on the angiogenesis ability of vascular endothelial cells detected by angiogenesis experiment, and D is the statistical result graph of B.

[0031] Figure 3Schematic diagram of the construction of renal ischemia / reperfusion model for wild-type mice (+ / +) and SAA1 knockout mice (— / —).

[0032] Figure 4 This is a graph showing the results of using biochemical methods to detect changes in serum creatinine levels in different mice.

[0033] Figure 5 The Western Blot method was used to detect the expression changes of SAA1 and fibrosis molecules in different mice and the statistical results; A is a photograph of the Western Blot detection results, and B to F are the statistical results of the changes in the molecular protein levels in A.

[0034] Figure 6 The figures are the test results of extracellular matrix deposition, expression of vascular marker Endomucin and interstitial leakage; A is a photograph of extracellular matrix deposition such as renal interstitial collagen fibers detected by immunohistochemical staining and Masson's staining, B to D are the statistical results in A; E is the result of detecting the expression of vascular marker Endomucin (EMCN) by immunohistochemical staining and interstitial leakage of FITC-dextran (Dextran, 150kD) detected by in vivo vascular permeability experiment.

[0035] Figure 7 Figure 2 is a diagram showing the preparation and detection results of the ureteral obstruction model; A is a schematic diagram of the construction of the ureteral obstruction model in wild-type mice (+ / +) and SAA1 gene knockout mice (- / -); B to D are the Western Blot method used to detect the changes in SAA1 and fibrosis molecules and the statistical results; E to H are the immunohistochemical staining and Masson staining methods used to detect the deposition of extracellular matrix such as renal interstitial collagen fibers and the statistical results.

[0036] Figure 8 These are photographs of the expression of the vascular marker EMCN in the ureteral obstruction model detected by immunohistochemical staining and the interstitial leakage of FITC-dextran detected by in vivo vascular permeability experiment.

[0037] Fig. 9 The figure is a graph showing the detection result of agarose gel electrophoresis for double enzyme digestion identification; wherein, lane M is DNA Marker, lane 1 is the product of double enzyme digestion with Bam HI and Hind III, and lane 2 is the product of double enzyme digestion with EcoR I and Hind III.

[0038] Fig.10 The figure is the expression and purification result diagram of hSAA1-LTB recombinant fusion protein; wherein, A is the expression level of hSAA1-LTB recombinant fusion protein in engineered bacteria, and B is the purification chromatogram of hSAA1-LTB recombinant fusion protein.

[0039] Fig.11 A diagram showing the preparation of hSAA1 recombinant protein vaccine and the comparative results of immunogenicity; wherein A is a photo of the appearance of the emulsified vaccine prepared with hSAA1-LTB recombinant fusion protein and adjuvant ISA201VG; B is a photo of the appearance of the adsorbed vaccine prepared with hSAA1-LTB recombinant fusion protein and adjuvant aluminum hydroxide; C is a schematic diagram of the immunization strategy of two different dosage forms of SAA1 recombinant protein vaccines; and D is the result of serum antibody detection of immunized mice.

[0040] Fig.12 Schematic diagram for vaccine immunization strategy and mouse kidney disease model construction.

[0041] Fig.13 This is the result of detecting anti-SAA1 antibodies in the sera of mice in each group using the Western blot method.

[0042] Fig.14 The biochemical test results of serum creatinine (A) and serum urea nitrogen (B) in mice are shown in Figure 2.

[0043] Fig.15 The results of Western Blot method detection of expression changes of fibrosis molecules; A is a photograph of the Western Blot detection results, and B and C are the statistical results of the changes in the protein levels of fibrosis molecules in A.

[0044] Fig.16 The results of immunohistochemical staining and Masson's staining to detect the deposition of extracellular matrix such as renal interstitial collagen fibers; A is a photograph of the staining results, and B to D are statistical results.

[0045] Fig.17 This is the result of detecting the expression of vascular marker EMCN by immunohistochemical staining.

[0046] Fig.18 The figure is a result diagram of the expression change of intercellular adhesion molecule-1 (ICAM-1), a marker of endothelial injury, detected by Western Blot method; wherein A is a photograph of the Western Blot detection result, and B is the statistical result of A.

[0047] Fig.19 Schematic diagram for vaccine immunization strategy and mouse chronic kidney disease model construction.

[0048] Fig. 20 The graphs show the expression changes of fibrosis molecules and vascular injury molecules detected by Western Blot method; A is a photograph of the Western Blot detection results, and B to C are the statistical results of A.

[0049] Fig.21 These are the results of immunohistochemical staining and Masson's staining to detect extracellular matrix deposition such as renal interstitial collagen fibers and the expression of the vascular marker EMCN; A is a photograph of the staining results, and B to D are statistical results. DETAILED DESCRIPTION

[0050] In order to enable those skilled in the art to better understand the scheme of the present invention, the technical scheme in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are partial embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the protection scope of the present invention.

[0051] Example 1: SAA1 induces vascular endothelial cell injury in vitro

[0052] 1. Experimental Materials

[0053] Cells: Human Umbilical Vein Endothelial Cells (HUVEC), ATCC, USA, catalog number PCS-100-010.

[0054] Culture medium: DMEM high glucose medium containing 10% (v / v) FBS.

[0055] Culture conditions: 37°C, 5% CO2 incubator.

[0056] 2. Experimental methods

[0057] (1) Detection of injury, inflammation and other indicators: HUVECs in good condition were seeded in 6-well cell culture plates and cultured overnight. After starvation for 12 hours, the serum-free medium was replaced and the SAA1 human recombinant protein was added to the cell culture medium of the treatment group for 48 hours. The protein was collected and Western blot experiments were performed to detect various injury indicators.

[0058] (2) Transwell migration assay: Pipette 700 μL of complete culture medium (containing 10% FBS) into the lower chamber of the Transwell plate. Pipette 200 μL of 1.5×10 6 / mL HUVEC cell suspension was inoculated in the upper chamber of the Transwell plate. The cell suspension did not contain serum. The cell suspension of the treatment group was added with SAA1 human recombinant protein and cultured in a 37°C, 5% CO2 incubator for 12 hours. The chamber was removed and washed twice with PBS buffer; fixed in 4% paraformaldehyde for 15 minutes, washed three times with PBS buffer; stained in hematoxylin staining solution for 15 minutes, washed three times with PBS buffer; and the non-migrated cells on the upper layer were gently wiped off with a cotton swab. After the chamber was air-dried, it was photographed under an inverted microscope to count the migrated cells.

[0059] (3) Angiogenesis experiment: A 96-well cell culture plate containing matrix gel was prepared and human umbilical vein endothelial cell suspension (total number of cells was about 3×10 4 The cells were inoculated on the matrix gel, and the SAA1 human recombinant protein was added to the cell suspension of the treatment group and cultured in a 37°C, 5% CO2 incubator for 4-6 hours. When the HUVEC tube morphology was better, photos were taken under an inverted microscope and the number of tubes was counted.

[0060] 3. Experimental results

[0061] (1) SAA1 induces endothelial cell damage, apoptosis, and the appearance of inflammatory and fibrotic phenotypes

[0062] The experimental results are as follows Figure 1 As shown in A, HUVECs treated with SAA1 (20 ng / mL) for 48 h showed upregulation of endothelial injury (ICAM-1 and ET-1), apoptosis (FADD and Bax), as well as inflammation (COX2 and TNF-α) and fibrosis (FN) markers. Figure 1 B~H in Figure 1 Statistical results of A in .

[0063] (2) SAA1 inhibits endothelial cell migration and angiogenesis

[0064] The experimental results are as follows Figure 2 As shown in A and C in Figure 3, SAA1 can inhibit the migration ability of HUVEC in a concentration-dependent manner. When the concentration of SAA1 reaches 40 ng / mL, the migration ability of HUVEC is inhibited by more than 50%; at the same time, the tube-forming ability of HUVEC is also inhibited by more than 50% (as shown in Figure 3). Figure 2 B and D in FIG.

[0065] Example 2 Knockout of the SAA1 gene has a protective effect on ischemia / reperfusion-induced renal disease

[0066] 1. Experimental Animals

[0067] SAA1 knockout mice were constructed by Saiye Biotechnology Co., Ltd. and kept in the SPF animal room of the Experimental Animal Center of Nanfang Hospital of Southern Medical University, with free movement and diet. The experiments were carried out in strict accordance with the experimental animal management regulations of the Experimental Animal Center of Nanfang Hospital of Southern Medical University, and all animal experiments were reviewed and approved by the Ethics Committee of Southern Medical University.

[0068] SAA1 knockout mice were obtained by breeding, and wild-type mice of the same litter were used as controls. Male SAA1 knockout mice and wild-type mice aged 7-10 weeks and weighing 20-26 g were randomly divided into sham operation group and ischemia / reperfusion group, 6 mice per group.

[0069] 2. Experimental methods

[0070] (1) Sham operation: The mice were weighed and anesthetized with 1% sodium phenobarbital. A midline abdominal incision was made. After local disinfection, the skin, subcutaneous tissue, muscular layer and peritoneum were cut layer by layer. The left renal pedicle was exposed for 35 minutes and then sutured layer by layer.

[0071] (2) Unilateral ischemia / reperfusion: Anesthesia and surgical approach were the same as above. After exposing the left renal pedicle, the renal artery was clamped with a micro-hemostatic clamp at 37.5°C for 35 min. The hemostatic clamp was released, the recovery of renal blood flow was observed, and the abdominal cavity was closed by suture layer by layer. Right nephrectomy was performed on the 10th day after surgery.

[0072] 3. Experimental process

[0073] SAA1 knockout mice and wild-type mice were subjected to unilateral renal ischemia / reperfusion surgery and sacrificed on the 11th day after surgery to collect serum and kidney tissue samples. Serum creatinine levels were detected by biochemical analyzer; the expression of SAA1 and fibrosis indexes in kidney tissue was detected by Western Blot; the expression of fibrosis indexes and endothelial cell markers was detected by immunohistochemical staining; the deposition of renal interstitial collagen fibers was detected by Masson staining; and the in vivo vascular permeability experiment was used to detect the leakage of 150kDFITC-dextran injected into the tail vein into the kidney tissue.

[0074] 4. Experimental results

[0075] Knocking out the SAA1 gene delayed the decline in renal function caused by renal ischemia / reperfusion injury, alleviated renal fibrosis and restored capillary network density. The specific results are as follows:

[0076] Figure 3 Schematic diagram of renal ischemia / reperfusion surgery in SAA1 wild-type and gene knockout mice. Figure 4 The results showed that simply knocking out the gene had no significant effect on serum creatinine in mice, but could significantly inhibit the increase in serum creatinine caused by ischemia / reperfusion injury. Figure 5Western blot detection and statistical results showed that knocking out SAA1 could inhibit the increase of fibrosis indicators caused by ischemia / reperfusion injury, such as TNC, FN, Col-I and α-SMA; Figure 6 The immunohistochemistry, Masson staining and statistical results of A to D in Figure 3 showed that knocking out SAA1 could significantly inhibit the deposition of extracellular matrix such as FN and collagen fibers; Figure 6 The results of immunohistochemical staining of E and vascular permeability experimental tests showed that knocking out the SAA1 gene could significantly restore the capillary network, reduce the leakage of glucan from the blood vessels and the aggregation of glucan in the renal interstitium.

[0077] Example 3: Knockout of the SAA1 gene has a protective effect on kidney disease induced by ureteral obstruction

[0078] 1. Experimental Animals

[0079] After breeding and identification, 12 SAA1 knockout mice and 12 wild-type control mice of the same litter were obtained. Male, 7-8 weeks old, weighing 20-22g, each was randomly divided into two groups, sham operation, unilateral ureteral obstruction, 6 mice / group.

[0080] 2. Treatment of each group

[0081] (1) Sham operation: The mice were weighed and anesthetized with 1% sodium phenobarbital. A midline abdominal incision was made. After local disinfection, the skin, subcutaneous tissue, muscular layer and peritoneum were cut layer by layer, and the left ureter was exposed and then sutured layer by layer.

[0082] (2) Unilateral ureteral obstruction: Anesthesia and surgical approach were the same as above. The left ureter was exposed, the surrounding fat tissue was stripped, and the ureter was ligated close to the renal hilum. After the operation, the layers were sutured layer by layer, and the mouse was returned to the cage after awakening from anesthesia. The sample was collected 7 days later.

[0083] 3. Experimental process

[0084] SAA1 knockout mice and wild-type control mice were subjected to unilateral ureteral ligation surgery and sacrificed on the 7th day after surgery to collect serum and kidney tissue samples. Western Blot was used to detect the expression of SAA1 and fibrosis indicators in kidney tissue; immunohistochemical staining was used to detect the expression of fibrosis indicators and endothelial cell markers; Masson staining was used to detect the deposition of renal interstitial collagen fibers; and in vivo vascular permeability experiments were used to detect the leakage of 150kD FITC-dextran injected into the tail vein into the kidney tissue.

[0085] 4. Experimental results

[0086] Knockout of SAA1 delayed ureteral obstruction-induced renal fibrosis and restored capillary network density. The specific results are as follows:

[0087] Figure 7 A in the figure is a schematic diagram of unilateral ureteral ligation surgery for SAA1 wild-type and gene knockout mice; Figure 7 Western blot detection and statistical results from B to D in the figure showed that knocking out SAA1 could significantly inhibit the increase of fibrosis indicators (such as Col-I) caused by ureteral obstruction; Figure 7 The immunohistochemistry, Masson staining, and statistical results from E to H in Figure 2 showed that knocking out SAA1 could significantly inhibit the deposition of extracellular matrix such as FN and collagen fibers; Figure 8 The results of immunohistochemical staining and vascular permeability experimental tests showed that knocking out the SAA1 gene could significantly restore the capillary network and reduce the leakage of glucan from the blood vessels and the aggregation of glucan in the renal interstitium.

[0088] Example 4: Design, preparation and screening of hSAA1 recombinant protein vaccine

[0089] 1. Experimental Materials

[0090] Bacterial strain: E. coli BL21 (DE3); Culture conditions: 37° C., 180 rpm shaking culture.

[0091] Experimental animals: Male C57 / BL6 mice, 7-8 weeks old, weighing 20-22 g, were purchased from Beijing Weitong Lihua Company. During the experiment, they were kept in the SPF animal room of the Experimental Animal Center of Nanfang Hospital of Southern Medical University and were allowed to move freely and eat freely.

[0092] 2. Experimental methods

[0093] (1) Gene synthesis and vector construction

[0094] The hSAA1-LTB gene sequence was synthesized by Sino-US Taihe Biotechnology (Beijing) Co., Ltd., with restriction sites EcoR I and Hind III at both ends. The expression vector pQEV is derived from the commercialized pRSET A vector (purchased from Qiyun Biotechnology, item number: QP1412). The Xpress antigen epitope and enterokinase cleavage recognition sequence on the N-terminal fusion peptide of the pRSET A vector were removed by PCR, and the order of the multiple cloning site arrangement was optimized. Its nucleotide sequence is shown in SEQ ID NO.3. The pQEV vector can make gene cloning more convenient and the expressed foreign protein purer. The hSAA1-LTB gene and the expression vector pQEV were double-digested with EcoR I and Hind III and then ligated with T4 DNA to construct the recombinant plasmid pQEV-hSAA1-LTB, which was transformed into competent cells E.coli BL21 (DE3), and the positive clones were picked for expansion and culture. Enzyme digestion identification and DNA sequencing showed that the obtained gene sequence was correct.

[0095] (2) Expression and purification of hSAA1-LTB recombinant protein

[0096] The engineered bacteria containing the recombinant plasmid pQEV-hSAA1-LTB were cultured at 37°C and 180rpm for 4 hours, and then 0.5mM propyl-β-D-thiogalactoside (IPTG) was added to induce the expression of the target protein; after 3 hours, the bacteria were collected by centrifugation; the cells were resuspended in PBS and then high-pressure homogenized to break the cells, centrifuged at 11000rpm for 15min, and the precipitate was collected. The target protein existed in the form of inclusion bodies. The harvested inclusion bodies were dissolved in 8M urea, purified by Sepharose SP FF cation chromatography column, and then renatured for vaccine preparation. The purified product was subjected to 12% SDS-PAGE gel electrophoresis and Coomassie Brilliant Blue staining to analyze the purity.

[0097] (3) Preparation of SAA1 recombinant protein vaccine

[0098] ① Preparation of ISA 201VG adjuvant emulsified vaccine

[0099] The renatured hSAA1-LTB antigen protein was quantified by the BCA method and diluted with PBS to a final concentration of 10 μg / mL. According to a 50 / 50 mass ratio (volume ratio of 45:55), 10 mL of antigen solution and ISA 201VG adjuvant were respectively heated in a water bath to 32°C. Under low shear stirring at 1100 rpm, the aqueous phase (i.e., antigen solution) was slowly added to the adjuvant phase, stirred at a constant temperature for 3 minutes, and then transferred to 4°C for cooling for 1 hour. Dispense in vials, 0.5 mL / bottle.

[0100] ②Preparation of aluminum hydroxide adjuvant adsorbed vaccine

[0101] The renatured hSAA1-LTB antigen protein was quantified by the BCA method and diluted with PBS to a final concentration of 20 μg / mL. According to the volume ratio of 15 / 85, 15 mL of aluminum hydroxide adjuvant was slowly added to 85 mL of antigen protein solution under stirring conditions of 150 rpm, and stirred at a constant temperature of 20-25°C for 90 minutes for full adsorption. After completion, it was divided into vials, 0.5 mL / bottle.

[0102] (4) Comparison of vaccination and immunogenicity

[0103] Male adult C57 / BL6 mice were then divided into three groups, 6 mice / group, namely control group, emulsified vaccine group, and adsorbed vaccine group. A two-immunization strategy was adopted, and the prepared vaccine was inoculated into the left lower limb of the mice, intramuscularly, 100 μL / time; the second vaccination was performed 3 weeks after the first vaccination, and serum was collected in the 4th week, and the anti-SAA1 antibodies in the serum of each group of mice were detected by Western blot method.

[0104] 3. Experimental results

[0105] (1) Construction and identification of recombinant plasmids

[0106] The gene fragment encoding hSAA1-LTB fusion protein antigen was synthesized and restriction sites EcoR I and Hind III were added at both ends, respectively. The sequence is shown in SEQ ID NO.1, wherein positions 15 to 326 are DNA sequences encoding hSAA1, positions 327 to 374 are Linker DNA sequences, and positions 375 to 683 are DNA sequences encoding hLTB; the corresponding encoded amino acid sequence is shown in SEQ ID NO.2; the synthesized fragment was cloned into the expression vector pQEV to obtain the recombinant plasmid pQEV-hSAA1-LTB; Fig. 9 The recombinant plasmid pQEV-hSAA1-LTB was identified by double enzyme digestion with EcoR I, Hind III and Bam HI, Hind III respectively. The target bands with molecular weight of about 700bp and 300bp were observed by agarose gel electrophoresis, which was consistent with the theoretical results. The DNA sequencing results of the recombinant plasmid pQEV-hSAA1-LTB proved that the sequence of the inserted fragment was correct and had no mutation.

[0107] (2) Expression and purification results of hSAA1-LTB recombinant fusion protein

[0108] like Fig.10 A in the figure shows that after IPTG induction, the engineered bacteria containing the recombinant plasmid pQEV-hSAA1-LTB produce a new protein band at about 27KDa, the molecular weight of which is consistent with the theoretical molecular weight of the target protein, and the expression amount accounts for about 30% of the total bacterial protein; further, after purification by a cation chromatography column, the purity of the target protein can reach 95%; Fig.10 B in the figure is the purification chromatogram of hSAA1-LTB recombinant protein. It can be seen that the impurity protein exists in the column peak and the target protein exists in the elution peak.

[0109] (3) Preparation and immunogenicity comparison of SAA1 recombinant protein vaccine

[0110] like Fig.11 In A, the hSAA1-LTB recombinant protein solution was emulsified with the ISA201VG adjuvant, and the prepared vaccine emulsion was stable without stratification, had a milky white appearance, and a viscosity of 60.23 cP; Fig.11 B in the figure represents the hSAA1-LTB recombinant protein solution that is fully adsorbed with aluminum hydroxide adjuvant to form a stable colloidal suspension adsorbed vaccine with a clear color. Fig.11C in the figure represents the above two vaccine strategies for immunizing mice. The serum of mice in each group was collected 4 weeks after the first immunization, and Western blot was performed using commercial SAA1 recombinant protein as antigen and mouse serum as primary antibody. The results are shown in the figure. Fig.11 In D, a positive band was detected in the serum of mice vaccinated with the emulsified vaccine, but no positive band was detected in the serum of mice vaccinated with the adsorbed vaccine, indicating that the emulsified vaccine can induce mice to produce antibodies against SAA1. Subsequent experiments were carried out using the emulsified vaccine, and it was named SAA1 recombinant protein vaccine.

[0111] Example 5: SAA1 recombinant protein vaccine protects against renal disease induced by ischemia / reperfusion injury

[0112] 1. Experimental Animals

[0113] Male C57 / BL6 mice, 7-8 weeks old, weighing 20-22 g, were purchased from Beijing Weitong Lihua Company and kept in the SPF animal room of the Experimental Animal Center of Nanfang Hospital of Southern Medical University during the experiment, with free movement and diet. 24 mice were randomly divided into 4 groups: sham operation, immune control + ischemia / reperfusion, low-dose vaccine immunization + ischemia / reperfusion, and high-dose vaccine immunization + ischemia / reperfusion, 6 mice / group.

[0114] 2. Treatment of each group

[0115] (1) Sham operation: The mice were weighed and anesthetized with 1% sodium phenobarbital. A midline abdominal incision was made. After local disinfection, the skin, subcutaneous tissue, muscular layer and peritoneum were cut layer by layer. The left renal pedicle was exposed for 35 minutes and then sutured layer by layer. The strategy was the same as before.

[0116] (2) Immunization control + unilateral ischemia / reperfusion: Two immunizations were performed using a control vaccine (PBS was used to replace the antigen solution, and PBS + adjuvant ISA 201VG was prepared as the control vaccine according to Example 4 "① Preparation of ISA 201VG adjuvant emulsified vaccine"). 100 μL / time; unilateral renal ischemia / reperfusion surgery was performed 4 weeks after the first immunization, and right nephrectomy was performed 10 days after the surgery. The immunization and surgical strategies were the same as before.

[0117] (3) Low-dose vaccine immunization + ischemia / reperfusion group: Two immunizations were performed with SAA1 recombinant protein vaccine (10 μg / mL) prepared by emulsification with ISA 201VG adjuvant, 100 μL / time; unilateral renal ischemia / reperfusion surgery was performed 4 weeks after the first immunization. The immunization and surgical strategies were the same as before.

[0118] (4) High-dose vaccine immunization + ischemia / reperfusion group: Two immunizations were performed with SAA1 recombinant protein vaccine (20 μg / mL) prepared by emulsification with ISA 201VG adjuvant, 100 μL / time; unilateral renal ischemia / reperfusion surgery was performed 4 weeks after the first immunization. The immunization and surgical strategies were the same as before.

[0119] 3. Experimental process

[0120] The mice were killed on the 11th day after surgery, and the serum and kidney tissues were collected. Western blot was used to detect the anti-SAA1 antibody in the serum of each group of mice; the serum creatinine and urea nitrogen levels were detected by biochemical methods; the protein was extracted from the kidney tissue homogenate, and the expression of fibrosis indexes and endothelial injury marker ICAM-1 was detected by Western Blot; the expression of fibrosis indexes and endothelial cell marker EMCN was detected by immunohistochemical staining; and the deposition of renal interstitial collagen fibers was detected by Masson staining.

[0121] 4. Experimental results

[0122] SAA1 recombinant protein vaccine protects against ischemia / reperfusion-induced renal disease. Fig.12 Schematic diagram of the experimental design; Western blot detection was performed using commercial SAA1 recombinant protein as antigen and mouse serum as primary antibody. The results are shown in Fig.13 In the sera of mice immunized with two different doses (10 μg / mL and 20 μg / mL), SAA1 antibodies were detected without significant differences, while no antibodies were produced in non-immunized mice. Furthermore, the results of renal function tests showed that SAA1 recombinant protein vaccine could reduce the increase of serum creatinine and urea nitrogen induced by ischemia / reperfusion and improve renal function, and there was no significant correlation with the immunization dose. Fig.14 Subsequent testing will focus on the low-dose (10 μg / mL) vaccine group. Fig.15 A, Western blot results showed that the expression of fibrosis molecules TNC and Col-I in the kidney tissue of vaccine-immunized mice was significantly reduced. Fig.15 B and C in the figure are statistical results; immunohistochemical staining, Masson staining and statistical results showed that the deposition of extracellular matrix such as FN, α-SMA and collagen fibers in the renal tissue of the vaccine immunization group was also significantly reduced, such as Fig.16 , suggesting that vaccine immunization can effectively delay renal fibrosis. Further, in order to evaluate the changes in renal blood vessels, immunohistochemistry was used to detect the expression of endothelial cell marker EMCN. The results are as follows Fig.17The expression level of EMCN in the vaccine treatment group was significantly higher than that in the ischemia / reperfusion group, indicating a richer and more complete capillary network; Western blot results also showed that the expression of ICAM-1, a marker of endothelial injury, was significantly reduced in the immunotherapy group. Fig.18 The above results suggest that SAA1 recombinant protein vaccine can effectively reduce ischemia / reperfusion-induced renal injury, improve renal function, delay renal fibrosis and restore capillary network structure after immunization.

[0123] Example 6: SAA1 recombinant protein vaccine protects against kidney disease induced by ureteral obstruction

[0124] 1. Experimental Animals

[0125] Male C57 / BL6 mice, 7-8 weeks old, weighing 20-22 g, were purchased from Beijing Weitong Lihua Company and kept in the SPF animal room of the Experimental Animal Center of Nanfang Hospital of Southern Medical University during the experiment, with free movement and diet. 18 mice were randomly divided into 3 groups: sham operation, immune control + ureteral obstruction, and vaccine immunization + ureteral obstruction, 6 mice / group.

[0126] 2. Treatment of each group

[0127] (1) Sham operation: The mice were weighed and anesthetized with 1% sodium phenobarbital. A midline abdominal incision was made. After local disinfection, the skin, subcutaneous tissue, muscular layer and peritoneum were cut layer by layer, and the left ureter was exposed and then sutured layer by layer.

[0128] (2) Immunization control + ureteral obstruction: The control vaccine (PBS was used to replace the antigen solution, and PBS + adjuvant ISA 201VG was prepared as the control vaccine according to Example 4 "① Preparation of ISA 201VG adjuvant emulsified vaccine") was used for two immunizations, 100 μL / time; unilateral ureteral obstruction surgery was performed 4 weeks after the first immunization. The immunization and surgical strategies were the same as before.

[0129] (3) Vaccine immunization + ureteral obstruction: SAA1 recombinant protein vaccine (10 μg / mL) prepared by emulsification with ISA201VG adjuvant was used for two immunizations, 100 μL / time, with the same strategy as before; unilateral ureteral obstruction surgery was performed 4 weeks after the first immunization. The immunization and surgical strategies were the same as before.

[0130] 3. Experimental process

[0131] The mice in each group were killed on the 7th day after unilateral ureteral ligation, and serum and kidney tissue samples were collected. Western Blot and immunohistochemical staining were used to detect the expression changes of renal tissue fibrosis and endothelial cell injury molecules and markers.

[0132] 4. Experimental results

[0133] SAA1 vaccine immunization has a significant protective effect against ureteral obstruction-induced renal disease, which reduces vascular endothelial cell damage and delays renal fibrosis. Fig.19 Design for experiments; Fig. 20 Western blot and statistical results showed that in the unilateral ureteral obstruction-induced nephropathy model, vaccine immunotherapy could significantly reduce the expression of Col-I, a fibrosis molecule, and ICAM-1, an endothelial cell injury molecule, in the mouse kidney tissue. Furthermore, immunohistochemistry and Masson staining results also confirmed that vaccine immunization could reduce the deposition of extracellular matrix such as α-SMA and collagen fibers, restore the expression of endothelial cell marker EMCN, and increase the density of capillary network ( Fig.21 ).

[0134] In summary, SAA1 is an important vascular injury factor that induces vascular endothelial cell injury, inflammation, apoptosis, and endothelial-mesenchymal transition, and inhibits vascular endothelial cell migration and angiogenesis. In vivo experiments have confirmed that knocking out the SAA1 gene or actively immunizing with SAA1 recombinant protein vaccine can improve renal fibrosis induced by ischemia / reperfusion or ureteral obstruction, restore capillary network density, improve renal tissue microcirculation, and delay the progression of renal disease. Therefore, SAA1 is expected to become a new target for the prevention and treatment of kidney disease, and SAA1 recombinant protein vaccine immunization is a new means and strategy for the clinical prevention and treatment of kidney disease.

[0135] The best embodiment of the present invention has been described in detail, but it should be clear that the technical solution of the present invention is not limited to the above specific implementation details. Under the guidance of the technical idea of ​​the present invention, any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. Application of SAA1 as a target in the preparation of drugs for the prevention and treatment of kidney diseases.

2. The application of drugs that can inhibit SAA1 expression or block the recognition of SAA1 and receptors in the prevention and treatment of kidney diseases.

3. Use of the drug capable of inhibiting SAA1 expression or blocking SAA1 from recognizing with receptors according to claim 2 in preventing and treating kidney diseases, characterized in that: The drug is a recombinant protein vaccine targeting the prevention and treatment target SAA1.

4. A recombinant protein vaccine targeting SAA1, characterized in that: The invention comprises a fusion protein hSAA1-LTB which is sequentially connected with human SAA1, a connecting peptide and an immune adjuvant molecule, the Escherichia coli thermolabile enterotoxin B subunit.

5. The recombinant protein vaccine targeting SAA1 according to claim 4, characterized in that: Adjuvants are also included.

6. The recombinant protein vaccine targeting SAA1 according to claim 5, characterized in that: In the vaccine targeting SAA1, a fusion protein hSAA1-LTB solution with a concentration of 5 to 100 μg / mL and an emulsified adjuvant are mixed in a mass ratio of 40 to 60:40 to 60.

7. The recombinant protein vaccine targeting SAA1 according to claim 5 or 6, characterized in that: The adjuvant is an emulsified adjuvant.

8. The recombinant protein vaccine targeting SAA1 according to any one of claims 4 to 6, characterized in that: The amino acid sequence of the fusion protein hSAA1-LTB is shown in SEQ ID NO.

2.

9. The recombinant protein vaccine targeting SAA1 according to claim 8, characterized in that: The nucleic acid sequence of the DNA encoding the fusion protein hSAA1-LTB is shown in SEQ ID NO.

1.

10. Use of the recombinant protein vaccine targeting SAA1 according to any one of claims 4 to 9 in the preparation of drugs for the prevention and treatment of kidney diseases.

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