ApoA-I anti-sugar functional mutant as well as preparation method and application thereof

Abstract

The invention relates to an apoA-I anti-sugar functional mutant as well as a preparation method and application thereof. The mutant is obtained by constructing an apoAE125C / E136C recombinant protein and crosslinking the recombinant protein into a dimer. The apoA-I anti-sugar functional mutant has superexcellent anti-sugar characteristic and biological safety, can remarkably improve the blood fat of diabetic mice, reduce ectopic fat deposition of the liver and inhibit formation of atheromatous plaques, and has a good market application prospect.

Description

Technical Field

[0001] The invention belongs to the field of treatment of diabetic cardiovascular diseases, and particularly relates to an apoA-I anti-sugar functional mutant and a preparation method and application thereof. Background Art

[0002] In August 2023, the European Society of Cardiology (ESC) released the "2023 European Society of Cardiology (ESC) Guidelines for the Management of Cardiovascular Disease in Diabetes", ushering in the first year of "diabetic cardiology". According to the guidelines, statins or PCSK9 inhibitors either inhibit LDL-C synthesis or promote LDL-C degradation, which is the cornerstone of the treatment of atherosclerosis. It is worth noting that both statins and PCSK9 inhibitors are based on lowering LDL-C, a cardiovascular risk factor. In fact, in addition to lowering serum total cholesterol (TC) and low-density lipoprotein cholesterol (LDL-C) levels, patients with coronary heart disease can also benefit by increasing the cardiovascular protective factor-high-density lipoprotein cholesterol (HDL-C). A large-scale meta-study showed that in patients receiving statin therapy, even if their low-density lipoprotein cholesterol (LDL-C) levels were very low, HDL-C and ApoA-I (the main functional protein component of HDL) levels were still independent of LDL-C and closely related to cardiovascular risk, among which an increase in ApoA-I levels could also reduce the risk of major cardiovascular events. These findings suggest that increasing plasma apolipoprotein AI levels may serve as a complementary therapy to statins or PCSK9, further improving patients' blood lipids through synergistic effects.

[0003] HDL (high-density lipoprotein) is the only confirmed cardiovascular protective factor. Apolipoprotein apoA-I accounts for more than 70% of HDL protein content and is the executor of HDL functions. It plays a role in maintaining cholesterol homeostasis, promoting cholesterol reverse transport, anti-inflammatory, anti-oxidation and anti-atherosclerosis and other cardiovascular protective functions. However, in diabetes, the main protein component apoA-I in HDL (accounting for more than 70% of the entire HDL protein component) will undergo non-enzymatic glycosylation modification, which not only causes HDL to lose its normal protective function, but also shows a negative effect of promoting atherosclerosis. At present, little is known about the non-enzymatic glycosylation modification of apoA-I at home and abroad, and there is no apoA-I modification aimed at anti-sugar and cardiovascular disease intervention in diabetic patients. Summary of the invention

[0004] The technical problem to be solved by the present invention is to provide an apoA-I anti-sugar functional mutant and its preparation method and application. The mutant has excellent anti-sugar properties and biosafety, can significantly improve the blood lipids of diabetic mice, reduce ectopic fat deposition in the liver and inhibit the formation of atherosclerotic plaques, and has good market application prospects.

[0005] The present invention provides an apoA-I anti-sugar functional mutant, by constructing an apoA E125C / E136C The recombinant protein is cross-linked into a dimer.

[0006] Furthermore, the apoA E125C / E136C The amino acids at positions 125 and 136 of the recombinant protein corresponding to the natural apoA-I amino acid sequence are not glutamic acid but cysteine, as shown in SEQ ID NO:1.

[0007] Further, the cross-linking refers to the use of a cross-linking agent in apoA E125C / E136C Cysteine ​​125 of recombinant protein A and apoA E125C / E136C The cysteine ​​at position 136 of recombinant protein B forms a thioether bond cross-link.

[0008] Furthermore, the cross-linking agent is bismaleimidohexane.

[0009] The present invention also provides a method for preparing an apoA-I anti-sugar functional mutant, comprising the following steps:

[0010] (1) apoA E125C / E136C The plasmid of the recombinant protein is transformed into competent cells, followed by expression and purification;

[0011] (2) In apoA E125C / E136C The recombinant protein solution is added with a cross-linking agent for cross-linking to obtain an apoA-I anti-sugar functional mutant.

[0012] The present invention also provides a use of the apoA-I anti-sugar functional mutant as claimed in claim 1 in preparing a drug for treating diabetic cardiovascular disease.

[0013] Furthermore, the drug also contains pharmaceutically acceptable carriers and / or excipients.

[0014] Furthermore, the pharmaceutically acceptable carrier and / or excipient includes at least one of a diluent, a binder, a surfactant, a humectant, an adsorption carrier, a lubricant, a filler, and a disintegrant.

[0015] Beneficial Effects

[0016] The apoA-I anti-sugar functional mutant of the present invention has excellent anti-sugar properties and biosafety, can significantly improve blood lipids in diabetic mice, reduce ectopic fat deposition in the liver and inhibit the formation of atherosclerotic plaques, and has good market application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 AF shows the transformation process and anti-glycemic properties of the apoA-I anti-glycemic functional mutants of the present invention.

[0018] Figure 2 AF shows that the apoA-I anti-glycemic functional mutant of the present invention can significantly improve the abnormal reverse transport function of apoA-I caused by glycation.

[0019] Figure 3 AF shows that the apoA-I anti-sugar functional mutant of the present invention can significantly improve the formation of atherosclerotic plaques in diabetic atherosclerosis model mice.

[0020] Figure 4 AC shows that the apoA-I anti-glycemic functional mutant of the present invention can significantly improve diabetic liver fat deposition and liver function damage.

[0021] Figure 5 AD shows that the apoA-I anti-sugar functional mutant of the present invention has good biosafety. DETAILED DESCRIPTION

[0022] The present invention will be further described below in conjunction with specific embodiments. It should be understood that these embodiments are only used to illustrate the present invention and are not intended to limit the scope of the present invention. In addition, it should be understood that after reading the content taught by the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms fall within the scope limited by the appended claims of the application equally.

[0023] Example 1

[0024] 1. Experimental Methods

[0025] 1.1 Construction of apoA-I anti-glycemic functional mutant apoA-I Ruijin

[0026] apoA-I Ruijin The functional apoA-I dimer protects the key glycosylation sites from being glycosylated. Its construction is divided into two steps: ① Construction of apoA (E125C / E136C) The recombinant protein, the sequence of which is shown in SEQ ID NO: 1; ② using the cross-linking agent bismaleimidohexane (BMH) in apoA E125C / E136C Cysteine ​​125 of recombinant protein A and apoAE125C / E136C The cysteine ​​at position 136 of recombinant protein B forms a thioether bond cross-link.

[0027] 1.2apoA (E125C / E136C) Expression and purification of recombinant proteins

[0028] 1. Transformation and small-scale culture identification

[0029] 1) Transformation: The constructed apoA-containing Ruijin The plasmid of the gene was transformed into BL21(DE3) competent cells.

[0030] 2) Plate coating: Spread the transformed competent cells evenly onto LB plates (containing 50 μg / mL kanamycin sulfate), and then invert them in a 37°C incubator overnight.

[0031] 3) Small shaking: Select a single clone from the transformed plate and inoculate it into 10 mL of LB medium (containing 50 μg / mL kanamycin sulfate), and culture it at 37°C and 200 rpm with vigorous shaking. When the OD600 reaches 0.6-0.8, add IPTG to a final concentration of 0.3 mM to the medium, and then culture it at 15°C for 16 hours.

[0032] 4) SDS-PAGE analysis to identify the results of induced expression: Take the induced culture medium, centrifuge at 12000rpm for 15min, remove the supernatant, add binding buffer to resuspend the precipitate, then add protein binding buffer to heat the sample at 100℃ for 10min, centrifuge and take the supernatant for electrophoresis.

[0033] 2. Large-scale cultivation of recombinant proteins

[0034] 1) Shake vigorously: Add 10 mL of the bacterial suspension to 1 L of LB medium (containing 50 μg / mL kanamycin sulfate), shake vigorously at 37°C and 200 rpm, and culture until the OD600 reaches 0.6-0.8. Add 0.3 mM IPTG to the culture medium, and then shake at 15°C and 200 rpm for 16 hours.

[0035] 2) Collecting bacteria: Transfer the culture solution after shaking to a centrifuge tube, centrifuge at 4000 rpm and 4°C for 20 minutes, discard the supernatant, collect the cultured bacteria, and then add 30 mL of binding buffer to resuspend the bacteria.

[0036] 3) Bacterial lysis: Add lysozyme (final concentration: 1 mg / mL) to the resuspended bacterial solution. After incubation on ice for 30 minutes, freeze and thaw three times to observe that the bacterial solution becomes thicker. Then add 10 μL of universal nuclease and react on ice for 30 minutes. The viscosity of the solution can be observed to decrease.

[0037] 4) Centrifugation: Centrifuge the solution after bacterial lysis at 4°C and 20,000 rpm for 40 min, collect the supernatant, and store it at -80°C for later use.

[0038] 3. Affinity chromatography on Ni column

[0039] 1) Equilibration: Use binding buffer to equilibrate the Ni column for 5-10 column volumes at a flow rate of 1 mL / min.

[0040] 2) Loading: Before loading, filter the bacterial supernatant with a 0.22 μm filter and load at a flow rate of 1 mL / min. Collect the effluent and mark it as “loading” for SDS-PAGE to observe the binding ability of the recombinant protein to the Ni column.

[0041] 3) Washing: After loading the sample, wash the Ni column with 100 mL of washing buffer at a flow rate of 1 mL / min. Collect the effluent and mark it as "wash" for SDS-PAGE. Observe that the imidazole concentration in the washing buffer is appropriate. After washing, balance the Ni column with binding buffer at a flow rate of 1 mL / min for 5-10 column volumes and then elute.

[0042] 4) Elution: Use elution buffer for elution at a flow rate of 1 mL / min, gradually increase the elution buffer gradient from 0 to 100% within 10 column volumes, collect the eluate below the elution peak, and mark the order.

[0043] 5) SDS-PAGE verification: Take 10 μL sample from each tube of eluate, then add protein binding buffer and heat the sample at 100°C for 10 min. After centrifugation, take the supernatant for electrophoresis.

[0044] 4. Molecular sieve chromatography

[0045] 1) Equilibration: Use binding buffer to equilibrate one column volume of Hiload Superdex (200 pg) molecular sieve at a flow rate of 1 mL / min.

[0046] 2) Sample processing: Before loading, use a 3kD ultrafiltration tube to concentrate the protein concentration to about 10 mg / mL, then centrifuge at 12000 rpm and 4°C for 15 minutes and take the supernatant.

[0047] 3) Sample loading: Add 4.5 mL of sample to the loop on the purifier, elute 1 column volume with binding buffer at a flow rate of 1 mL / min, and collect the sample under the elution peak.

[0048] 4) SDS-PAGE verification: Take 10 μL sample from each tube of eluate, then add protein binding buffer and heat the sample at 100°C for 10 min, then centrifuge and take the supernatant for electrophoresis to observe the expression and purification of the recombinant protein.

[0049] 1.3apoA Ruijin Cross-linking preparation of recombinant proteins

[0050] Required reagents:

[0051] a) Coupling buffer: PBS (pH 6.5-7.2, the solution also needs to contain 10 mM EDTA);

[0052] b) Cross-linker stock solution: BMH dissolved in DMSO (20 mM);

[0053] c) Quenching solution: dithiothreitol (DTT) solution;

[0054] d) apoA I (E125C / E136C) Solution (the total time from protein solution purification to cross-linking should not exceed 12h).

[0055] Experimental procedures

[0056] 1) Prepare the above solution and measure apoA_I (E125C / E136C) For the protein solution, the protein solution buffer was replaced with coupling buffer using a 3 kd ultrafiltration tube (Millipore), and the protein solution was diluted to 0.2 mM (5.6 mg / mL).

[0057] 2) Add the cross-linker stock solution to the solution in step 1) to make the working concentration of the cross-linker 0.64 mM.

[0058] 3) Place solution b on a shaker at 4°C to react for 2 hours.

[0059] 4) Add 10 mM DTT, incubate at room temperature for 15 min, centrifuge at 12000 rpm, 4°C for 10 min, and purify the protein solution after removing the precipitate by molecular sieve (Superdex 200, Cytiva) to further obtain cross-linked apoA-I (E125C / E136C)

[0060] Protein, apoA-I Ruijin .

[0061] 2. Results

[0062] 1) Construction of anti-sugar functional mutant apoA-I Ruijin

[0063] In order to protect the key functional sites of apoA-I from being destroyed, an apoA-I anti-sugar functional mutant apoA-I was designed. Ruijin , by introducing cysteine ​​to replace glutamic acid residues at positions 125 and 136, and using the cross-linking agent bis-maleimidohexane (BMH) to form thioether bonds to stabilize apoA-I dimers ( Figure 1 A) The results showed that apoA-I Ruijin It has the same protein secondary structure as natural apoA-I, but can significantly reduce the glycation modification induced by glycation reactants and has good anti-glycemic properties. Figure 1 BC circular dichroism showed that apoA-I Ruijin It does not significantly change the secondary structure of the protein. Figure 1 D Size exclusion chromatography shows that in its native state, apoA-I Ruijin It exists mainly in the form of dimers. Figure 1 E SDS-PAGE shows that apoA-I Ruijin The dimer form is still the main form, suggesting that BMH is used as a medium, and two molecules of apoA-I Ruijin Forms a stable thioether bond. Figure 1 F Heat map showing native apoA-I and apoA-I Ruijin In vitro anti-glycemic effect: as the concentration of the glycating reagent gradually increased, the glycating degree of natural apoA-I was significantly enhanced, and it showed significant anti-glycemic ability.

[0064] 2) Anti-glycemic functional mutant apoA-I Ruijin Can significantly improve the abnormal reverse transport function of apoA-I caused by glycation

[0065] As an HDL function executor, apoA-I mainly lowers blood lipids through the reverse cholesterol transport process. Ruijin Whether it can counteract the glycation-induced reverse cholesterol transport dysfunction was evaluated using in vivo and in vitro reverse cholesterol transport experiments. Figure 2 The reverse cholesterol transport function test showed that as the concentration of glycation reactants gradually increased, the ability of natural apoA-I to induce reverse cholesterol transport decreased significantly, while apoA-I Ruijin The ability to promote reverse cholesterol transport does not change much with changes in the glycosylation environment ( Figure 2 B) Similarly, Figure 2 In vivo reverse cholesterol transport experiments showed that compared with exogenous administration of glycated apoA-I, administration of glycated apoA-I Ruijin The apoA-I knockout mice (excluding the influence of endogenous apoA-I in mice) showed a significantly enhanced ability to promote cholesterol reversal ( Figure 2 DF).

[0066] 3) Anti-glycemic functional mutant apoA-I Ruijin Can significantly improve the formation of atherosclerotic plaques in diabetic atherosclerosis model mice

[0067] In order to evaluate the anti-glycemic function of apoA-I mutant Ruijin In order to protect the diabetic arteriosclerotic plaque from formation, we constructed a control group with intraperitoneal injection of albumin + cross-linker (control), natural apoA-I (4 mg / kg) and apoA-I Ruijin (4mg / kg) high cholesterol fed diabetes apoE - / - Mouse model. After drug intervention every other day for 3 months, it was found that compared with native apoA-I, apoA-I Ruijin Can significantly reduce plaque formation in diabetic patients ( Figure 3 AB), reduce plaque vulnerability ( Figure 3 CE), and significantly improved the abnormal serum lipid profile ( Figure 3 F).

[0068] 4) Anti-sugar functional mutant apoA-I Ruijin Can significantly improve ectopic fat deposition in diabetic liver and improve liver function

[0069] Oil red staining of liver showed that apoA-I Ruijin Can significantly reduce the deposition of liver lipids in diabetic patients ( Figure 4 AB); Improve liver function ( Figure 4 C).

[0070] 5) Anti-sugar functional mutant apoA-I Ruijin Good biosafety

[0071] After continuous intraperitoneal injection of saline, albumin + cross-linker, native apoA-I (4 mg / kg) and apoA-I Ruijin One month after administration (4 mg / kg), the biosafety of the above proteins was evaluated. HE staining showed that compared with the saline group C57BL mice, albumin + cross-linker, native apoA-I and apoA-I Ruijin Will not cause significant pathological changes in the kidneys, liver and heart ( Figure 5 AD). Complete blood cell counts showed that the number of white blood cells, lymphocytes, and neutrophils in the four groups of mice was not significantly different; at the same time, there was no difference in plasma IgM, IgG, and IgE levels among the four groups, suggesting that apoA-I RuijinIt does not increase inflammation or cause immune response, and has the same good biosafety as natural apoA-I protein.

[0072] 3. Conclusion

[0073] 1) apoA-I Ruijin It can significantly improve the formation of atherosclerotic plaques in diabetic atherosclerosis model mice.

[0074] 2) apoA-I Ruijin It can significantly improve diabetic liver fat deposition and liver function damage.

[0075] 3) apoA-I Ruijin It can have good biosafety and will not induce tissue lesions, inflammation or abnormal immune responses.

Claims

1. An apoA-I anti-sugar functional mutant, characterized in that: By constructing apoA E125C / E136C The recombinant protein is cross-linked into a dimer.

2. The apoA-I anti-sugar functional mutant according to claim 1, characterized in that: apoA E125C / E136C The amino acids at positions 125 and 136 of the recombinant protein corresponding to the natural apoA-I amino acid sequence are not glutamic acid but cysteine, as shown in SEQ ID NO:

1.

3. The apoA-I anti-sugar functional mutant according to claim 1, characterized in that: The cross-linking refers to the use of a cross-linking agent in apoA E125C / E136C Cysteine ​​125 of recombinant protein A and apoA E125C / E136C The cysteine ​​at position 136 of recombinant protein B forms a thioether bond cross-link.

4. The apoA-I anti-sugar functional mutant according to claim 3, characterized in that: The cross-linking agent is bismaleimidohexane.

5. A method for preparing an apoA-I anti-sugar functional mutant, comprising the following steps: (1) apoA E125C / E136C The plasmid of the recombinant protein is transformed into competent cells, followed by expression and purification; (2) In apoA E125C / E136C The recombinant protein solution is added with a cross-linking agent for cross-linking to obtain an apoA-I anti-sugar functional mutant.

6. Use of the apoA-I anti-sugar functional mutant as claimed in claim 1 in the preparation of a drug for treating diabetic cardiovascular disease.

7. The use according to claim 6, characterized in that: The medicine further comprises a pharmaceutically acceptable carrier and / or excipient.

8. The use according to claim 6, characterized in that: The pharmaceutically acceptable carrier and / or excipient includes at least one of a diluent, a binder, a surfactant, a humectant, an adsorption carrier, a lubricant, a filler, and a disintegrant.

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