Long-acting recombinant human alpha-galactosidase A-Fc fusion protein and preparation method thereof

Abstract

The invention discloses a long-acting recombinant human alpha-galactosidase A-Fc fusion protein and a preparation method thereof, and the preparation method of the long-acting recombinant human alpha-galactosidase A-Fc fusion protein comprises the following steps: constructing a transgenic mouse by using a gene segment GLA-Fc of the recombinant human alpha-galactosidase A-Fc fusion protein; the method comprises the following steps: secreting milk containing recombinant human alpha-galactosidase A-Fc fusion protein through a transgenic mouse, and purifying the milk. The recombinant human alpha-galactosidase A-Fc fusion protein prepared by the method disclosed by the invention is high in yield and low in cost; in the process of purifying milk, a sodium citrate buffer solution is used for degreasing, and a specific affinity chromatography and gel chromatography combined mode is used for separation and purification, so that the recombinant human alpha-galactosidase A-Fc fusion protein with a long half-life period can be obtained, and the recombinant human alpha-galactosidase A-Fc fusion protein has market popularization and application prospects.

Description

Technical Field

[0001] The present invention specifically relates to a long-acting recombinant human alpha-galactosidase A-Fc fusion protein and a preparation method thereof. Background Art

[0002] Fabry disease is caused by α-galactosidase A (GLA) deficiency, which causes Gb3 and Lyso-Gb3 to accumulate in nerves, skin, kidneys, heart and other tissues, leading to multiple organ dysfunction. The most widely used treatment for Fabry disease is enzyme replacement therapy using recombinant human GLA. However, the recombinant human GLA currently used generally has a short half-life, which leads to a high frequency of medication for patients, affecting patient compliance.

[0003] Recombinant human α-galactosidase A-Fc fusion protein is a protein (abbreviated as GLA-Fc) that is formed by fusing human α-galactosidase A (GLA) with the Fc fragment of immunoglobulin G (IgG) through genetic engineering technology. This fusion protein is designed to enhance the stability of GLA and prolong its half-life, thereby improving its efficacy in the treatment of Fabry disease. Patent CN202380022935.7 obtains recombinant α-galactosidase A-Fc fusion protein through recombinant cell fermentation culture. However, studies have suggested that different host cells (such as Escherichia coli, CHO cells, plant cells, etc.) have different expression and modification capabilities for therapeutic recombinant proteins, which may lead to differences in the structure, activity, and stability of the fusion protein (Shen Luo et al., Benchmark Glycan Profle of Therapeutic Monoclonal Antibodies Produced by Mammalian Cell Expression Systems); in some host cells, endogenous proteases may degrade recombinant proteins, resulting in reduced yields; in CHO cells, glycosylation modification may be affected by culture conditions (such as carbon source type, pH value, etc.), and different culture conditions may increase the difficulty of separating and purifying therapeutic recombinant proteins from CHO cell fermentation broth (Cao Mengyuan, Upstream process regulation strategy for CHO cells expressing easily degradable proteins).

[0004] The production of fusion proteins in animal mammary bioreactors is characterized by high yield and low cost, and can provide a new expression route for the supply of α-galactosidase A, a drug for the treatment of Fabry disease. However, the mechanism of protein processing and modification by mammary cells is not yet fully understood; the total content of components such as fat and lactose in milk is relatively high, which makes it difficult to purify the target protein. Currently, there are no reports on expressing recombinant human α-galactosidase A-Fc fusion protein in transgenic animals to increase the yield of recombinant human α-galactosidase A-Fc fusion protein and reduce production costs. Summary of the invention

[0005] In order to solve the above problems, the present invention provides a long-acting recombinant human α-galactosidase A-Fc fusion protein, the amino acid sequence of which is shown in SEQ ID NO.8.

[0006] The present invention also provides a gene fragment encoding the aforementioned fusion protein, and its nucleotide sequence is shown as SEQ ID NO.4.

[0007] The present invention also provides a method for preparing the aforementioned fusion protein, comprising the following steps:

[0008] Inserting a gene fragment with a nucleotide sequence such as that shown in SEQ ID NO.4 into a plasmid to obtain a recombinant plasmid; taking the recombinant plasmid, digesting it with a restriction endonuclease, injecting the linearized insert fragment after digestion into the fertilized egg of an animal, and culturing it to a morula or blastocyst; taking the surviving morula or blastocyst and transplanting it into the uterus of a surrogate animal by non-surgical cervical embryo transfer technology for gestation; after the offspring is born, collecting tissue samples to identify the transgenic animals through PCR, raising, mating, giving birth, collecting the milk of the transgenic animals that gave birth, and purifying it to obtain the transgenic animals.

[0009] Furthermore, the plasmid is a pBC1 vector; the restriction endonucleases are Sal I and Not I; the animals include rabbits, cattle, sheep, and mice; and the mice include ICR mice.

[0010] The present invention also provides a method for purifying the aforementioned fusion protein, comprising the following steps:

[0011] Take animal milk containing the above-mentioned fusion protein, add sodium citrate solution to mix, centrifuge, take the supernatant to filter, perform affinity chromatography on the filtrate, perform gel filtration chromatography on the affinity chromatography fluid, collect the gel filtration chromatography fluid to obtain the product.

[0012] Furthermore, the volume ratio of the latex to the sodium citrate solution is 1:0.5-1:1.5; the pH value of the sodium citrate solution is 5.5±0.5, and the concentration is 10-50mM.

[0013] Furthermore, the affinity chromatography is performed by loading the filtrate onto a chromatography column filled with Protein A, eluting with citric acid buffers of pH 3.2 and 2.8 in sequence, collecting 20 mM citric acid buffer of pH 3.2 to obtain an affinity chromatography fluid.

[0014] Furthermore, the gel filtration chromatography is performed by loading the affinity chromatography liquid onto a chromatography column filled with gel, eluting with a phosphate buffer containing NaCl at a pH of 6.8-7.4, and starting to collect when the absorbance rises to 11.75 mAU at a wavelength of 280 nm, and ending the collection when the absorbance drops to 11.27 mAU to obtain a gel filtration chromatography liquid; the pH value of the phosphate buffer containing NaCl is preferably 7.0-7.2.

[0015] Furthermore, the gel includes a composite matrix of dextran and agarose, or porous silica gel; the composite matrix includes Cytiva Superdex 200, Nano-W Super 200, and Boglon Chromdex 200PG; the concentration of phosphate in the phosphate buffer is 25mM, and the concentration of NaCl is 150mM; the pore size of the porous silica gel is 25nm, and the particle size is 13μm; the phosphate is disodium hydrogen phosphate-sodium dihydrogen phosphate.

[0016] Furthermore, the animal milk is milk prepared according to the aforementioned method.

[0017] The present invention utilizes the gene fragment GLA-Fc of human α-galactosidase A-Fc fusion protein to construct transgenic mice, prepares milk containing human α-galactosidase A-Fc fusion protein by transgenic mice, and then purifies the milk to obtain human α-galactosidase A-Fc fusion protein. The human α-galactosidase A-Fc fusion protein prepared by the method of the present invention has high yield and low cost; in the process of purifying the milk, sodium citrate buffer is used for delipidation, and a specific affinity chromatography and gel chromatography are used for separation and purification. The obtained human α-galactosidase A-Fc fusion protein has a long half-life, high stability, and has market promotion and application prospects.

[0018] Obviously, according to the above contents of the present invention, in accordance with common technical knowledge and customary means in the art, without departing from the above basic technical ideas of the present invention, other various forms of modification, replacement or change may be made.

[0019] The following is a further detailed description of the above contents of the present invention through specific implementation methods in the form of embodiments. However, this should not be understood as the scope of the above subject matter of the present invention being limited to the following examples. All technologies implemented based on the above contents of the present invention belong to the scope of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 PCR analysis and identification results of transgenic mice (left: PCR analysis results, middle: transgenic mice with different copy numbers; right: low copy refers to less than 2 copies, high copy refers to more than 8 copies);

[0021] Figure 2 Purification profile of PBS buffer defatting;

[0022] Figure 3 Purification profile of sodium citrate buffer;

[0023] Figure 4 Chromatographic profile of affinity chromatography in purification route 1;

[0024] Figure 5 Chromatographic profile of SP chromatography in purification route 1;

[0025] Figure 6 Chromatographic profile of gel filtration chromatography in purification route 1;

[0026] Figure 7 SEC-HPLC results of affinity chromatography in purification route 1;

[0027] Figure 8 SEC-HPLC results of SP chromatography in purification route 1;

[0028] Fig. 9 SEC-HPLC results of gel filtration chromatography in purification route 1;

[0029] Fig.10 Chromatographic profile of affinity chromatography in purification route 2;

[0030] Fig.11 Chromatographic profile of gel filtration chromatography in purification route 2;

[0031] Fig.12 SEC-HPLC results of gel filtration chromatography in purification route 2;

[0032] Fig.13 SEC-HPLC results of GLA-Fc stability (left: GLA-Fc with Tween20 added, GLA-Fc+Tween20); right: GLA-Fc without Tween20 added, GLA-Fc-Tween20);

[0033] Fig.14 Activity results of GLA-Fc stability (left: GLA-Fc with Tween20 added, GLA-Fc+Tween20); right: GLA-Fc without Tween20 added (GLA-Fc-Tween20);

[0034] Fig.15Pharmacokinetic characteristics of α-Gal A-Fc protein and Replagal in mouse serum. The half-life of α-Gal A-Fc protein is approximately 471 minutes; the half-life of Replagal is approximately 11 minutes. One group of mice received a single intravenous injection of 1.633 mg / kg α-GalA-Fc protein, while another group of mice received an intravenous injection of Replagal at a concentration of 1 mg / mL (the molar ratio between the injected α-Gal A-Fc protein and Replagal was 1:1). Serum samples were collected at different time points after injection, and α-Gal-A enzyme activity was evaluated using pNP-Gal as a synthetic substrate. The results are presented as the mean ± SD of three measurements (n = 3). DETAILED DESCRIPTION

[0035] The raw materials, reagents and equipment in the specific embodiments of the present invention are all known products, which are purchased from the market. The fusion protein involved is a fusion protein GLA-Fc in which the C-terminus of GLA is fused to the Fc segment of IgG1. The sequence information is as follows:

[0036] Nucleotide sequence of GLA-Fc (SEQ ID NO.4):

[0037]

[0038]

[0039] Note: The capitalized part is the GLA nucleic acid sequence (SEQ ID NO.1); the capitalized bold part is the linker nucleic acid sequence (SEQ ID NO.2); the capitalized underlined part is the IgG1 Fc nucleic acid sequence (SEQ ID NO.3)

[0040] GLA-Fc protein sequence (SEQ ID NO.8)

[0041]

[0042]

[0043] Note: The capitalized part is the GLA protein sequence (SEQ ID NO.5); the capitalized bold part is the linker sequence (SEQ ID NO.6); the capitalized underlined part is the Fc protein sequence (SEQ ID NO.7).

[0044] Example 1 Preparation of milk containing recombinant human α-galactosidase A-Fc (GLA-Fc) fusion protein

[0045] The gene fragment with the nucleotide sequence as shown in SEQ ID NO.4 is inserted into the pBC1 plasmid to obtain a recombinant plasmid; the recombinant plasmid is digested with restriction endonucleases Sal I and Not I, and the linearized insert fragment after digestion is injected into the fertilized egg of ICR mice and cultured into morula or blastocyst; the surviving morula or blastocyst is transplanted into the uterus of surrogate ICR mice by non-surgical cervical embryo transfer technology, the ear tissue or tail tissue of the surviving offspring is taken and PCR is used to identify the transgenic ICR mice, the mice are raised, mated, and litters are produced, and the milk of the transgenic ICR mice produced is taken to obtain the transgenic ICR mice.

[0046] Example 2 Purification of recombinant human α-galactosidase A-Fc (GLA-Fc) fusion protein

[0047] Take the milk obtained in Example 1, add 20mM sodium citrate solution with pH 5.5 at a volume ratio of 1:1.15, centrifuge at 16000×g for 40min, take the supernatant and filter it with a 0.45um membrane, perform affinity chromatography on the filtrate, perform gel filtration chromatography on the affinity chromatography solution, collect the gel filtration chromatography solution, and obtain;

[0048] The affinity chromatography is to load the filtrate onto a chromatography column filled with Protein A, and then elute with 20 mM citric acid buffer at pH 3.2 and 2.8, and collect the 20 mM citric acid buffer at pH 3.2 to obtain the affinity chromatography solution. The specific operation steps are as follows:

[0049]

[0050] Gel filtration chromatography is an affinity chromatography liquid loaded on a chromatography column filled with porous silica gel (pore size 25nm, particle size 13μm), eluted with 25mM phosphate buffer containing 150mM NaCl at pH 7.0, and the elution peak during elution was detected by a UV-visible spectrophotometer (wavelength 280nm). When the absorbance rises to 11.75mAU, collection begins, and when it drops to 11.27mAU, collection ends to obtain a gel filtration chromatography liquid. The specific operation steps are as follows:

[0051]

[0052] The beneficial effects of the present invention are described below through test examples.

[0053] Experimental Example 1 Preparation of the GLA-Fc fusion protein of the present invention

[0054] 1. Using transgenic animals to express GLA-Fc fusion protein

[0055] 1.1 Construction of transgenic expression cassette

[0056] α-Gal A-Fc is a recombinant protein (also called fusion protein) composed of human GLA (from Genebank, ID: NM_000169.3), a linker, and the Fc region of human IgG1. The gene fragment GLA-Fc expressing the recombinant human α-Gal A-Fc protein is sequentially connected by GLA (SEQ ID NO.1), a linker (SEQ ID NO.2), and Fc (SEQ ID NO.3). The GLA-Fc gene fragment was inserted into the pBC1 vector (Invitrogen) to construct the recombinant plasmid pBC1-GLA-Fc.

[0057] 1.2 Construction of GLA-Fc transgenic mice

[0058] The pBC1-GLA-Fc vector was digested with restriction endonucleases (Sal I and Not I) to remove the prokaryotic sequence. The linearized insert was injected into the fertilized eggs of ICR mice by microinjection, and then cultured and developed into morula or blastocysts. The surviving morula or blastocysts were transplanted into the unilateral uterus of the surrogate mother mouse by non-surgical cervical embryo transfer (TCET). Finally, the surrogate mother mouse successfully became pregnant and gave birth to live pups. After the pups were born, tissue samples (mouse tail or mouse ear tissues) were collected and identified by PCR analysis. Transgenic mice ( Figure 1 ).

[0059] The transgenic mice were raised, mated, and gave birth. The milk of the transgenic mother mice was collected, and the activity of the target protein in the milk was detected by an α-galactosidase activity detection method. The activity range of the target protein was about 9.5-14.2 mmol / h / ml, which met the collection standard. The milk was used as a raw material to extract the GLA-Fc fusion protein (SEQ ID NO.8).

[0060] 2. Extraction and purification of GLA-Fc fusion protein from mouse milk

[0061] At present, GLA-Fc fusion protein is mainly expressed by recombinant CHO cells, and GLA-Fc fusion protein is extracted from CHO fermentation broth. The components of mouse milk are quite different from those of CHO fermentation broth. Therefore, when extracting and purifying GLA-Fc fusion protein from mouse milk, in addition to the common method of chromatography purification, it is also necessary to consider methods for removing animal milk components. In the study, centrifugation was first used to remove fat from mouse milk, and the skimmed mouse milk was filtered and then explored through chromatography steps.

[0062] 2.1 Degreasing method

[0063] Select PBS (10-50mM phosphate, 0.05-0.3M NaCl, pH7.4±0.2) or sodium citrate buffer (10-50mM sodium citrate, pH5.5±0.5) to dilute mouse milk (volume ratio of mouse milk: buffer 1:0.5-1:1.5), centrifuge for 40-60min (2-8℃, 12000-20000×g), collect the supernatant and filter. Delipidation evaluation of the two buffer systems was performed by affinity chromatography. The conditions of affinity chromatography are as follows:

[0064]

[0065] Note: PBS buffer is prepared from disodium hydrogen phosphate-sodium dihydrogen phosphate according to the phosphate concentration and buffer pH value, the same below.

[0066] The protein elution peak of elution buffer 1 was detected and collected by UV-Vis spectrophotometer (wavelength 280 nm), and the protein yield was calculated. Figures 2-3 and Table 1.

[0067] Table 1 Yield of purified products from mouse milk after treatment with different dilutions

[0068]

[0069] From the results, it can be seen that when the dilution buffer system of mouse milk is sodium citrate buffer (10-50mM sodium citrate, pH5.5±0.5), the degreasing effect is better and the yield of the purified product collected is relatively high, thus confirming that the dilution buffer system for subsequent experiments is sodium citrate buffer.

[0070] 2.2 Purification method

[0071] According to the molecular characteristics of GLA-Fc, three different chromatographic combinations of Protein A filler, cationic filler (SP) and gel were used to purify the defatted mouse milk.

[0072] 2.2.1 Purification route 1

[0073] After the mouse milk was combined, it was diluted with buffer (20 mM sodium citrate, pH 5.5) at a volume ratio of 1:1.15, centrifuged at 16000×g for 40 min, the supernatant was collected, and filtered through a 0.45 um membrane for sample loading.

[0074] ①Affinity chromatography

[0075] Filler: Protein A, affinity chromatography operating conditions are as follows:

[0076]

[0077] The protein elution peak in elution buffer 1 was detected and collected by UV-visible spectrophotometer (wavelength 280 nm), labeled as GLA-Fc PH3.2-XT, and concentrated by ultrafiltration for subsequent further chromatography.

[0078] ②Cation exchange chromatography (SP chromatography)

[0079] Filler: SP, cation exchange chromatography operating conditions are as follows:

[0080]

[0081] The eluates of the two elution steps were detected by a UV-visible spectrophotometer (wavelength 280 nm), and the elution products were collected according to the UV absorption of the two elution steps. Finally, the elution product of the first elution step was collected and marked as: GLA-Fc SP-XT, which was then concentrated by ultrafiltration for subsequent further chromatography;

[0082] ③Gel filtration chromatography

[0083] Filler: Cytiva Superdex 200, gel filtration chromatography operating conditions are as follows:

[0084]

[0085] The elution peak was detected by UV-visible spectrophotometer, and the eluted protein separation peak was collected in a fixed volume collection mode. The two collected solutions 1B-10 and 1B-11 (corresponding to 280nm UV absorption of 2.38-54.94mAU) were combined and labeled as: GLA-Fc SEC 1B-10 / 1B-11.

[0086] ④ Detection of eluent at each chromatography stage

[0087] The chromatograms of affinity chromatography, SP chromatography and gel chromatography at each stage of the chromatography process are shown in Figures 4 to 6 From the chromatograms of each stage, it can be seen that the fusion protein can be effectively eluted by affinity chromatography; although a single protein peak can be obtained by SP chromatography, the purity analysis shows that the fusion protein cannot be effectively separated from aggregates and other impurities; the fusion protein can be effectively separated by gel filtration, and the fusion protein is in the last partial separation peak.

[0088] The eluate from affinity chromatography (GLA-Fc PH3.2-XT), the eluate from SP chromatography (GLA-Fc SP-XT), and the fractions collected from gel filtration chromatography (GLA-Fc SEC 1B-10 / 1B-11) were tested using SEC-HPLC according to General Chapter 3122 of the Chinese Pharmacopoeia. The results are shown in Figures 7 to 9The test results suggest that: ① SP chromatography cannot reduce the aggregate level of the target protein; ② Gel filtration chromatography can significantly remove the target protein aggregates, and a two-step chromatography method of affinity chromatography and gel filtration chromatography can be tried to purify the target protein with a purity of >90%.

[0089] 2.2.2 Purification route 2

[0090] After the mouse milk was combined, it was diluted with buffer (20 mM sodium citrate, pH 5.5) at a volume ratio of 1:1.15, centrifuged at 16000×g for 40 min, the supernatant was collected, and filtered through a 0.45 um membrane for sample loading.

[0091] ①Affinity chromatography

[0092] Filler: Protein A. Affinity chromatography operating conditions are as follows.

[0093]

[0094] The protein elution peak of elution buffer 1 is detected and collected by UV-visible spectrophotometer, and the collected elution peak is concentrated by ultrafiltration for subsequent further chromatography;

[0095] ②Gel filtration chromatography

[0096] Filler: porous silica gel (pore size 25 nm, particle size 13 μm), gel filtration chromatography operating conditions are as follows:

[0097]

[0098] The elution peak was detected by UV-visible spectrophotometer, and the eluted protein separation peak was collected in a fixed volume collection mode. The collected liquids from 2G9 to 2H7 (corresponding to 280nm UV absorption of 11.75-11.27mAU) were combined and labeled as: GLA-Fc SEC 2G9-2H7.

[0099] ③ Detection of eluent at each chromatography stage

[0100] Chromatographic profiles during affinity chromatography and gel chromatography are shown in Figures 10-11 The gel filtration chromatography fractions (GLA-Fc SEC 2G9-2H7) were collected for SEC-HPLC detection. The results are shown in Fig.12 The test results suggest that the target protein aggregates can be significantly removed by capturing the mouse milk with affinity chromatography and then purifying it with gel filtration chromatography. The target protein with a purity of >95% can be obtained by two-step chromatography purification.

[0101] The above results show that the GLA-Fc fusion protein obtained from mouse milk containing GLA-Fc fusion protein is highly pure and can reach more than 95% after being defatted using sodium citrate buffer and then subjected to affinity chromatography and gel filtration chromatography.

[0102] Experimental Example 2 Biological Activity and Pharmacokinetic Analysis of the GLA-Fc Fusion Protein of the Present Invention

[0103] 1. GLA-Fc activity detection

[0104] According to the marketed products, a synthetic substrate that can be catalyzed and hydrolyzed by human recombinant α-GLA is used to determine its enzyme activity. The chromogenic substrate is p-nitrophenyl-α-D-pyranogalactoside, and the enzyme activity is detected by quantitatively analyzing the chromogenic group p-nitrophenol produced by the enzymatic hydrolysis of the substrate.

[0105] 1.1 GLA-Fc fusion protein activity detection

[0106] Dilution of standard solution: dilute the p-nitrophenol standard solution with distilled water to 200, 100, 50, 25, 12.5, 6.25, 0 mmol / mL. Refer to the following table for the dilution of standard solution:

[0107]

[0108] Sample determination: Add the reagents in the kit based on the chromogenic principle of nitrophenyl-α-D-pyranogalactoside provided by Beijing Solebow Technology Co., Ltd. to a 1.5mL EP tube in sequence. The specific method of adding the reagents is as follows:

[0109]

[0110] Enzyme activity of purified product:

[0111] The GLA-Fc fusion protein (20240626) purified by the method of purification route 2 in Experimental Example 1 was mixed with Tween 20 at a mass ratio of 7.5:1. The activity test results showed that the specific activity of the sample GLA-Fc plus Tween 20, pH 6.0, was 8.5×10 5 U / mg.

[0112] 1.2 GLA-Fc stability

[0113] In order to confirm the effect of Tween 20 on the stability of GLA-Fc purified by the purification route 2 in Experimental Example 1 stored at 2-8°C, the purity of GLA-Fc with or without Tween 20 added after 34 days was tested by SEC-HPLC, and its activity was determined by the protein activity detection method under "1.1". The results are shown in Figures 13-14 .

[0114] The results showed that after adding Tween 20, GLA-Fc was stored at 2-8°C for at least 6 weeks and still met the requirements of purity>95% and stable activity; GLA-Fc without Tween 20 could only meet the requirements of purity>95% within 3 weeks, and the activity fluctuated greatly, and could not meet the requirements of stability. This shows that the GLA-Fc fusion protein expressed by transgenic animals has higher stability after being defatted, extracted and purified by the present invention with the addition of Tween 20.

[0115] 2. Pharmacokinetic analysis

[0116] The pharmacokinetic characteristics of the two drugs were evaluated by a single tail vein injection of α-Gal A-Fc protein (the injected product contained Tween 20 at a ratio of 0.023% (w / v) and a ratio of 1:7.5 to GLA-Fc, and GLA-Fc was prepared according to the method of purification route 2 in Experimental Example 1) or Replagal. The dosage was calculated by molar ratio (to ensure that the activity of the administered α-GalA was consistent). The α-Gal A-Fc group received 1.633 mg / kg of α-Gal A-Fc protein, while the Replagal group received 1 mg / kg of Replagal. Fig.15 For the pharmacokinetic characteristics, Fig.15 It can be seen that at a dose of 1.633 mg / kg, the half-life of α-GalA-Fc protein is 471 minutes (about 8 hours), while Replagal is only 11 minutes. Under the same experimental conditions, Replagal retains about 15% of its initial activity within 15 minutes after injection, and α-Gal A-Fc protein retains about 70% of its initial activity; 30 minutes after injection, Replagal has almost no enzyme activity, while α-Gal A-Fc protein still maintains about 50% of its activity, and it is not until 24 hours after injection that α-Gal A-Fc protein has almost no detectable enzyme activity. This shows that the circulation half-life of α-Gal A-Fc protein is significantly longer than that of the commercial product Replagal.

[0117] In summary, the present invention utilizes the gene fragment GLA-Fc of human α-galactosidase A-Fc fusion protein to construct transgenic mice, prepares milk containing human α-galactosidase A-Fc fusion protein by transgenic mice, and then defattes the milk with sodium citrate buffer, separates and purifies it by combining specific affinity chromatography and gel chromatography, and obtains human α-galactosidase A-Fc fusion protein with long half-life and high stability. The human α-galactosidase A-Fc fusion protein prepared by the method of the present invention has high yield, good quality, low cost, and market promotion and application prospects.

Claims

1. A long-acting recombinant human α-galactosidase A-Fc fusion protein, characterized in that: Its amino acid sequence is shown in SEQ ID NO.

8.

2. A gene fragment encoding the fusion protein of claim 1, characterized in that: Its nucleotide sequence is shown in SEQ ID NO.

4.

3. A method for preparing the fusion protein according to claim 1, characterized in that: The steps include: Inserting a gene fragment with a nucleotide sequence such as that shown in SEQ ID NO.4 into a plasmid to construct a recombinant plasmid; taking the recombinant plasmid, digesting it with a restriction endonuclease, injecting the linearized insert fragment after digestion into the fertilized egg of an animal, and culturing it into a morula or blastocyst; taking the surviving morula or blastocyst and transplanting it into the uterus of a surrogate animal by non-surgical cervical embryo transfer technology for gestation; after the offspring is born, collecting tissue samples and identifying the transgenic animals through PCR, raising, mating, giving birth, collecting the milk of the transgenic animals that gave birth, and purifying it.

4. The preparation method according to claim 3, characterized in that: The plasmid is pBC1 plasmid; the restriction endonucleases are SalⅠ and NotⅠ; the animals include rabbits, cattle, sheep, and mice; and the mice include ICR mice.

5. A method for purifying the fusion protein according to claim 1, characterized in that: The method comprises the following steps: Take animal milk containing the fusion protein of claim 1, add sodium citrate solution to mix, centrifuge, take the supernatant to filter, perform affinity chromatography on the filtrate, perform gel filtration chromatography on the affinity chromatography fluid, collect the gel filtration chromatography fluid to obtain the product.

6. The purification method according to claim 5, characterized in that: The volume ratio of the latex to the sodium citrate solution is 1:0.5-1:1.5; the pH value of the sodium citrate solution is 5.5±0.5, and the concentration is 10-50mM.

7. The purification method according to claim 5, characterized in that: The affinity chromatography is performed by loading the filtrate onto a chromatography column filled with Protein A, eluting with citric acid buffers of pH 3.2 and 2.8 in sequence, collecting 20 mM citric acid buffer of pH 3.2 to obtain an affinity chromatography solution.

8. The purification method according to claim 5, characterized in that: The gel filtration chromatography is performed by loading the affinity chromatography liquid onto a chromatography column filled with gel, eluting with a phosphate buffer containing NaCl at pH 6.8-7.4, and collecting when the absorbance rises to 11.75 mAu at a wavelength of 280 nm and ending when the absorbance drops to 11.27 mAu to obtain a gel filtration chromatography liquid.

9. The purification method according to claim 8, characterized in that: The gel comprises a composite matrix of dextran and agarose, or porous silica gel; the composite matrix comprises Cytiva Superdex 200, Nano-Wave NWSuper 200, and Boglong Chromdex 200PG; the concentration of phosphate in the phosphate buffer is 25mM, and the concentration of NaCl is 150mM.

10. The purification method according to claim 5, characterized in that: The animal milk is prepared according to the method according to any one of claims 3 to 4.

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