Recombinant human heparin n-deacetylase encoding gene, recombinant carrier, recombinant strain and application thereof

Abstract

The present application relates to the field of molecular biology, in particular to a recombinant human heparin N-deacetylase encoding gene, a recombinant vector, a recombinant strain and application thereof, the recombinant human heparin N-deacetylase constructed in the expression process shows excellent stability and activity, has the ability of specific catalysis of deacetylation of acetyl group in heparin precursor, and can effectively generate deacetylated heparin precursor. This property significantly improves the conversion efficiency and reduces the generation of by-products. The present application optimizes the codon of the recombinant human heparin N-deacetylase, and uses the promoter P AOX1 or P GAP to regulate the expression, so that the expression level of the recombinant human heparin N-deacetylase in Pichia pastoris is significantly improved, and the obtained GS115-AOX1-hNDase and GS115-PGAP-hNDase strains successfully realize the secretory expression of the target gene, providing a new strategy and method for the biosynthesis of heparin.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of molecular biology, in particular to a recombinant human heparin N-deacetylase encoding gene, a recombinant vector, a recombinant strain and application thereof. BACKGROUND

[0002] Heparin is an important natural polysaccharide belonging to the glycosaminoglycan family, which is widely present in animals and mainly secreted by the liver and mast cells. Due to its excellent anticoagulant properties, heparin is widely used in the clinic for the prevention and treatment of various blood-related diseases, such as deep vein thrombosis and pulmonary embolism. However, the clinical application of heparin faces some challenges, especially the issue of its source. Traditionally, heparin is mainly extracted from the intestinal mucosa of pigs and cows, which not only raises ethical and safety concerns about animal sources, but also easily leads to batch-to-batch quality instability and supply shortages. In addition, the poor stability of heparin and the difficulty in dose control further limit its widespread application.

[0003] The main methods for heparin deacetylation include biological enzyme method and chemical method. Biological enzyme method usually uses specific enzymes (such as heparin deacetylase, NDase) to remove acetyl groups in heparin molecules through catalytic reaction, thereby effectively generating different heparin derivatives, which are often used as key substrates for the synthesis and modification of heparin polysaccharides, and also affect biological activity. NDase exists in the form of N-deacetylase / N-sulfotransferase in living organisms, and current research mainly focuses on its molecular characteristics, functions and application potential.

[0004] Compared with biological enzyme method, chemical method mainly uses chemical reagents for deacetylation process, but has some disadvantages. First, chemical deacetylation reaction lacks selectivity, which may lead to deacetylation at non-specific sites, generating multiple by-products, which increases the complexity and cost of subsequent separation and purification. Second, chemical method often requires high temperature and acid-base environment, which may lead to degradation of heparin molecules, reducing the quality of the final product. In addition, the use of harmful chemical reagents pollutes the environment and increases the requirements for safety and environmental protection. At the same time, the overall cost of chemical method is usually high, mainly driven by the cost of required equipment and operation process. In addition, this method is sensitive to changes in reaction conditions, which may lead to poor reproducibility between experiments, affecting the consistency and reliability of production. These shortcomings prompt researchers to explore more selective and environmentally friendly methods such as biological enzyme method.

[0005] Most of the successful human N-deacetylase is derived from the eukaryotic expression system, and is mainly expressed in the form of complete N-deacetylase / N-sulfotransferase. However, due to the large molecular weight of the complete bifunctional protein, it is difficult to express and secrete. Some studies have attempted to express the complete N-deacetylase / N-sulfotransferase in Pichia pastoris, but the expression amount is still low. The independent N-sulfotransferase (NST) has been successfully expressed heterologously, but the single activity expression of NDase has not been reported yet.

[0006] In summary, the current source of human N-deacetylase is still very limited, which cannot meet the needs of clinical and scientific research. Therefore, it is necessary to develop a new expression strategy to improve the expression of this protein. SUMMARY

[0007] The present application is to solve the above problems, and aims to provide a recombinant human heparin N-deacetylase encoding gene, a recombinant vector, a recombinant strain and the application thereof.

[0008] In a first aspect of the present application, a recombinant human heparin N-deacetylase encoding gene based on Pichia pastoris is provided, characterized in that the nucleotide sequence of the recombinant human heparin N-deacetylase encoding gene is shown in SEQ ID NO. 1.

[0009] In some embodiments of the first aspect, the recombinant human heparin N-deacetylase encoding gene is regulated by a promoter P AOX1 or P GAP .

[0010] In a second aspect of the present application, a recombinant vector is provided, characterized in that it comprises the recombinant human heparin N-deacetylase encoding gene based on Pichia pastoris according to the first aspect.

[0011] In some embodiments of the second aspect, the recombinant human heparin N-deacetylase encoding gene is inserted into the pPICZ alpha A as a starting plasmid, and the recombinant vector comprises a promoter P AOX1 , and the nucleotide sequence of the P AOX1 is shown in SEQ ID No. 2.

[0012] In some embodiments of the second aspect, the recombinant human heparin N-deacetylase encoding gene is inserted into the pPICZ alpha A as a starting plasmid, and the recombinant vector comprises a promoter P GAP , and the nucleotide sequence of the P GAP is shown in SEQ ID No. 3.

[0013] In a third aspect of the present application, a recombinant strain is provided, characterized in that it comprises the recombinant vector according to any one of the second aspect.

[0014] In a fourth aspect, the present application provides a method for preparing a recombinant strain, comprising the following steps:

[0015] (1) transforming the recombinant vector into the Pichia pastoris strain to obtain a transformed strain;

[0016] (2) screening the transformed strain using an antibiotic and testing the expression level of the recombinant human heparin N-deacetylase to obtain the recombinant strain.

[0017] In some embodiments of the fourth aspect, the Pichia pastoris is selected from one of the following: Pichia pastoris X-33 strain, Pichia pastoris GS115 strain, Pichia pastoris KM71 strain, and Pichia pastoris SMD1168 strain.

[0018] In some embodiments of the fourth aspect, the antibiotic is Zeocin, and the method for testing the expression level of the recombinant human heparin N-deacetylase is SDS-page method.

[0019] In a fifth aspect, the present application provides a method for preparing a recombinant human heparin N-deacetylase, comprising culturing the recombinant strain of the fourth aspect at 25-28℃ and pH 5.5-6.5 to obtain the recombinant human heparin N-deacetylase from the cells or culture of the recombinant strain.

[0020] In a sixth aspect, the present application provides an application of a recombinant human heparin N-deacetylase, comprising using the recombinant human heparin N-deacetylase of the fifth aspect to specifically catalyze the deacetylation of acetyl groups in heparin precursors to form deacetylated heparin precursors.

[0021] By implementing the above technical solutions, the present application has the following advantages:

[0022] The recombinant human heparin N-deacetylase constructed by the present application has excellent stability and activity during expression, has the ability to specifically catalyze the deacetylation of acetyl groups in heparin precursors, and can effectively generate deacetylated heparin precursors. This property significantly improves the transformation efficiency and reduces the generation of by-products.

[0023] Compared with traditional chemical methods, the biological enzyme method adopted by the present application has obvious advantages, higher selectivity, and environmental friendliness, can reduce the generation of by-products and pollutants in the production process, thereby effectively reducing the production cost. Further, the present application combines Pichia pastoris as an expression system for recombinant human heparin N-deacetylase, which has the advantages of high expression, simple operation, and low cost. Compared with more complex insect cells and mammalian cells, Pichia pastoris is more suitable for large-scale industrial production.

[0024] In the expression of recombinant human heparin N-deacetylase by Pichia pastoris, the codon optimization is needed according to the amino acid sequence of the target protein to obtain a more favorable DNA sequence for translation. The present application optimizes the codon of recombinant human heparin N-deacetylase, and uses the promoter P AOX1 or P GAP to regulate its expression, so that the expression level of recombinant human heparin N-deacetylase in Pichia pastoris is significantly improved. The finally obtained GS115-AOX1-hNDase and GS115-PGAP-hNDase strains successfully realize the secretory expression of the target gene, providing a new strategy and method for the biosynthesis of heparin. BRIEF DESCRIPTION OF DRAWINGS

[0025] Figure 1 Figure for the results of general primer colony PCR identification of recombinant expression strains GS115-AOX1-hNDase and GS115-PGAP-hNDase in Example 1;

[0026] Figure 2 Figure for protein expression analysis of recombinant expression strains GS115-AOX1-hNDase and GS115-PGAP-hNDase in Example 2;

[0027] Figure 3 Figure for SDS-page analysis of hNST and mASTIV protein purification in Example 3;

[0028] Figure 4 Figure for the reaction mechanism of N-sulfotransferase-ASTIV combined with NDase in Example 4;

[0029] Figure 5 Figure for the standard curve of PNP in Example 4;

[0030] Figure 6 Figure for protein expression analysis of recombinant expression strains GS115-AOX1-hNDase and GS115-PGAP-hNDase at different fermentation temperatures in Example 5;

[0031] Figure 7 Figure for liquid chromatography analysis of heparin precursor (K5 polysaccharide) standard and partially N-sulfated K5 polysaccharide standard in Example 6;

[0032] Figure 8 Figure for liquid chromatography analysis of heparin precursor polysaccharide before and after the reaction of NDase catalyzing the generation of deacetylated heparin precursor polysaccharide in Example 6;

[0033] Figure 9 Figure for liquid chromatography analysis of heparin precursor before and after the reaction of NDase combined with NST one-pot method in Example 7. DETAILED DESCRIPTION

[0034] In order to make the technical means, creative features, purposes and effects of the present application easy to understand, the present application is specifically described below in combination with examples and drawings.

[0035] Example 1

[0036] (1) Construction of hNDase recombinant expression vector GS115-AOX1-hNDase:

[0037] The base sequence of the Pichia pastoris codon bias optimized coding (hNDase) was designed and artificially synthesized, and was inserted into the Pichia pastoris methanol-induced secretory plasmid pPICZαA to construct the plasmid pPICZαA-hNDase. The hNDase base sequence after codon optimization is shown in SEQ ID No. 2.

[0038] After linearizing the above plasmid pPICZαA-hNDase with Sal I enzyme, the hNDase gene was inserted into the genome of Pichia pastoris GS115 by electric transformation with the parameters of voltage 1500V and resistance 200Ω, and then 1 ml of 4-degree pre-cooled 1 mol / L D-sorbitol was quickly added, and then the mixture was pre-cultured at 30°C in a shaker at 220 rpm for 2 h, and then was plated on a 100 μg / ml high-concentration Zeocin YPD plate. A high-copy strain was screened out using a 1.5 mg / ml high-concentration Zeocin YPD plate as the starting strain GS115-AOX1-hNDase.

[0039] (2) Construction of hNDase recombinant expression vector GS115-PGAP-hNDase:

[0040] The constructed plasmid pPICZαA-hNDase was subjected to the following operations, and a homologous recombination kit was used to replace the promoter, and a constitutive promoter PGAP was used to replace the inducible promoter P GAP in pPICZαA-hNDase, to obtain the recombinant plasmid pPGAPZαA-hNDase. AOX1 , wherein the sequence of P AOX1 is shown in SEQ ID No. 3, and the sequence of P GAP is shown in SEQ ID No. 4.

[0041] The specific operation of introducing the plasmid into the strain to achieve the strain containing the target plasmid is as follows: the pGAPZA type plasmid is linearized by using the restriction endonuclease Avr II, the purified linear plasmid is mixed uniformly with the competent Pichia pastoris in an electroporation cup, and then placed on ice for 15 min; the linear plasmid is inserted into the Pichia pastoris genome by electroporation (1500 V, 250 Ω), 1 mL of 4-degree pre-cooled 1 mol / L D-sorbitol is quickly added, and pre-cultured at 30 °C for 2 h; then, the culture is spread on a YPD plate containing 100 μg / mL of bleomycin and grown for 3 days; and a high-concentration Zeocin (1.5 mg / ml) YPD plate is used to screen a high-copy strain as the starting strain GS115-PGAP-hNDase.

[0042] (3) Confirmation of construction of GS115-AOX1-hNDase and GS115-PGAP-hNDase

[0043] Colony PCR identification is performed by using the universal primers a-factor: 5'-TACTATTGCCAGCATTGCTGC-3' and 3AOX: 5'-GCAAATGGCATTCTGACATCC-3', to confirm that the related genes are integrated into Pichia pastoris, and the strains GS115-AOX1-hNDase and GS115-PGAP-hNDase are successfully constructed, as shown in FIG. 1, wherein lanes 1-2 are the PCR products of the strain GS115-AOX1-hNDase; lanes 3-4 are the PCR products of the strain GS115-PGAP-hNDase; and the expected PCR product size is 1624 bp. Figure 1

[0044] Example 2:

[0045] Strain construction and expression of hNDase recombinant protein:

[0046] SDS-PAGE is used to verify the product of the shake flask level culture.

[0047] The specific conditions of the shake flask culture of GS115-AOX1-hNDase are as follows: a single colony is picked and cultured in YPD at 30 °C and 220 rpm for 24 h to obtain a seed liquid; the GS115-AOX1-hNDase is transferred to BMGY medium and cultured for 24 h until the OD600 is 15; then, the culture is resuspended in BMGY, and 0.5% of methanol is added every 12 h, and the culture is performed for a total of 144 h.

[0048] ​The specific conditions of GS115-PGAP-hNDase shake flask culture are as follows: picking single colony in YPD at 30 ℃ 220 rpm for 24 h as seed liquid, transferring GS115-PGAP-hNDase to YPD culture medium for 144 h, and adding 2% glucose every 24 h.

[0049] The results are shown in Figure 2 As shown in Table 1, the strains GS115-AOX1-hNDase and GS115-PGAP-hNDase constructed in Example 1 both achieved successful secretion expression of the target gene, that is, the plasmid can be normally integrated into the target strain, and the target gene can also be stably present.

[0050] Example 3:

[0051] Expression and purification of N-sulfotransferase and arylthiosulfotransferase

[0052] The base sequence of E. coli codon bias optimization coding human N-sulfotransferase domain (hNST, genebank: NP_001287992.1; 599-882) was designed and artificially synthesized, and inserted into T7 promoter vector pET-32a(+). After verifying the sequence, heat shock transformation was performed on E. coli BL21(DE3) competent cells to obtain hNST expression engineering bacteria. Single colonies were picked from LB medium containing 50 μg / ml ampicillin and inoculated into LB liquid medium containing 50 μg / ml ampicillin. After 12 h of 37℃ shaking bed 200 rpm shaking culture, the culture was transferred to 3 L liquid TB medium for expansion culture, and 37℃ shaking bed 200 rpm shaking culture was continued for 12 h. When the optical density OD600 of the culture reached 0.6, the temperature was reduced to 16℃, and IPTG solution with a final concentration of 0.2~0.8 mM was added for induction expression for 16~20 h. The culture was centrifuged at 4000 rpm for 25 min, and the supernatant was discarded. The bacterial cells were stored at -20℃ for standby.

[0053] The collected bacteria were added with 20 mL cell lysate (20 mM imidazole, 50 mM NaH2PO4, 300 mM NaCl, pH 8.0) per 4 g, 1 mg / mL lysozyme was added before use, and the cells were broken by ultrasonic at 4 ℃ for 20 min, centrifuged at 12,000 rpm for 25 min, and the supernatant was the crude enzyme solution. 100 mL Ni-NTA filler was added to the glass chromatography column, the filler was settled, 20 mL lysate was equilibrated, and the crude enzyme solution was loaded at a rate of 1 ml / min. 20 mL cell lysate was used to elute impurities, the target protein was eluted with elution buffer (250 mM imidazole, 50 mM NaH2PO4, 300 mM NaCl, pH 8.0), the protein concentration was determined, and dialysis was performed to remove imidazole, thereby obtaining hNST with high purity, which was stored at 4 ℃ for standby.

[0054] The base sequence of E. coli codon bias optimized coding mouse aryl sulfotransferase (mASTIV, genebank: NP_114022.1) was designed and artificially synthesized, and inserted into pCold promoter vector pCold II. After verifying the sequence, it was heat-shocked and transformed into E. coli BL21(DE3) competent cells to obtain hNST expression engineering bacteria. Single colonies were picked from LB medium containing 50 μg / ml ampicillin, inoculated into LB liquid medium containing 50 μg / ml ampicillin, and cultured at 37℃ with shaking at 200 rpm for 12 h. Then, it was transferred to 3 L liquid TB medium for large-scale culture, and continued to be cultured at 37℃ with shaking at 200 rpm for 12 h. When the optical density OD600 of the culture reached 0.6, the temperature was reduced to 16℃, and IPTG solution with a final concentration of 0.1-1.0 mM was added for induction expression for 16-20 h. The culture was centrifuged at 4000 rpm for 25 min, and the supernatant was discarded. The bacteria were stored at -20℃ for standby. The purity of the purified protein was analyzed by SDS-page.

[0055] For every 4 g of collected bacterial cells, 20 mL of cell lysis buffer (20 mM imidazole, 50 mM NaH2PO4, 300 mM NaCl, pH 8.0) was added. Just before use, 1 mg / mL lysozyme was added. The cells were lysed by sonication at 4 °C for 20 min, followed by centrifugation at 12,000 rpm for 25 min. The supernatant was the crude enzyme solution. 100 mL of Ni-NTA packing material was added to a glass chromatography column. After the packing material settled, 20 mL of lysis buffer was added for equilibration. The crude enzyme solution was loaded at a rate of 1 mL / min. 20 mL of cell lysis buffer was used to elute impurities, and the target protein was eluted with elution buffer (250 mM imidazole, 50 mM NaH2PO4, 300 mM NaCl, pH 8.0). The protein concentration was determined, and the solution was dialyzed to remove imidazole, yielding highly pure mASTIV, which was stored at 4 °C for later use. The purity of the purified protein was analyzed using SDS-PAGE.

[0056] Depend on Figure 3 It can be seen that hNST and mASTIV expression and purification were successful, with protein purity exceeding 90%. The proteins were flash-frozen and stored at -20℃ for later use.

[0057] Example 4: Determination of NDase activity using N-sulfotransferase-ASTIV combination

[0058] The activity of NDase was determined by combining N-sulfotransferase and ASTIV, taking advantage of the maximum UV absorption of PNP (4-nitrophenol) at 405 nm. The absorbance was measured using a microplate reader. The specific reaction mechanism is described in [link to relevant documentation]. Figure 4 A PNP standard curve was prepared for calculating NDase activity.

[0059] The enzyme activity assay system comprises the following components: 10–20 mM potassium p-nitrobenzenesulfonate, 0.5 mM PAPS, 50 μg ASTIV, 50 μg NST, and 1 mg / mL heparin precursor polysaccharide, dissolved in 20 mM pH 7.4 HEPES buffer, which is the enzyme activity assay stock solution. Add the stock solution to the microplate at a ratio of enzyme solution / crude enzyme solution: enzyme activity assay stock solution = 1:3. After incubation at 37°C for 30 min, add 20% 1 M NaOH, scan the absorbance at 405 nm, and calculate the generated PNP using the standard curve. Unit enzyme activity is defined as the amount of enzyme required to catalyze the generation of 1 μmol of PNP per minute under the above reaction conditions. That is, 1 U = 1 μmol / min. The PNP standard curve is shown below. Figure 5 .

[0060] Example 5: Detection of optimal expression conditions for the strain

[0061] The activity of products cultured in shake flasks was detected using N-sulfotransferase-ASTIV coupling.

[0062] By adjusting the temperature and pH, protein expression levels under different temperature and pH conditions were measured. Fermentation temperature, pH, and time were adjusted, and the fermentation supernatant was collected by centrifugation as a crude enzyme solution. Protein activity was then measured using N-sulfotransferase-ASTIV to determine the optimal expression conditions for the strain. Results are as follows: Figure 6 As shown, Figure 6 -A: Lanes 1-5 show the test results of the supernatant of GS115-AOX1-hNDase fermented at 20 ℃, 25 ℃, 28 ℃, 30 ℃, and 32 ℃, respectively; Figure 6 -B: Lanes 1-5 show the test results of the supernatant of GS115-PGAP-hNDase fermented at 20 ℃, 25 ℃, 28 ℃, 30 ℃, and 32 ℃, respectively.

[0063] Depend on Figure 6 It can be seen that the induced promoter P AOX1 The optimal conditions for fermentation production of hNDase are 25℃, pH 6.0, and the enzyme activity reaches its peak (28.83 U / mL) after 96 hours of methanol induction. The constitutive promoter P... GAP The optimal conditions for fermentation production of hNDase are 28℃ and pH 6.0. Enzyme activity reached its peak after 48 hours using glucose as the carbon source, but only reached 20.64 U / mL. Therefore, this study selected the inducible promoter P... AOX1 Fermentation to produce hNDase.

[0064] Based on the enzyme activity expression level, the methanol-induced promoter P AOX1 At this time, there is a trend of a significant increase in enzyme activity, especially when using the constitutive promoter P. GAP At this time, the enzyme activity is low, but no external inducer is needed for induction, and it has a more stable and safer effect compared to inducible promoters.

[0065] Example 6:

[0066] Production of deacetylated heparin precursor polysaccharide using NDase modification of heparin precursor:

[0067] The crude enzyme solution prepared in Example 5 was used as a catalyst to carry out the conversion reaction of the substrate heparin polysaccharide precursor.

[0068] The crude enzyme solution obtained in Example 5 was added to the reaction system after activity determination, which contained 50-200 U NDase, 10 mM CaCl2, 1 mg / mL heparin polysaccharide precursor, dissolved in 50 mM pH 7.5 Tris-HCl buffer, and reacted at 30°C for 16 h with constant shaking, and the reaction was terminated by heating at 99°C for 5 min. After centrifugation at 12000 rpm for 30 min, the supernatant was added to heparinase for enzymatic hydrolysis for 4 h, and then analyzed by high performance liquid chromatography (HPLC) for the amount of substrate and product. The liquid chromatogram of the standard sample is shown in Figure 7 wherein, Figure 7 -A is the liquid chromatogram of the heparin precursor (K5 polysaccharide) standard sample, Figure 7 -B is the liquid chromatogram of the N-sulfated K5 polysaccharide standard sample.

[0069] The NH2 conversion rate was determined by liquid chromatography before and after the reaction, and the conversion rate was >80%. The liquid chromatogram is shown in Figure 8 , Figure 8 -A is the liquid chromatogram of the reaction 0 h, Figure 8 -B is the liquid chromatogram of the reaction 8 h, Figure 8 -C is the liquid chromatogram of the reaction 20 h.

[0070] Example 7:

[0071] One-pot modification of heparin precursor to produce desacetyl sulfate heparin polysaccharide using NDase combined with NST:

[0072] The protein obtained in Example 4 was mixed with the crude enzyme solution obtained in Example 5 and added to the reaction system, which contained 50-200 U NDase, 100-400 U NST, 50 mM PAPS, 10 mM CaCl2, 1 mg / mL heparin polysaccharide precursor, dissolved in 50 mM pH 7.5 Tris-HCl buffer, and reacted at 30°C for 16 h with constant shaking, and the reaction was terminated by heating at 99°C for 5 min. After centrifugation at 12000 rpm for 30 min, the supernatant was added to heparinase for enzymatic hydrolysis for 4 h, and then analyzed by high performance liquid chromatography (HPLC) for the amount of substrate and product. The NH2 conversion rate was determined by liquid chromatography before and after the reaction, and the conversion rate was >90%. The liquid chromatograms before and after the reaction are shown in Figure 9 , Figure 9 -A is the liquid chromatogram before the reaction, Figure 9 -B is the liquid chromatogram after the reaction.

[0073] The above embodiments are preferred cases of the present application and do not limit the protection scope of the present application.

Claims

1. A recombinant human heparin N-deacetylase encoding gene based on Pichia pastoris, characterized in that, The nucleotide sequence of the gene encoding the recombinant human heparin N-deacetylase is shown in SEQ ID NO.

1.

2. The recombinant human heparin N-deacetylase encoding gene according to claim 1, characterized in that, The recombinant human heparin N-deacetylase encoding gene is activated via promoter P. AOX1 or P GAP Regulation.

3. A recombinant vector, characterized in that, Includes the recombinant human heparin N-deacetylase encoding gene based on Pichia pastoris as described in claim 2.

4. The recombinant vector as described in claim 3, characterized in that, Using pPICZαA as the starting plasmid, a recombinant human heparin N-deacetylase encoding gene was inserted. The recombinant vector contains the promoter P. AOX1 The P AOX1 The nucleotide sequence is shown in SEQ ID No.

2.

5. The recombinant vector as described in claim 3, characterized in that, Using pPICZαA as the starting plasmid, a recombinant human heparin N-deacetylase encoding gene was inserted. The recombinant vector contains the promoter P. GAP The P GAP The nucleotide sequence is shown in SEQ ID No.

3.

6. A recombinant bacterial strain, characterized in that, It includes the recombinant vector according to any one of claims 3-5.

7. The method for preparing the recombinant strain according to claim 6, characterized in that, Includes the following steps: The recombinant vector was transformed into Pichia pastoris strain to obtain the transformed strain; The transformed strains were screened using antibiotics, and further screened by testing the expression level of recombinant human heparin N-deacetylase to obtain the recombinant strains.

8. The method for preparing the recombinant strain according to claim 7, characterized in that, The Pichia pastoris is selected from one of the following strains: Pichia pastoris X-33, Pichia pastoris GS115, Pichia pastoris KM71, and Pichia pastoris SMD1168.

9. A method for preparing recombinant human heparin N-deacetylase, characterized in that, The recombinant strain of claim 8 is cultured at 25-28°C and pH 5.5-6.5 to obtain the recombinant human heparin N-deacetylase from the cells or culture of the recombinant strain.

10. The application of a recombinant human heparin N-deacetylase, characterized in that, The recombinant human heparin N-deacetylase of claim 9 is used to specifically catalyze the deacetylation of acetyl groups in heparin precursors to form deacetylated heparin precursors.

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