Recombinant human heparin N-deacetylase coding gene, recombinant vector, recombinant strain and application thereof

Abstract

The invention relates to the field of molecular biology, in particular to a recombinant human heparin N-deacetylase coding gene, a recombinant vector, a recombinant strain and application of the recombinant human heparin N-deacetylase coding gene, the recombinant vector, the recombinant strain and the application of the recombinant human heparin N-deacetylase coding gene. The catalyst has the capability of specifically catalyzing deacetylation of acetyl groups in heparin precursors, and can effectively generate deacetylated heparin precursors. By means of the characteristic, the conversion efficiency is remarkably improved, and generation of by-products is reduced. The recombinant human heparin N-deacetylase is subjected to codon optimization, and the expression of the recombinant human heparin N-deacetylase is regulated and controlled by using a promoter PAOX1 or PGAP, so that the expression level of the recombinant human heparin N-deacetylase in pichia pastoris is remarkably improved, the obtained GS115-AOX1-hNDase and GS115-PGAP-hNDase strains successfully realize the secretory expression of target genes, and the recombinant human heparin N-deacetylase can be used for preparing the recombinant human heparin N-deacetylase. And a new strategy and a new method are provided for biosynthesis of heparin.

Description

Technical Field

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

[0002] Heparin is an important natural polysaccharide belonging to the glycosaminoglycan class. It is widely present in animals and is mainly secreted by the liver and mast cells. Due to its excellent anticoagulant properties, heparin is widely used in clinical practice to prevent and treat various blood-related diseases, such as deep vein thrombosis and pulmonary embolism. However, the clinical application of heparin faces some challenges, especially its source. Traditionally, heparin is mainly extracted from the intestinal mucosa of pigs and cattle, which not only raises ethical and safety issues about animal sources, but also easily leads to unstable quality and insufficient supply between batches. In addition, problems such as heparin's poor stability and difficulty in dosage control further limit its widespread application.

[0003] The main methods of heparin deacetylation include bioenzymatic and chemical methods. Bioenzymatic methods usually use specific enzymes (such as heparin deacetylase, NDase) to remove the acetyl group in the heparin molecule through catalytic reactions, thereby effectively generating different heparin derivatives, which are often used as key substrates for the synthesis and modification of heparin polysaccharides and affect biological activity. NDase exists in the body as a bifunctional N-deacetylase / N-sulfotransferase, and current research focuses on its molecular characteristics, functions and application potential.

[0004] Compared with the bio-enzymatic method, the chemical method mainly uses chemical reagents for the deacetylation process, but it has some disadvantages. First, the chemical deacetylation reaction lacks selectivity, which may lead to deacetylation of non-specific sites and generate a variety of by-products, which increases the complexity and cost of subsequent separation and purification. Secondly, the chemical method often requires higher temperatures and acid-base environments, which may lead to the degradation of heparin molecules and reduce 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 the chemical method is usually high, mainly driven by the cost of the required equipment and operating procedures. In addition, the method is sensitive to changes in reaction conditions, which may lead to poor repeatability between experiments, affecting the consistency and reliability of production. These shortcomings have prompted researchers to explore more selective and environmentally friendly methods such as bio-enzymatic methods.

[0005] Most of the human N-deacetylases that have been successfully expressed so far are derived from eugenic expression systems, and are mainly expressed in the form of complete N-deacetylase / N-sulfotransferase. However, due to the large molecular weight of the complete bifunctional protein, secretory expression is difficult. Some studies have attempted to express the complete N-deacetylase / N-sulfotransferase in Pichia pastoris, but the expression level is still low. Independent N-sulfotransferase (NST) has now been successfully heterologously expressed, but the independent active expression of NDase has not yet been reported.

[0006] In summary, the current sources of human N-deacetylase are still very limited and cannot meet the needs of clinical and scientific research. Therefore, it is very necessary to develop new strategies to increase the expression of this protein. Summary of the invention

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

[0008] In a first aspect of the present invention, a recombinant human heparin N-deacetylase encoding gene based on Pichia pastoris is provided, wherein 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 expressed via promoter P AOX1 or P GAP Regulation.

[0010] The second aspect of the present invention provides a recombinant vector, characterized in that it comprises the Pichia pastoris-based recombinant human heparin N-deacetylase encoding gene described in the first aspect.

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

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

[0013] The third aspect of the present invention provides a recombinant strain, characterized in that it contains the recombinant vector described in any one of the second aspects.

[0014] A fourth aspect of the present invention provides a method for preparing a recombinant strain, characterized in that it comprises the following steps: (1) transforming the recombinant vector into a Pichia pastoris strain to obtain a transformed strain; (2) The transformed strains are screened using antibiotics and the expression level of recombinant human heparin N-deacetylase is tested to obtain the recombinant strains.

[0015] In some embodiments of the fourth aspect, the Pichia pastoris is selected from one of Pichia pastoris X-33 strain, Pichia pastoris GS115 strain, Pichia pastoris KM71 strain and Pichia pastoris SMD1168 strain.

[0016] In some embodiments of the fourth aspect, the antibiotic is Zeocin, and the method for testing the expression level of recombinant human heparin N-deacetylase is the SDS-page method.

[0017] The fifth aspect of the present invention provides a method for preparing a recombinant human heparin N-deacetylase, characterized in that the recombinant strain described in the fourth aspect is cultured at 25-28°C and pH 5.5-6.5, and the recombinant human heparin N-deacetylase is obtained from the cells or culture of the recombinant strain.

[0018] The sixth aspect of the present invention provides an application of a recombinant human heparin N-deacetylase, characterized in that the recombinant human heparin N-deacetylase described in the fifth aspect is used to specifically catalyze the deacetylation of the acetyl group in a heparin precursor to form a deacetylated heparan precursor.

[0019] By implementing the above technical solution, the present invention has the following beneficial effects: The recombinant human heparin N-deacetylase constructed by the present invention exhibits excellent stability and activity during the expression process, has the ability to specifically catalyze the deacetylation of the acetyl group in the heparin precursor, and can effectively generate the deacetylated heparan precursor. This characteristic significantly improves the conversion efficiency and reduces the generation of by-products.

[0020] Compared with the traditional chemical method, the bioenzyme method adopted in the present invention has obvious advantages, has higher selectivity and environmental friendliness, can reduce the generation of by-products and pollutants in the production process, thereby effectively reducing production costs. Further, the present invention combines Pichia pastoris as an expression system for recombinant human heparin N-deacetylase, which has significant advantages such as high expression amount, simple operation and low cost. Compared with more complex insect cells and mammalian cells, Pichia pastoris is more suitable for large-scale industrial production.

[0021] When using Pichia pastoris to express recombinant human heparin N-deacetylase, codon optimization is required according to the amino acid sequence of the target protein to obtain a DNA sequence that is more conducive to translation. AOX1 or P GAP By regulating its expression, the expression level of recombinant human heparin N-deacetylase in Pichia pastoris was significantly increased. The resulting GS115-AOX1-hNDase and GS115-PGAP-hNDase strains successfully achieved secretory expression of the target gene, providing a new strategy and method for the biosynthesis of heparin. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 The universal primer colony PCR identification result of the recombinant expression strains GS115-AOX1-hNDase and GS115-PGAP-hNDase in Example 1 is shown; Figure 2 This is a protein expression analysis diagram of the recombinant expression strains GS115-AOX1-hNDase and GS115-PGAP-hNDase in Example 2; Figure 3 This is the SDS-page analysis diagram of hNST and mASTIV protein purification in Example 3; Figure 4 This is a diagram of the reaction mechanism of the combined use of N-sulfotransferase and ASTIV to determine NDase in Example 4; Figure 5 It is the PNP standard curve diagram in Example 4; Figure 6 This is a graph showing protein expression analysis of the recombinant expression strains GS115-AOX1-hNDase and GS115-PGAP-hNDase at different fermentation temperatures in Example 5; Figure 7 This is a liquid chromatography analysis chart of the heparin precursor (K5 polysaccharide) standard and the partially N-sulfonated K5 polysaccharide standard in Example 6; Figure 8 This is a liquid chromatography analysis diagram before and after the reaction of NDase catalyzing heparin precursor polysaccharide to generate deacetylheparan precursor polysaccharide in Example 6; Fig. 9 This is a liquid chromatography analysis diagram before and after the reaction of modifying the heparin precursor by the one-pot method of NDase combined with NST in Example 7. DETAILED DESCRIPTION

[0023] In order to make the technical means, creative features, objectives and effects achieved by the present invention easier to understand, the present invention is described in detail below in conjunction with embodiments and drawings.

[0024] Embodiment 1: (1) Construction of hNDase recombinant expression vector GS115-AOX1-hNDase: The base sequence encoding (hNDase) optimized by Pichia pastoris codon preference was designed and artificially synthesized, and inserted into the Pichia pastoris methanol-induced secretion plasmid pPICZαA to construct the plasmid pPICZαA-hNDase. The base sequence of hNDase after codon optimization is shown in SEQ ID No.2.

[0025] After linearizing the plasmid pPICZαA-hNDase using Sal I enzyme, the hNDase gene was inserted into the genome of Pichia pastoris GS115 by electroporation with the voltage set to 1500 V and the resistance set to 200 Ω. 1 ml of 1 mol / L D-sorbitol precooled at 4 degrees was quickly added, and the plate was pre-cultured at 220 rpm in a 30°C shaker for 2 h. The plate was then spread on a YPD plate with a high concentration of Zeocin of 100 μg / ml. A high-copy strain was selected using a YPD plate with a high concentration of Zeocin of 1.5 mg / ml as the starting strain GS115-AOX1-hNDase.

[0026] (2) Construction of hNDase recombinant expression vector GS115-PGAP-hNDase: The constructed plasmid pPICZαA-hNDase was subjected to the following operations: a homologous recombination kit was used to replace the promoter, and a constitutive promoter P GAP Replace the inducible promoter P in pPICZαA-hNDase AOX1 , where P AOX1 The sequence is shown in SEQ ID No. 3, P GAP The sequence is shown in SEQ ID No. 4, and the recombinant plasmid pPGAPZαA-hNDase was obtained.

[0027] The specific operation of introducing plasmid into strain to achieve the target plasmid in strain is as follows: pGAPZA type plasmid is linearized by restriction endonuclease AvrⅡ, the purified linear plasmid and competent Pichia pastoris are mixed evenly in an electroporation cup, and placed on ice for 15 min, and the linear plasmid is inserted into the Pichia pastoris genome by electroporation (1500 V, 250 Ω), 1 mL of 4 degrees pre-cooled 1 mol / L D-sorbitol is quickly added, and pre-cultured at 30 ℃ for 2 h, and spread on YPD plates containing 100 μg / mL zeocin for 3 days, and a high copy strain is screened out using a YPD plate with a high concentration of Zeocin of 1.5 mg / ml as the starting strain GS115-PGAP-hNDase.

[0028] (3) Construction and confirmation of GS115-AOX1-hNDase and GS115-PGAP-hNDase Colony PCR identification was performed using universal primers a-factor: 5´-TACTATTGCCAGCATTGCTGC-3´, 3AOX: 5´-GCAAATGGCATTCTGACATCC-3´, and the relevant genes were confirmed to be integrated into Pichia pastoris. The strain GS115-AOX1-hNDase and strain GS115-PGAP-hNDase were successfully constructed. Figure 1 As shown, lanes 1-2 are the PCR products of strain GS115-AOX1-hNDase; lanes 3-4 are the PCR products of strain GS115-PGAP-hNDase; the expected PCR product size is 1624 bp.

[0029] Embodiment 2: Construction and expression of hNDase recombinant protein strains: The products of the shake flask culture were verified by SDS-PAGE.

[0030] The specific conditions for shaking flask culture of GS115-AOX1-hNDase are as follows: a single colony was picked and cultured in YPD at 30°C and 220rpm for 24 h as seed solution, GS115-AOX1-hNDase was transferred to BMGY medium and cultured for 24 h until OD600 reached 15, then resuspended in BMGY, 0.5% methanol was added every 12 h, and cultured for a total of 144 h.

[0031] The specific conditions for shaking flask culture of GS115-PGAP-hNDase are as follows: a single colony was picked and cultured in YPD at 30 ℃ and 220 rpm for 24 h as seed solution, and GS115-PGAP-hNDase was transferred to YPD medium and cultured for 144 h, with 2% glucose added every 24 h.

[0032] The results are as follows Figure 2 As shown, the strains GS115-AOX1-hNDase and GS115-PGAP-hNDase constructed in Example 1 both achieved successful secretory expression of the target gene, that is, the plasmid could be normally integrated into the target strain, and the target gene could also exist stably.

[0033] Embodiment 3: Expression and purification of N-sulfotransferase and arylsulfotransferase The base sequence encoding the human N-sulfotransferase domain (hNST, genebank: NP_001287992.1; 599-882) was designed and synthesized based on the codon preference optimization of Escherichia coli, and then inserted into the T7 promoter vector pET-32a (+). After sequence verification, heat shock transformation was performed into E. coli BL21 (DE3) competent cells to obtain hNST expression engineered bacteria. A single colony was picked from the LB plate medium containing 50 μg / ml ampicillin and inoculated into LB liquid medium containing 50 μg / ml ampicillin. After shaking culture at 37°C and 200 rpm for 12 h, the culture was transferred to 3 L liquid TB medium for expansion culture. Shaking culture was continued at 37°C and 200 rpm for 12 h. When the optical density OD600 of the culture reached 0.6, the temperature was lowered to 16°C, and IPTG solution with a final concentration of 0.2~0.8 mM was added to induce expression for 16~20 h. The culture was centrifuged at 4000 rpm for 25 min, the supernatant culture was discarded, and the bacteria were stored at -20°C for later use.

[0034] For every 4 g of collected bacteria, 20 mL of cell lysis buffer (20 mM imidazole, 50 mM NaH2PO4, 300 mM NaCl, pH 8.0) was added. 1 mg / mL lysozyme was added before use. The cells were ultrasonically disrupted for 20 min at 4 °C and centrifuged at 12,000 rpm for 25 min. The supernatant was the crude enzyme solution. 100 mL of Ni-NTA filler was added to the glass chromatography column. After the filler settled, 20 mL of lysis buffer was used for balance. The crude enzyme solution was loaded at a rate of 1 ml / min. 20 mL of cell lysis buffer was used to elute the 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 dialyzed to remove imidazole to obtain hNST with high purity. It was stored at 4 °C for future use.

[0035] The base sequence encoding mouse arylsulfoxide transferase (mASTIV, genebank: NP_114022.1) was designed and synthesized based on the codon preference optimization of Escherichia coli, and then inserted into the pCold promoter vector pCold II. After sequence verification, heat shock transformation was performed into E. coli BL21 (DE3) competent cells to obtain hNST expression engineered bacteria. Single colonies were picked from LB plate medium containing 50 μg / ml ampicillin and inoculated into LB liquid medium containing 50 μg / ml ampicillin. After shaking culture at 37°C and 200 rpm for 12 h, the culture was transferred to 3 L liquid TB medium for expansion culture. Shaking culture was continued at 37°C and 200 rpm for 12 h. When the optical density OD600 of the culture reached 0.6, the temperature was lowered to 16°C, and IPTG solution with a final concentration of 0.1~1.0 mM was added to induce expression for 16~20 h. The culture was centrifuged at 4000 rpm for 25 min, the supernatant culture was discarded, and the bacteria were stored at -20°C for later use. The purity of the purified protein was analyzed by SDS-page.

[0036] For every 4 g of collected bacteria, 20 mL of cell lysis buffer (20 mM imidazole, 50 mM NaH2PO4, 300 mM NaCl, pH 8.0) was added. 1 mg / mL lysozyme was added before use. The cells were ultrasonically disrupted for 20 min at 4 °C and centrifuged at 12,000 rpm for 25 min. The supernatant was the crude enzyme solution. 100 mL of Ni-NTA filler was added to the glass chromatography column. After the filler settled, 20 mL of lysis buffer was used for balance, and the crude enzyme solution was loaded at a rate of 1 ml / min. 20 mL of cell lysis buffer was used to elute the 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 dialyzed to remove imidazole to obtain mASTIV with high purity, which was stored at 4 °C for future use. The purity of the purified protein was analyzed by SDS-page.

[0037] Depend on Figure 3 It can be seen that hNST and mASTIV were successfully expressed and purified, and the protein purity reached more than 90%. They were quickly frozen and stored at -20°C for future use.

[0038] Example 4: Determination of NDase activity by combining N-sulfotransferase and ASTIV The NDase activity was determined by using the N-sulfotransferase-ASTIV method, taking advantage of the fact that PNP (4-nitrophenol) has a maximum UV absorption at 405 nm, and measuring the absorbance using a microplate reader. The specific reaction mechanism is shown in Figure 4. Make a PNP standard curve for calculating NDase activity.

[0039] The enzyme activity assay system contains the following components: 10~20 mM potassium p-nitrobenzene sulfonate, 0.5 mM PAPS, 50 μg ASTIV, 50 μg NST, 1 mg / mL heparin precursor polysaccharide, dissolved in 20 mM pH = 7.4 HEPES buffer, which is the enzyme activity detection mother solution. Add it to the ELISA plate at a ratio of enzyme solution / crude enzyme solution: enzyme activity detection mother solution = 1:3, incubate at 37°C for 30 min, add 20% 1 M NaOH, scan the absorbance at 405 nm, and use the standard curve to calculate the generated PNP. The unit enzyme activity is defined as the amount of enzyme required to catalyze the generation of 1 μmol PNP per minute under the above reaction conditions. That is, 1 U = 1 μmol / min. See the PNP standard curve for details. Figure 5 .

[0040] Example 5: Detection of optimal expression conditions of strains Activity detection of shake flask culture products using N-sulfotransferase-ASTIV combination By adjusting the temperature and pH, the protein expression levels under different temperature and pH conditions were measured, and the fermentation temperature, pH, and time were adjusted. The fermentation supernatant was centrifuged and used as a crude enzyme solution to measure protein activity using N-sulfotransferase-ASTIV to obtain the expression conditions of the strain below the optimal expression level. The results are as follows Figure 6 As shown, Figure 6 -A: Lanes 1-5 are the test results of the supernatant of GS115-AOX1-hNDase fermented at 20 ℃, 25 ℃, 28 ℃, 30 ℃, and 32 ℃; Figure 6 -B: Lanes 1-5 are the test results of the supernatant of GS115-PGAP-hNDase fermented at 20℃, 25℃, 28℃, 30℃, and 32℃, respectively.

[0041] Depend on Figure 6 Available, inducible promoter P AOX1 The optimal conditions for the fermentation production of hNDase were 25℃ and pH 6.0. The enzyme activity reached the highest level at 96h after methanol induction, reaching 28.83 U / mL. GAP The optimal conditions for the fermentation production of hNDase were 28°C and pH 6.0. The enzyme activity reached the highest level after 48 h using glucose as the carbon source, but it only reached 20.64 U / mL. Therefore, the inducible promoter P was selected in this study. AOX1 Fermentation production of hNDase.

[0042] The enzyme activity expression level was obtained by using methanol-inducible promoter P AOX1When the constitutive promoter P GAP When the promoter is activated, the enzyme activity is low, but no external inducer is required for induction, which makes it more stable and safer than the inducible promoter.

[0043] Embodiment 6: Production of heparan deacetylase precursor polysaccharide using NDase-modified heparin precursor: 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.

[0044] The crude enzyme solution obtained in Example 5 was further tested for activity and then added to a reaction system comprising 50-200 U NDase, 10 mM CaCl2, and 1 mg / mL heparin polysaccharide precursor dissolved in 50 mM pH 7.5 Tris-HCl buffer, and the reaction was shaken at 30°C for 16 h, and the reaction was terminated by heating at 99°C for 5 min. The reaction solution was centrifuged at 12,000 rpm for 30 min, and the supernatant was added with heparinase for enzymatic hydrolysis for 4 h, and then the substrate and product amounts were analyzed by high performance liquid chromatography (HPLC). The liquid chromatography analysis of the standard product is shown in FIG. Figure 7 ,in, Figure 7 -A is the liquid chromatography analysis chart of heparin precursor (K5 polysaccharide) standard, Figure 7 -B is the liquid chromatography analysis chart of N-sulfonated K5 polysaccharide standard.

[0045] The NH2 conversion rate was measured by liquid chromatography before and after the reaction, and the conversion rate was >80%. Figure 8 , Figure 8 -A is the liquid chromatography analysis chart of reaction 0 h, Figure 8 -B is the liquid chromatography analysis chart after 8 h reaction, Figure 8 -C reaction 20 h liquid chromatography analysis.

[0046] Embodiment 7: One-pot modification of heparin precursor using NDase and NST to produce deacetylated heparan sulfate polysaccharide: 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, and 1 mg / mL heparin polysaccharide precursor, dissolved in 50 mM pH 7.5 Tris-HCl buffer, and the reaction was carried out at 30°C for 16 h by constant temperature oscillation, and the reaction was terminated by heating at 99 degrees for 5 min. The reaction solution was centrifuged at 12000 rpm for 30 min, and the supernatant was added with heparinase for enzymatic hydrolysis for 4 h, and then the substrate and product amount were analyzed by high performance liquid chromatography (HPLC). The NH2 conversion rate was determined by liquid chromatography before and after the reaction, and the conversion rate was >90%. The liquid chromatography analysis before and after the reaction is shown in FIG. Fig. 9 , Fig. 9 -A is the liquid chromatography analysis chart before the reaction, Fig. 9 -B is the liquid chromatography analysis chart after the reaction.

[0047] The above-mentioned embodiments are preferred examples of the present invention and are not intended to limit the protection scope of the present invention.

Claims

1. A recombinant human heparin N-deacetylase encoding gene based on Pichia pastoris, characterized in that: The nucleotide sequence of the recombinant human heparin N-deacetylase encoding gene 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 expressed via promoter P AOX1 or P GAP Regulation.

3. A recombinant vector, characterized in that: The invention comprises the recombinant human heparin N-deacetylase encoding gene based on Pichia pastoris as claimed in claim 2.

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

2.

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

3.

6. A recombinant strain, characterized in that: Contains the recombinant vector according to any one of claims 3 to 5.

7. The method for preparing the recombinant strain according to claim 6, characterized in that: The following steps are involved: Transforming the recombinant vector into a Pichia pastoris strain to obtain a transformed strain; The transformed strains are screened using antibiotics and the expression level of the recombinant human heparin N-deacetylase is tested 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 according to 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. An application of recombinant human heparin N-deacetylase, characterized in that: The recombinant human heparin N-deacetylase according to claim 9 is used to specifically catalyze the deacetylation of the acetyl group in the heparin precursor to form a deacetylated heparan precursor.

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