Recombinant Batroxobin and Method for Its Preparation on an Industrial Scale with High Expression Level

Abstract

The present invention relates to recombinant batroxobin and a method for its preparation on an industrial scale with high expression level. Specifically, the method for preparing recombinant batroxobin according to the present invention is to artificially synthesize the structural gene sequence of batroxobin, then use Pichia pastoris to clone and express to screen and obtain the seed liquid of a high-expression engineering strain, and then ferment and culture to prepare recombinant batroxobin according to the following operations: preparation of primary seed liquid, preparation of secondary seed liquid, inoculation and pre-culture in a fermenter, glycerol fed-batch culture, methanol fed-batch induction culture, concentration to obtain a fermentation concentrate containing recombinant batroxobin and optionally purifying it. This recombinant batroxobin can be clinically used for acute cerebral infarction, for improving ischemic symptoms caused by various occlusive vascular diseases (such as thromboangiitis obliterans, deep phlebitis, pulmonary embolism, etc.), and for improving peripheral and microcirculation disorders (such as sudden deafness, motion sickness). The expression level of batroxobin by the method of the present invention is high, and industrial-scale production can be easily achieved.

Description

Technical Field

[0001] The present invention relates to a recombinant batroxobin and a method for preparing the same. More specifically, the present invention relates to a method for preparing a recombinant protein by using genetic engineering technology and a yeast culture production method, which can easily obtain a high-expression recombinant batroxobin on an industrial production scale. The recombinant batroxobin obtained by the method of the present invention has good performance. Background Art

[0002] The marketed varieties of snake venom thrombin-like enzyme preparations are: batroxobin from the venom of Bothrops atrox Bothrops moojeni ); ancrod (Ancrod, Viprinex, Arwin) from the venom of Calloselasma rhodostoma; defibrase from the venom of Deinagkistrodon acutus Agkistrodon acutus ). From 1985 to 2000, multi-component preparations containing snake venom thrombin-like enzymes from different snake venoms were circulated in China, and most of them were called agkistrodon acutus antithrombotic enzyme. Since 1997, domestic defibrase has replaced agkistrodon acutus antithrombotic enzyme.

[0003] Since the agkistrodon acutus has been listed as a national second-class endangered protected animal, the resources of natural snake venom are extremely limited. In addition, due to well-known reasons, such as factors related to the variation of snake venom raw materials, such as the growth environment of snakes, snake age, venom collection season, etc., and the influence of impurities in snake venom and the limitation of raw material production, it is extremely difficult to industrially produce this natural batroxobin on a stable scale so that it can be successfully applied to clinical practice as a safe, effective, and controllable drug, resulting in the current related products being difficult to meet the clinical medication needs. Therefore, it is of great practical significance to express snake venom thrombin-like enzymes by genetic engineering technology and prepare recombinant batroxobin with biological activity equivalent to that of natural batroxobin.

[0004] Recombinant batroxobin is a single, glycosylated, single-chain polypeptide containing 231 amino acids. As a linear glycoprotein compound, it has a carbohydrate content of approximately 5%, and its N-terminal amino acid is valine. Batroxobin is a serine protease secreted by the venom gland of Bothrops atrox. It can release fibrinopeptide A by cleaving 16-Arg-17-Gly in the fibrinogen A (α) chain and form a clot by aggregating fibrin I monomers or Des-AA monomers. However, the specificity of further fibrinogen degradation varies depending on the species source of the enzyme. In contrast to thrombin, it does not affect platelet function. Therefore, BX is used to study the final stage of blood coagulation. Since it is insensitive to heparin, it can detect fibrinogen polymerization disorders even in the presence of heparin. In addition, it is used to treat thrombotic diseases under the name of defibrase (Pentapharm).

[0005] Japanese scholars first successfully expressed the batroxobin gene in Escherichia coli in 1987 (J. Biol. Chem. 262: 3132-3135). Chinese scholars Li Zhaofa et al. (China Patent Authorization Announcement No. CN100564532C, Patent No. 200610165344.3) expressed biologically active batroxobin protein in yeast. In addition, CN113862246A (Application No. 202111186090.4, Gretterson) discloses a method for inducing the expression of recombinant batroxobin in Pichia pastoris and its purification, including using a continuous fermentation method during the fermentation process and using a mixed carbon source to induce Pichia pastoris, through fermentation processes such as glycerol fed-batch culture and methanol / glucose mixed induction culture. Additionally, CN116334052 (Application No. 202310399752.9, Shanghai Tengrui) discloses a fermentation method for batroxobin, including the following steps: taking the preserved strain and inoculating it into a first-stage seed medium. The formula of the first-stage seed medium is: yeast powder, 1.0%; polypeptone, 2.0%; glucose, 2.0%. After being prepared with purified water and sterilized for standby; under sterile conditions, the first-stage seed liquid is inoculated into a seed tank containing a second-stage medium, and the inoculation ratio is 5%-11%. The formula of the second-stage seed medium is: yeast powder, 1.0%; polypeptone, 2.0%; glucose, 2.0%. After being prepared with purified water and sterilized for standby; when the OD600 of the second-stage seed liquid is between 5 and 7, 4 ml / L of the optimized basic fermentation medium is added to the fermentation tank under sterile conditions, and the second-stage seed liquid is transferred into the fermentation tank under sterile conditions. After culturing for 4 hours, the optimized glycerol fed-batch medium is added to the fermentation tank, and the pH value is adjusted to 6.5 for methanol induction; the concentration of the glycerol fed-batch medium is 10 ml / L; the supernatant is collected and purified to obtain recombinant batroxobin; the components of the optimized basic fermentation medium are: K2SO4: 0.91%; MgSO4: 0.36%; CaSO4·2H2O: 0.059%; 85% H3PO4: 2.5% V / V; KOH: 0.206%; glycerol: 4%; antifoam: 0.05%-0.1%; PTM1: 0.4% V / V, casamino acids: 0.5%; the components of the optimized glycerol fed-batch medium are: 50% glycerol + 12 mL / L PTM1 + 0.5% casamino acids; during the methanol induction stage, the optimized methanol induction medium is used for induction. The components of the optimized methanol induction medium are: (100% methanol + 12 mL / L PTM1) 95% + 4.5% sorbitol + 0.5% casamino acids.

[0006] However, there is still a need in the art for new methods for preparing recombinant batroxobin, especially methods for stably obtaining high expression levels of recombinant batroxobin on an industrial production scale. Summary of the Invention

[0007] One of the objectives of the present invention is to provide a recombinant batroxobin and its preparation method, in particular to provide a method for stably obtaining a recombinant batroxobin with a high expression level on an industrial production scale. It has unexpectedly been found that by using the method of the present invention, a recombinant batroxobin with a high expression level can be prepared on an industrial production scale. The present invention has been completed based on such findings.

[0008] For this purpose, the first aspect of the present invention provides a method for preparing recombinant batroxobin, which comprises artificially synthesizing the structural gene sequence of batroxobin, then using Pichia pastoris to clone and express to screen and obtain a seed solution of a high-expression engineering strain, and then fermenting and culturing to prepare recombinant batroxobin according to the following operations:

[0009] (1) Preparation of primary seed solution: Inoculate the screened engineering strain into YPD medium and culture it in a shaking flask until the OD600 reaches 7-9 to obtain the primary seed solution;

[0010] (2) Preparation of secondary seed solution: Inoculate the primary seed solution into YPD medium and culture it in a shaking flask until the OD600 reaches 7-9 to obtain the secondary seed solution;

[0011] (3) Inoculation and pre-culture in a fermenter: Inoculate the secondary seed solution into the fermentation medium in the fermenter and culture it under the initial culture conditions of 30 °C, pH 5.8-6.2, rotation speed of 150-220 rpm, and air ventilation volume of 0.8-1.2 vvm. During the fermentation process, gradually increase the rotation speed and air ventilation volume, and control the dissolved oxygen within the range of 15-25%, until the rotation speed is increased to 650-750 rpm and the air ventilation volume is increased to 1.8-2.5 vvm, and then continue to culture until the carbon source is exhausted, and then carry out glycerol feeding culture;

[0012] (4) Glycerol feeding culture: Start to drip-feed 50% glycerol containing 10-14 ml / L of PTM1 solution at a rate of 5 ml / h / L for feeding, gradually increase the feeding speed to 45-55 ml / h / L within 30 minutes, adjust the rotation speed and air ventilation volume during this period to control the dissolved oxygen within the range of 15-25%, and continue to culture until the wet weight of the cells reaches within the range of 250-270 g / L, and then enter the next methanol feeding;

[0013] (5) Methanol feeding induction culture: Lower the temperature of the fermentation broth to 25 °C, adjust the pH to 2.8-3.2, and drip-feed methanol containing 10-14 ml / L of PTM1 solution at a rate of 2.8-3.5 ml / h / L, and maintain this methanol feeding speed until the fermentation ends. During this period, adjust the rotation speed and air ventilation volume to control the dissolved oxygen within the range of 15-25%, and start to introduce oxygen to make the ventilation volume up to 0.28-0.32 vvm after the rotation speed and air ventilation volume both reach the maximum; after the fermentation ends, centrifuge the fermentation broth and collect the supernatant;

[0014] (6) Ultrafilter the centrifuged supernatant using an ultrafiltration membrane package with a molecular weight cut-off of 10K, and concentrate it until the volume of the liquid on the membrane is 1 / 10 of the initial supernatant volume, obtaining a fermentation concentrate containing recombinant batroxobin, and optionally purify it.

[0015] According to the method of the first aspect of the present invention, the titer of the batroxobin fermentation concentrate obtained in step (6) is greater than 40 BU / ml, for example, greater than 50 BU / ml, for example, greater than 60 BU / ml, for example, greater than 70 BU / ml, for example, greater than 80 BU / ml.

[0016] According to the method of the first aspect of the present invention, the composition of the YPD medium used is: 1% peptone, 0.5% yeast extract, 2% glucose, water, and the pH is adjusted to 6.0 ± 0.2 with hydrochloric acid or sodium hydroxide.

[0017] According to the method of the first aspect of the present invention, the composition of the fermentation medium used is: 13 ml / L of 85% phosphoric acid, 0.46 g / L of calcium sulfate dihydrate, 9.1 g / L of potassium sulfate, 7.46 g / L of magnesium sulfate heptahydrate, 2.06 g / L of potassium hydroxide, 30.0 g / L of glycerol, 4.35 ml / L of PTM1 solution, and the balance is water.

[0018] According to the method of the first aspect of the present invention, the composition of the PTM1 solution used is: 6 g / L of copper sulfate, 0.088 g / L of sodium iodide, 3 g / L of manganese sulfate, 0.2 g / L of sodium molybdate, 0.02 g / L of boric acid, 0.5 g / L of cobalt chloride, 20 g / L of zinc chloride, 65 g / L of ferrous sulfate, 0.2 g / L of biotin, and the balance is water.

[0019] According to the method of the first aspect of the present invention, the composition of the PTM1 solution used is: 6 g / L of copper sulfate, 0.088 g / L of sodium iodide, 3 g / L of manganese sulfate, 0.2 g / L of sodium molybdate, 0.02 g / L of boric acid, 0.5 g / L of cobalt chloride, 20 g / L of zinc chloride, 65 g / L of ferrous sulfate, 0.2 g / L of biotin, 1.2 g / L of inositol, 2.5 g / L of calcium pantothenate, and the balance is water.

[0020] According to the method of the first aspect of the present invention, when artificially synthesizing the structural gene sequence of batroxobin, in order to recombine the synthesized target gene into the yeast secreted expression vector pPICZα, an Xho I restriction enzyme site is added to the 5′ end of the gene, a stop codon TAATGA and a SacII restriction enzyme site are added to the 3′ end of the gene.

[0021] According to the method of the first aspect of the present invention, when artificially synthesizing the structural gene sequence of batroxobin, the synthesized target gene and the pPICZα vector are digested with Xho I and SacII, electrophoresed and recovered, the target gene is recombined into pPICZα, and transformed into Escherichia coli Top10F', and screening is carried out. Escherichia coli Top10F' transformed with pPICZα is cultured on an LB medium plate supplemented with Zeocin, single colonies are picked, cultured in a liquid medium, plasmids are extracted, detected by double digestion with Xho I and SacII, and α-factor priming and 3'AOX1 priming PCR detections are carried out, showing that the target gene is contained in pPICZα and can be used to transform Pichia pastoris.

[0022] According to the method of the first aspect of the present invention, when artificially synthesizing the structural gene sequence of batroxobin, the composition of the LB medium supplemented with Zeocin is: 1% tryptone, 0.5% yeast extract, 0.5% sodium chloride, 1.5% agar, the pH value is adjusted to 7.0, autoclaved, and 25 μg / mg of zeocin (bleomycin) is added during plating. According to the method of the first aspect of the present invention, when artificially synthesizing the structural gene sequence of batroxobin, the liquid medium: 1% tryptone, 0.5% yeast extract, 0.5% sodium chloride, the pH value is adjusted to 7.0, autoclaved.

[0023] According to the method of the first aspect of the present invention, when using Pichia pastoris to clone and express to screen for the seed liquid of a highly expressed engineering strain, pPICZα-Bg is linearized with the DNA restriction endonuclease SacI-HF™, and the multi-copy Pichia pastoris expression kit of Gibco is used. According to the method described in the kit, yeast host competent cells are prepared and transformed. The transformed cells are spread on YPDZ medium and grown at 30 °C for 4-5 days to form single colonies. Clones with large and plump colonies are selected for expression screening, and highly expressed engineering strains are screened out in test tubes and inoculated into shake flasks for proliferation as the engineering strain seed liquid.

[0024] According to the method of the first aspect of the present invention, the composition of the YPDZ medium is: 1% yeast extract, 2% peptone, 2% glucose, 2% agar, 200 μg / ml bleomycin.

[0025] According to the method of the first aspect of the present invention, in the preparation of the primary seed liquid in step (1): the screened highly expressed engineering strain is inoculated into a shake flask (such as 2 L) containing YPD medium (such as containing 500 ml) at an inoculation amount of 1‰, and cultured at 30 °C and 250 rpm until the OD600 reaches 7-9 as the primary seed liquid.

[0026] The method according to the first aspect of the present invention, wherein in the preparation of the secondary seed solution in step (2): inoculate the primary seed solution into a shake flask (e.g., 4 L) containing YPD medium (e.g., 1000 ml each in 3 flasks) at an inoculation amount of 10%, and culture in the shake flask at 30 °C and 250 rpm until the OD600 reaches 7 - 9 to obtain the secondary seed solution.

[0027] The method according to the first aspect of the present invention, wherein in step (3), when the secondary seed solution is inoculated into the fermentation medium in the fermenter, the volume ratio of the secondary seed solution to the fermentation medium is 1:8 - 12.

[0028] The method according to the first aspect of the present invention, wherein in step (5), during the period of maintaining the methanol feeding rate until the end of fermentation, samples are taken at regular intervals (e.g., every 5 h) to detect the expression level of recombinant batroxobin in the supernatant, and when the expression level no longer increases, it is taken as the end point of fermentation.

[0029] As a commonly used anticoagulant drug, the natural batroxobin product Batroxobin Injection has the following main clinical indications: Since the recombinant batroxobin obtained in the present invention also exhibits comparable biological activity to natural batroxobin, therefore, the second aspect of the present invention provides the use of the recombinant batroxobin prepared by the method according to any one of the first aspects of the present invention in the preparation of drugs for the following medical indications:

[0030] For acute cerebral infarction;

[0031] For improving ischemic symptoms caused by various occlusive vascular diseases (such as thromboangiitis obliterans, deep phlebitis, pulmonary embolism, etc.);

[0032] For improving peripheral and microcirculation disorders (such as sudden deafness, motion sickness).

[0033] In the method steps described in the present invention, although the specific steps described are different from the method steps in other parts of this article in some details or language descriptions, however, those skilled in the art can fully summarize the above - described method steps based on the detailed disclosure of the full text of the present invention.

[0034] Any embodiment of any aspect of the present invention can be combined with any other embodiment of the present invention as long as they do not conflict. In addition, in any embodiment of any aspect of the present invention, any technical feature can be applied to the corresponding technical feature in any other embodiment of the present invention as long as they do not conflict.

[0035] The present invention will be further described below.

[0036] All documents cited in the present invention are incorporated herein by reference in their entirety, and in case of any inconsistency between the meanings expressed by these documents and the present invention, the description of the present invention shall prevail. In addition, various terms and phrases used in the present invention have the general meanings known to those skilled in the art. Even so, the present invention still wishes to provide more detailed explanations and interpretations of these terms and phrases herein. In case of any inconsistency between the meanings of the terms and phrases mentioned and the known meanings, the meanings expressed by the present invention shall prevail.

[0037] The commercially available product of natural batroxobin has the trade name of Dongling Jingchun Keshuimei. It is a single-chain glycoprotein, a serine protease isolated from the venom of Bothrops atrox, and is a glycopeptide composed of 231 amino acids. Because it has the functions of decomposing fibrinogen and promoting the release of tissue-type plasminogen activator, the gene of this enzyme has been cloned and recombinantly expressed in the Pichia pastoris system, and some of its biochemical properties have also been studied. It can specifically cleave the bond at the Arg16-Gly17 position in the fibrinogen chain, releasing fibrinopeptide A. This enzyme does not activate factors, thereby converting fibrinogen in the blood into a loose soft clot, which is easily degraded by hydrolases, resulting in a decrease in the concentration of fibrinogen in the blood and playing the role of a defibrase. Based on these biochemical characteristics, natural batroxobin has been successfully developed into a defibrating drug and is used in clinical treatment of thrombotic diseases such as deep vein thrombosis, myocardial infarction, pulmonary embolism, and acute ischemic stroke.

[0038] The natural batroxobin used clinically is administered by intravenous drip, showing a one-compartment model. It can effectively reduce the content of fibrinogen in the blood, reduce the whole blood viscosity and plasma viscosity, decrease the vascular resistance, and increase the blood flow. For healthy adults, intravenous drip is administered at a dose of 10 BU every other day for a total of three times. The half-life is measured as follows: the first administration is 5.9 hours; the second administration is 3.0 hours; the third administration is 2.8 hours. Compared with the first administration, the half-life after the second administration shortens with the decrease in the fibrinogen concentration, and the half-life after administration is the same as that of the first administration after the fibrinogen concentration is restored. Animal experiments show that when 125I-batroxobin is intravenously injected into Wistar rats to examine the in vivo distribution, the results show that the distribution is higher in the liver and kidneys; it is also distributed in the blood, spleen, and lungs; the distribution is lower in the brain, fat, and muscles; there is no significant distribution difference between male and female genders; there is a phenomenon of transient liver function disorder in the fetus. After intravenous administration (10 BU) to healthy adults, most of the metabolites are excreted in the urine.

[0039] The present invention adopts genetic engineering methods to be able to prepare genetically engineered recombinant batroxobin with biological activity equivalent to that of natural batroxobin on an industrial scale and with a high expression level. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Figure 1: SDS electrophoresis diagram of the recombinant batroxobin obtained in the present invention. Detailed implementation mode

[0041] The present invention can be further described by the following examples. However, the scope of the present invention is not limited to the following examples. Those skilled in the art can understand that various changes and modifications can be made to the present invention without departing from the spirit and scope of the present invention. The present invention generally and / or specifically describes the materials and test methods used in the experiments. Although many materials and operation methods used to achieve the purpose of the present invention are well known in the art, the present invention still describes them in as much detail as possible here. The following examples further illustrate the present invention rather than limit the present invention.

[0042] Native batroxobin: The preparation Batroxobin Injection (Dongling Difu, National Medicine Approval Number H20030295, 0.5 ml: 5 BU, Torii) of native batroxobin was purchased through commercial channels. Its remaining expiration date was more than 13 months when used in the comparative test in the present invention.

[0043] Example 1: Structural gene sequence of synthetic batroxobin

[0044] The method for the structural gene sequence of synthetic batroxobin is known. For example, see Itoh, N., Tanaka, N., Mihashi, S. and Yamashina, I.; Molecular cloning and sequence analysis of cDNA for batroxobin, a thrombin-like snake venom enzyme; J. Biol. Chem. 262(7), 3132 - 3135 (1987). The nucleic acid sequence of batroxobin is also detailed in the Genbank database (GenBank: J02684.1, see in detail https: / / www.ncbi.nlm.nih.gov / nuccore / J02684.1. The present invention uses this sequence as the target gene to prepare recombinant batroxobin). In addition, the protein sequence of the batroxobin precursor is also detailed in the Genbank database (GenBank: AAA48552.1, see in detail https: / / www.ncbi.nlm.nih.gov / protein / 211024). The batroxobin precursor consists of 255 amino acids. Amino acids 1 - 18 are the signal peptide, and amino acids 25 - 255, a total of 231 amino acids, are the batroxobin protein sequence.

[0045] The structural gene sequence of batroxobin was artificially synthesized by a method common in the art, such as the method described in CN102258483B (CN2010101577855). To recombine the synthesized target gene into the yeast secretory expression vector pPICZα, an Xho I restriction enzyme site was added to the 5′ end of the gene, and the stop codon TAATGA and a SacII restriction enzyme site were added to the 3′ end of the gene.

[0046] The synthesized target gene and the pPICZα vector were digested with Xho I and SacII, electrophoresed, and recovered. The target gene was recombined into pPICZα and transformed into Escherichia coli Top10F′ for screening. Escherichia coli Top10F′ transformed with pPICZα was cultured on an LB medium plate supplemented with Zeocin, single colonies were picked, cultured in a liquid medium, plasmids were extracted, detected by double digestion with Xho I and SacII, and subjected to α-factor priming and 3′AOX1 priming PCR detection, showing that the target gene was contained in pPICZα and could be used to transform Pichia pastoris, such as Pichia Pastoris X-33 Yeast Glycerol Stock (Beyotime), KM71H Pichia pastoris (NTCC® Type Culture Collection Center), Pichia pastoris GS115 Glycerol Stock (Beyotime), etc. Unless otherwise specified, GS115 was used in the present invention. LB medium: 1% tryptone, 0.5% yeast extract, 0.5% sodium chloride, 1.5% agar, adjusted to pH 7.0, autoclaved, and 25 µg / mg of zeocin (bleomycin) was added during plating. Liquid medium: 1% tryptone, 0.5% yeast extract, 0.5% sodium chloride, adjusted to pH 7.0, autoclaved.

[0047] Example 2: Cloning, expression and screening of high-expression engineering bacteria

[0048] The pPICZα-Bg was linearized with DNA restriction endonuclease SacI-HF™ (purchased from New England Biolabs, #R3156). Using the Multi-Copy Pichia Expression Kit from Gibco (which contains 20 µg each of pPIC3.5K, pPIC9K, and pAO815 vectors, Pichia pastoris strain GS115 (his4), KM71 (arg4-his4-aox1::arg4), control strain GS115 albumin, control strain GS115 β-gal, Spheroplast transformation kit, and 2 µg each of 5´aox1, 3´AOX2, and α-factor sequencing primers, and can be used to produce and screen Pichia pastoris with more than one copy of the target gene), according to the method described in its instruction manual, yeast host competent cells were prepared and transformed. The transformed cells were spread on YPDZ medium (which was prepared from 1% yeast extract, 2% peptone, 2% glucose, 2% agar, and 200 µg / ml bleomycin). Single colonies appeared after growing at 30 °C for 4 - 5 days. Clones with large and plump colonies were selected for expression screening. High-expressing engineering bacteria were screened out in test tubes and inoculated into shake flasks for proliferation as the engineering bacteria seed solution.

[0049] Example 3: Preparation of recombinant batroxobin

[0050] There have been records in the art regarding the preparation method of recombinant batroxobin. For example, see CN116286408A (202111502192.2). The recombinant batroxobin was prepared according to the following operations in this example.

[0051] (1) Preparation of primary seed solution: The high-expressing engineering bacteria screened out in Example 2 were inoculated into a 2 L shake flask containing 500 ml of YPD medium at an inoculation amount of 1‰, and cultured at 30 °C and 250 rpm until the OD600 reached 7 - 9, as the primary seed solution;

[0052] (2) Preparation of secondary seed solution: The primary seed solution was inoculated into three 4 L shake flasks containing 1000 ml of YPD medium each at an inoculation amount of 10%, and cultured at 30 °C and 250 rpm until the OD600 reached 7 - 9, as the secondary seed solution.

[0053] (3) Fermenter inoculation and pre-culture: Inoculate 2 L of secondary seed liquid into a 30 L fermenter containing 20 L of fermentation medium for fermentation. The initial culture conditions are 30 °C, pH 6.0, rotation speed 200 rpm, and air ventilation rate 1 vvm. During the fermentation process, gradually increase the rotation speed and air ventilation rate, and control the dissolved oxygen within the range of 15 - 25%, until the rotation speed is increased to 750 rpm and the air ventilation rate is increased to 2 vvm, then continue the culture until the carbon source is exhausted (about 8.6 h, the dissolved oxygen increases significantly), and then carry out glycerol feeding culture;

[0054] (4) Glycerol feeding culture: Start to drip-feed 50% glycerol containing 12 ml / L of PTM1 solution at a rate of 5 ml / h / L, gradually increase the feeding speed to 50 ml / h / L within 30 min. During this period, adjust the rotation speed and air ventilation rate to control the dissolved oxygen within the range of 15 - 25%, and continue the culture for 6.5 h until the wet cell weight reaches within the range of 250 - 270 g / L (265 g / L), and the liquid material is about 23 L, then enter the next methanol feeding;

[0055] (5) Methanol feeding induction culture: Lower the temperature of the fermentation broth to 25 °C, adjust the pH to 3.0, drip-feed methanol containing 12 ml / L of PTM1 solution at a rate of 3 ml / h / L, and maintain this methanol feeding speed until the end of fermentation (take samples every 5 h to detect the expression level of recombinant batroxobin in the supernatant. When the expression level no longer increases, it is taken as the end point of fermentation, which takes 46 h). During this period, adjust the rotation speed and air ventilation rate to control the dissolved oxygen within the range of 15 - 25%, and start to introduce oxygen after both the rotation speed and air ventilation rate reach the maximum (the highest ventilation rate is 0.3 vvm); After the fermentation ends, centrifuge the fermentation broth and collect about 24 L of the supernatant;

[0056] (6) Ultrafilter the centrifuged supernatant with an ultrafiltration membrane package with a molecular weight cut-off of 10K, and concentrate it until the volume of the liquid on the membrane is reduced to 1 / 10 of the initial supernatant volume, obtaining 2375 ml of fermentation concentrate containing recombinant batroxobin.

[0057] Using the method of Example 4, the titer of the batroxobin fermentation concentrate obtained from step (3) to step (6) was measured to be 54.6 BU / ml, and the total titer was 129.7 kBU; If it is sub-packed into glass bottles according to the specification of 5 BU / ml per commercially available product, theoretically, it can be used to sub-pack 26,000 vials of recombinant batroxobin injection without considering subsequent purification losses; Even considering a 50% subsequent purification loss, the harvest amount of this recombinant batroxobin can still be used to sub-pack 13,000 vials of the final product recombinant batroxobin injection in the later preparation process, indicating that the method of the present invention can be used for the preparation of recombinant batroxobin on an industrial scale.

[0058] The composition of the above YPD medium: 1% peptone, 0.5% yeast extract, 2% glucose, water, adjusted to pH 6.0 ± 0.2 with hydrochloric acid or sodium hydroxide, prepared according to the conventional method;

[0059] The composition of the above fermentation medium: 13 ml / L of 85% phosphoric acid, 0.46 g / L of calcium sulfate dihydrate, 9.1 g / L of potassium sulfate, 7.46 g / L of magnesium sulfate heptahydrate, 2.06 g / L of potassium hydroxide, 30.0 g / L of glycerol, 4.35 ml / L of PTM1 solution, the balance being water, prepared according to the conventional method;

[0060] The composition of the above PTM1 solution: 6 g / L of copper sulfate, 0.088 g / L of sodium iodide, 3 g / L of manganese sulfate, 0.2 g / L of sodium molybdate, 0.02 g / L of boric acid, 0.5 g / L of cobalt chloride, 20 g / L of zinc chloride, 65 g / L of ferrous sulfate, 0.2 g / L of biotin, the balance being water; prepared according to the conventional method.

[0061] Example 4: Potency determination of batroxobin

[0062] Referring to the method for determining the activity of batroxobin in the national drug standard of commercially available batroxobin injection, the activity of the batroxobin fermentation concentrate obtained in step (6) of each example of the present invention was determined. The specific method is as follows.

[0063] (1) Preparation of test solution:

[0064] Diluent: Take 0.2 g of partially hydrolyzed gelatin, 1.0 g of dextran 70, 0.3 g of chlorobutanol, dissolve and dilute to 100 ml with tris(hydroxymethyl)aminomethane buffer solution, shake well, and it is ready;

[0065] Partially hydrolyzed gelatin: Take 50 g of refined gelatin and 2.5 g of calcium chloride, add 450 ml of water, heat to dissolve in a water bath at 50 °C, slowly adjust the pH value to 3.0 with phosphoric acid, then place it in an autoclave at 121 °C and 1.1 kg / cm 2 Heat for 90 minutes, take out and cool to room temperature, adjust the pH value to 6.5 with 1 mol / L sodium hydroxide solution, let it stand and take the supernatant, filter it through a 0.45 µm filter membrane, and freeze-dry the filtrate for later use;

[0066] Tris(hydroxymethyl)aminomethane buffer solution: Take 18.17 g of tris(hydroxymethyl)aminomethane and 0.8 g of sodium chloride, add 800 ml of water to dissolve, adjust the pH value to 7.2 with 1 mol / L hydrochloric acid solution, then add water to 1000 ml, shake well, and it is ready;

[0067] Fibrinogen solution: Take 0.2 g of partially hydrolyzed gelatin, 1.0 g of dextran 70, 0.3 g of chlorobutanol, dissolve with water, then add water to 100 ml, shake well. Separately take 8 mg of bovine fibrinogen and dissolve it with 2 ml of the above solution, and prepare it before use.

[0068] (2) Preparation of standard curve:

[0069] Take an appropriate amount of batroxobin reference standard, weigh accurately, and dilute accurately with diluent to prepare reference standard solutions containing 2.0, 4.0, 6.0, 8.0, and 10.0 BU per 1 ml respectively;

[0070] Take 0.2 ml of fibrinogen solution and place it in 5 small test tubes respectively. Keep the above test tubes in a 37°C water bath for 3 minutes;

[0071] Accurately pipette 0.1 ml of each pre-warmed reference standard solution respectively, quickly add it to each test tube containing fibrinogen solution, start timing, shake well immediately, place it in a 37°C water bath, and record the clotting time;

[0072] Repeat the determination 5 times for each concentration of the reference standard solution, calculate the average value and standard deviation (when the standard deviation is greater than 3.0 seconds, re-determination is required); use the logarithm of the reference standard solution concentration as the abscissa and the logarithm of the average clotting time as the ordinate to plot the standard curve.

[0073] (3) Determination:

[0074] For the commercially available batroxobin injection (Dongling Difu, 0.5 ml: 5 BU), accurately pipette 5 ml and place it in a 10-ml volumetric flask, dilute to the mark with diluent, and shake well to obtain the test solution (concentration is about 5 BU / ml);

[0075] For the batroxobin fermentation concentrate obtained in step (6) of Examples such as Example 3, determine its approximate potency through preliminary experiments, then accurately pipette an appropriate amount and add it to a volumetric flask, dilute to the mark with diluent to prepare a solution with a concentration of about 5 BU / ml as the test solution;

[0076] After pre-warming the test solution in a 37°C water bath, accurately pipette 0.1 ml, and according to the method under "(2) Preparation of the standard curve", add it to the test tube containing 0.2 ml of fibrinogen solution, determine the clotting time, calculate the average value of 5 times and the standard deviation (the standard deviation is the same as the requirements for the preparation of the standard curve), and obtain the potency of the test solution from the standard curve.

[0077] After determination, the potency of the commercially available batroxobin injection is 11.26 BU / ml, indicating that the method of the present invention is reliable when verified with commercially available products.

[0078] Example 5: Preparation of recombinant batroxobin

[0079] This example mainly refers to Example 3 of the present invention, but appropriately modifies the operating parameters of the preparation process.

[0080] (1) Preparation of primary seed liquid: The highly expressed engineered bacteria screened in Example 2 were inoculated into a 2 L shake flask containing 500 ml of YPD medium at an inoculation amount of 1‰, and cultured in a shaker at 30 °C and 250 rpm until the OD600 reached 7 - 9, which was used as the primary seed liquid;

[0081] (2) Preparation of secondary seed liquid: The primary seed liquid was inoculated into three 4 L shake flasks containing 1000 ml of YPD medium each at an inoculation amount of 10%, and cultured in a shaker at 30 °C and 250 rpm until the OD600 reached 7 - 9, which was used as the secondary seed liquid.

[0082] (3) Inoculation and pre - culture in fermenter: 2 L of the secondary seed liquid was inoculated into a 30 L fermenter containing 20 L of fermentation medium for fermentation. The initial culture conditions were 30 °C, pH 6.2, rotation speed 150 rpm, and air ventilation rate 1.2 vvm; during the fermentation process, the rotation speed and air ventilation rate were gradually increased, and the dissolved oxygen was controlled within the range of 15 - 25%, until the rotation speed was increased to 700 rpm and the air ventilation rate was increased to 2.5 vvm, and then continued to be cultured until the carbon source was exhausted (about 8.3 h, the dissolved oxygen increased significantly), and then glycerol feeding culture was carried out;

[0083] (4) Glycerol feeding culture: Start to drip 50% glycerol containing 10 ml / L of PTM1 solution at a rate of 5 ml / h / L for feeding, gradually increase the feeding speed to 55 ml / h / L within 30 min, adjust the rotation speed and air ventilation rate during this period to control the dissolved oxygen within the range of 15 - 25%, and continue to culture for 6.3 h until the wet cell weight reached within the range of 250 - 270 g / L (257 g / L), and the liquid material was about 23 L, then enter the next methanol feeding;

[0084] (5) Methanol feeding and induction culture: The temperature of the fermentation broth was lowered to 25 °C, the pH was adjusted to 3.2, and methanol containing 14 ml / L of PTM1 solution was dripped at a rate of 3.5 ml / h / L, and this methanol feeding speed was maintained until the end of fermentation (sampling was taken every 5 h to detect the expression level of recombinant batroxobin in the supernatant, and when the expression level no longer increased, it was taken as the end point of fermentation, which took 45 h), during which the rotation speed and air ventilation rate were adjusted to control the dissolved oxygen within the range of 15 - 25%, and oxygen was introduced after the rotation speed and air ventilation rate both reached the maximum (the highest ventilation rate was 0.32 vvm); after the fermentation ended, the fermentation broth was centrifuged, and about 24 L of the supernatant was collected;

[0085] (6) The centrifuged supernatant was ultra - filtered with an ultra - filtration membrane package with a molecular weight cut - off of 10K, and concentrated to 1 / 10 of the initial supernatant volume on the membrane, obtaining 2426 ml of the fermentation concentrate containing recombinant batroxobin.

[0086] Using the method of Example 4, the titer of the batroxobin fermentation concentrate obtained from step (3) to step (6) was measured to be 51.4 BU / ml, and the total titer was 124.7 kBU. The YPD medium, fermentation medium, and PTM1 solution used in this example were the same as those in Example 3.

[0087] Example 6: Preparation of recombinant batroxobin

[0088] This example mainly refers to Example 3 of the present invention, but the operating parameters of the preparation process are appropriately modified.

[0089] (1) Preparation of primary seed liquid: The highly expressed engineering bacteria screened in Example 2 were inoculated into a 2 L shake flask containing 500 ml of YPD medium at an inoculation amount of 1‰, and cultured at 30 °C and 250 rpm until the OD600 reached 7 - 9, serving as the primary seed liquid;

[0090] (2) Preparation of secondary seed liquid: The primary seed liquid was inoculated into three 4 L shake flasks each containing 1000 ml of YPD medium at an inoculation amount of 10%, and cultured at 30 °C and 250 rpm until the OD600 reached 7 - 9, serving as the secondary seed liquid.

[0091] (3) Inoculation and pre - culture in the fermenter: 2 L of the secondary seed liquid was inoculated into a 30 L fermenter containing 20 L of fermentation medium for fermentation. The initial culture conditions were 30 °C, pH 5.8, rotation speed 220 rpm, and air ventilation volume 0.8 vvm; during the fermentation process, the rotation speed and air ventilation volume were gradually increased, and the dissolved oxygen was controlled within the range of 15 - 25% until the rotation speed was increased to 650 rpm and the air ventilation volume was increased to 1.8 vvm, and then continued to be cultured until the carbon source was exhausted (about 9.1 h, the dissolved oxygen increased significantly), and then glycerol feeding culture was carried out;

[0092] (4) Glycerol feeding culture: Start to drip 50% glycerol containing 14 ml / L of PTM1 solution at a rate of 5 ml / h / L for feeding, gradually increase the feeding speed to 45 ml / h / L within 30 min, adjust the rotation speed and air ventilation volume during this period to control the dissolved oxygen within the range of 15 - 25%, continue to culture for 7 h until the wet cell weight reached within the range of 250 - 270 g / L (268 g / L), and the liquid material was about 23 L, and then enter the next methanol feeding;

[0093] (5) Methanol feeding induction culture: Lower the temperature of the fermentation broth to 25 °C, adjust the pH to 2.8, and drip methanol containing 10 ml / L of PTM1 solution at a rate of 2.8 ml / h / L until the end of fermentation (sample the supernatant every 5 h to detect the expression level of recombinant batroxobin. When the expression level no longer increases, it is regarded as the fermentation end point, which takes 49 h). During this period, adjust the rotation speed and air ventilation volume to control the dissolved oxygen within the range of 15 - 25%, and start to introduce oxygen after both the rotation speed and air ventilation volume reach the maximum (the maximum ventilation volume is 0.28 vvm); after fermentation, centrifuge the fermentation broth and collect about 24 L of the supernatant;

[0094] (6) Ultrafilter the centrifuged supernatant with an ultrafiltration membrane package with a molecular weight cut-off of 10K, and concentrate it until the volume of the liquid on the membrane is reduced to 1 / 10 of the initial supernatant volume, obtaining 2393 ml of the fermentation concentrate containing recombinant batroxobin.

[0095] Using the method of Example 4, the titer of the batroxobin fermentation concentrate obtained from step (3) to step (6) was measured to be 56.6 BU / ml, and the total titer was 135.4 kBU. The YPD medium, fermentation medium, and PTM1 solution used in this example were the same as those in Example 3.

[0096] Example 7: Preparation of recombinant batroxobin

[0097] This example mainly refers to Example 3 of the present invention, but makes appropriate modifications to the composition of the PTM1 solution.

[0098] (1) Preparation of primary seed liquid: Inoculate the highly expressing engineering bacteria screened in Example 2 into a 2 L shake flask containing 500 ml of YPD medium at an inoculation amount of 1‰, and culture it at 30 °C and 250 rpm until the OD600 reaches 7 - 9 as the primary seed liquid;

[0099] (2) Preparation of secondary seed liquid: Inoculate the primary seed liquid into three 4 L shake flasks each containing 1000 ml of YPD medium at an inoculation amount of 10%, and culture it at 30 °C and 250 rpm until the OD600 reaches 7 - 9 as the secondary seed liquid.

[0100] (3) Inoculation and pre-culture in the fermenter: Inoculate 2 L of the secondary seed liquid into a 30 L fermenter containing 20 L of fermentation medium for fermentation. The initial culture conditions are 30 °C, pH 6.0, rotation speed 200 rpm, and air ventilation volume 1 vvm; during the fermentation process, gradually increase the rotation speed and air ventilation volume, and control the dissolved oxygen within the range of 15 - 25% until the rotation speed is increased to 750 rpm and the air ventilation volume is increased to 2 vvm, and then continue to culture until the carbon source is exhausted (about 8.4 h, the dissolved oxygen increases significantly), and then carry out glycerol feeding culture;

[0101] (4) Glycerol feeding culture: Initially, 50% glycerol containing 12 ml / L of PTM1 solution was fed at a rate of 5 ml / h / L, and the feeding rate was gradually increased to 50 ml / h / L within 30 min. During this period, the rotation speed and air ventilation volume were adjusted to control the dissolved oxygen within the range of 15 - 25%. The culture was continued for 6.5 h until the wet cell weight reached the range of 250 - 270 g / L (261 g / L), and the liquid material was about 23 L, then entered the subsequent methanol feeding;

[0102] (5) Methanol feeding induction culture: The temperature of the fermentation broth was reduced to 25 °C and the pH was adjusted to 3.0. Methanol containing 12 ml / L of PTM1 solution was fed at a rate of 3 ml / h / L, and this methanol feeding rate was maintained until the end of fermentation (samples were taken every 5 h to detect the expression level of recombinant batroxobin in the supernatant. When the expression level no longer increased, it was taken as the end point of fermentation, which took 46 h). During this period, the rotation speed and air ventilation volume were adjusted to control the dissolved oxygen within the range of 15 - 25%, and oxygen was introduced after both the rotation speed and air ventilation volume reached the maximum (the maximum ventilation volume was 0.3 vvm); After fermentation, the fermentation broth was centrifuged, and about 24 L of the supernatant was collected;

[0103] (6) The centrifuged supernatant was ultrafiltered using an ultrafiltration membrane package with a molecular weight cut-off of 10K, and concentrated to 1 / 10 of the initial supernatant volume, obtaining 2411 ml of the fermentation concentrate containing recombinant batroxobin.

[0104] The composition of the YPD medium used in this example: 1% peptone, 0.5% yeast extract, 2% glucose, water, and the pH was adjusted to 6.0 ± 0.2 with hydrochloric acid or sodium hydroxide, prepared according to the conventional method;

[0105] The composition of the fermentation medium used in this example: 13 ml / L of 85% phosphoric acid, 0.46 g / L of calcium sulfate dihydrate, 9.1 g / L of potassium sulfate, 7.46 g / L of magnesium sulfate heptahydrate, 2.06 g / L of potassium hydroxide, 30.0 g / L of glycerol, 4.35 ml / L of PTM1 solution, and the balance water, prepared according to the conventional method;

[0106] The composition of the PTM1 solution used in this example: 6 g / L of copper sulfate, 0.088 g / L of sodium iodide, 3 g / L of manganese sulfate, 0.2 g / L of sodium molybdate, 0.02 g / L of boric acid, 0.5 g / L of cobalt chloride, 20 g / L of zinc chloride, 65 g / L of ferrous sulfate, 0.2 g / L of biotin, 1.2 g / L of inositol, 2.5 g / L of calcium pantothenate, and the balance water; Prepared according to the conventional method.

[0107] Using the method of Example 4, the titer of the batroxobin fermentation concentrate obtained from step (3) to step (6) was measured to be 87.3 BU / ml, and the total titer was 210.5 kBU. If it was subpackaged into glass bottles according to the specification of 5 BU / ml per commercially available product, theoretically, it could be used to subpackage 42,000 vials of recombinant batroxobin injection. Even considering a 50% loss during subsequent purification, the harvest of this recombinant batroxobin could still be used to subpackage 21,000 vials of the final product recombinant batroxobin injection in the later formulation preparation process, indicating that the method of the present invention can be used for the preparation of recombinant batroxobin on an industrial scale.

[0108] Example 8: Referring to the preparation process and operation parameters of Example 5, but the YPD medium, fermentation medium, and PTM1 solution used were the same as those in Example 7, including the composition of the PTM1 solution used: copper sulfate 6 g / L, sodium iodide 0.088 g / L, manganese sulfate 3 g / L, sodium molybdate 0.2 g / L, boric acid 0.02 g / L, cobalt chloride 0.5 g / L, zinc chloride 20 g / L, ferrous sulfate 65 g / L, biotin 0.2 g / L, inositol 1.2 g / L, calcium pantothenate 2.5 g / L, and the balance was water; it was prepared according to the conventional method. In step (6) of this Example 8, the volume of the batroxobin fermentation concentrate obtained was 2368 ml, the titer was 91.7 BU / ml, and the total titer was 217.1 kBU.

[0109] Example 9: Referring to the preparation process and operation parameters of Example 6, but the YPD medium, fermentation medium, and PTM1 solution used were the same as those in Example 7, including the composition of the PTM1 solution used: copper sulfate 6 g / L, sodium iodide 0.088 g / L, manganese sulfate 3 g / L, sodium molybdate 0.2 g / L, boric acid 0.02 g / L, cobalt chloride 0.5 g / L, zinc chloride 20 g / L, ferrous sulfate 65 g / L, biotin 0.2 g / L, inositol 1.2 g / L, calcium pantothenate 2.5 g / L, and the balance was water; it was prepared according to the conventional method. In step (6) of this Example 9, the volume of the batroxobin fermentation concentrate obtained was 2403 ml, the titer was 88.6 BU / ml, and the total titer was 212.9 kBU. From the results of Examples 7 to 9, it can be seen that, compared with Examples 3 and 5 to 6 of the present invention, unexpectedly, after adding an appropriate amount of inositol and calcium pantothenate to the PTM1 solution used in steps (3) to (6), the batroxobin titer in the fermentation concentrate can be significantly increased, and the expression level of recombinant batroxobin in the fermentation concentrate can be significantly increased.

[0110] The batroxobin fermentation concentrate obtained in step (6) of Example 3 and Examples 5 to 9 can be further purified by methods commonly used in the art such as hydrophobic column chromatography, ion exchange column chromatography, gel affinity column chromatography, and / or reverse phase liquid chromatography to obtain a glycoprotein recombinant batroxobin with a desired purity and meeting pharmaceutical requirements. In another experiment by the present inventors, an appropriate amount of the batroxobin fermentation concentrate obtained in step (6) of Example 7 was taken, and according to the method of Example 4 of CN108559740B (Chinese Patent No. 2018104523815), gel affinity column chromatography and reverse phase liquid chromatography were used for purification, and the glycoprotein purity of the purified recombinant protein was 98.6%. The SDS electrophoresis pattern is shown in Figure 1 , showing an apparent molecular weight of 33.8 kDa, which is consistent with the results of recombinant batroxobin reported in the literature (such as CN116286755B, 202310399963.2). The recombinant proteins obtained after purification of the batroxobin fermentation concentrates obtained in step (6) of Example 3, Examples 5 to 6, and Examples 8 to 9 also showed an apparent molecular weight in the range of 33.5 to 34.2 kDa by SDS electrophoresis, indicating that the products obtained in these experiments were all consistent with glycoprotein recombinant batroxobin.

[0111] Example 10: Three examples of preparing recombinant batroxobin were carried out respectively according to the preparation processes and operation parameters of Examples 7 to 9, but inositol was not added to the PTM1 solution used; when operating according to Example 7, the volume of the batroxobin fermentation concentrate obtained in step (6) was 2382 ml, the titer was 53.6 BU / ml, and the total titer was 127.7 kBU; when operating according to Example 8, the volume of the batroxobin fermentation concentrate obtained in step (6) was 2407 ml, the titer was 54.6 BU / ml, and the total titer was 131.4 kBU; when operating according to Example 9, the volume of the batroxobin fermentation concentrate obtained in step (6) was 2436 ml, the titer was 51.8 BU / ml, and the total titer was 126.2 kBU. These results indicate that when inositol is not added to the PTM1 solution, the expression level of recombinant batroxobin cannot be effectively increased.

[0112] Example 11:Three examples of preparing recombinant batroxobin were carried out according to the preparation processes and operation parameters of Examples 7 to 9 respectively, but calcium pantothenate was not added to the PTM1 solution used; when operating according to Example 7, the volume of the batroxobin fermentation concentrate obtained in step (6) was 2426 ml, the titer was 60.7 BU / ml, and the total titer was 147.3 kBU; when operating according to Example 8, the volume of the batroxobin fermentation concentrate obtained in step (6) was 2374 ml, the titer was 59.4 BU / ml, and the total titer was 141.0 kBU; when operating according to Example 9, the volume of the batroxobin fermentation concentrate obtained in step (6) was 2403 ml, the titer was 60.6 BU / ml, and the total titer was 145.6 kBU. These results indicate that the expression level of recombinant batroxobin cannot be effectively increased when calcium pantothenate is not added to the PTM1 solution.

[0113] Example 12: Effect of recombinant batroxobin on thrombus formation

[0114] It is known that natural batroxobin has an antithrombotic effect. In this example, a commercially available batroxobin injection (0.5 ml: 5 BU) was used as a control, and the method of a relevant literature (2010101577855) was referred to investigate the effect of the purified recombinant batroxobin (glycoprotein purity: 98.6%) obtained in Example 7 of the present invention on the formation of intravenous thrombosis in healthy SD rats. The specific operations are as follows: 30 male healthy SD rats (Institute of Zoology, Chinese Academy of Sciences, SCXK(Beijing)2021-0012, body weight 180-210 g) were randomly divided into a blank control group, a batroxobin injection group, and a recombinant batroxobin group, with 10 rats in each group; the doses of the batroxobin injection group and the recombinant batroxobin group were both 10 BU / kg. The drug was diluted to an appropriate concentration with water for injection, and 3 ml / kg of the drug solution was intravenously injected. The blank control group was injected with the same volume of 0.9% sodium chloride injection in the same manner; after administration, the animals were anesthetized with sodium pentobarbital, fixed supine, the skin was incised along the midline of the abdomen (about 2 cm), the abdominal cavity was opened, the inferior vena cava was separated, and after ligating the inferior vena cava with a No. 4 suture below the left renal vein, the abdominal wall was sutured; 3 h later, the abdominal cavity was reopened, the blood vessel was clamped with an artery clamp about 2 cm below the ligation site, the blood in this section of the lumen was aspirated, longitudinally cut open, the thrombus was taken out and its wet weight was weighed, and then the wet thrombus was placed in an oven at 40 °C for 18 h and then weighed for dry weight; the results were expressed as mean ± sd, and the t-test was used for comparison between groups. P < 0.05 indicated a significant difference; Results: The wet weight (mg) and dry weight (mg) of the blank control group were 15.46 ± 3.25 and 5.23 ± 2.14 respectively, the wet weight (mg) and dry weight (mg) of the batroxobin injection group were 4.73 ± 2.74** and 1.68 ± 1.47** respectively, and the wet weight (mg) and dry weight (mg) of the recombinant batroxobin group were 4.48 ± 2.38** and 1.42 ± 1.19** respectively, where ** indicated P < 0.01 compared with the blank control group. These results show that the recombinant purified batroxobin obtained in the present invention has an antithrombotic effect comparable to that of natural batroxobin. Although there is no statistical difference between the two, simply from the absolute value results, the recombinant batroxobin shows a better antithrombotic effect. Considering the production cost and the availability of resources, it is obvious that the recombinant batroxobin obtained in the present invention has obvious industrial advantages over natural batroxobin.

[0115] The spirit of the present invention has been elaborated in detail through the preferred embodiments of the present invention. Those skilled in the art understand that any modification, equivalent change and modification made to the above embodiments according to the technical essence of the present invention all fall within the protection scope of the present invention.

Claims

1. A method for preparing recombinant batroxobin, which comprises artificially synthesizing the structural gene sequence of batroxobin, then using Pichia pastoris to clone and express to obtain the seed solution of highly expressed engineered bacteria, and then fermenting and culturing to prepare recombinant batroxobin according to the following operations: (1) Preparation of primary seed solution: The selected engineered bacteria were inoculated into YPD medium and cultured in a shake flask until the OD600 reached 7-9, which was used as the primary seed solution; (2) Preparation of secondary seed solution: The primary seed solution was inoculated into YPD medium and cultured in a shake flask until the OD600 reached 7-9 to serve as the secondary seed solution; (3) Fermentation tank inoculation and pre-culture: The secondary seed liquid was inoculated into the fermentation medium of the fermentation tank, and cultured under the initial culture conditions of 30°C, pH 5.8-6.2, rotation speed 150-220 rpm, and air ventilation volume 0.8-1.2 vvm. During the fermentation process, the rotation speed and air ventilation volume were gradually increased, and the dissolved oxygen was controlled in the range of 15-25% until the rotation speed was increased to 650-750 rpm and the air ventilation volume was increased to 1.8-2.5 vvm. Then, the culture was continued until the carbon source was exhausted, and then glycerol fed-batch culture was carried out; (4) Glycerol feeding culture: Start feeding by adding 50% glycerol containing 10-14 ml / L of PTM1 solution at a rate of 5 ml / h / L. Gradually increase the feeding rate to 45-55 ml / h / L within 30 min. During this period, adjust the rotation speed and air ventilation volume to control the dissolved oxygen in the range of 15-25%. Continue to culture until the wet weight of the bacteria reaches the range of 250-270 g / L, and then enter the next methanol feeding; (5) Methanol feeding induction culture: the fermentation broth temperature was lowered to 25°C, the pH was adjusted to 2.8-3.2, and methanol containing 10-14 ml / L of PTM1 solution was added dropwise at a rate of 2.8-3.5 ml / h / L. The methanol feeding rate was maintained until the end of the fermentation. During this period, the rotation speed and air ventilation volume were adjusted to control the dissolved oxygen in the range of 15-25%. After the rotation speed and air ventilation volume reached the maximum, oxygen was introduced to achieve a maximum ventilation volume of 0.28-0.32 vvm. After the fermentation was completed, the fermentation broth was centrifuged and the supernatant was collected. (6) ultrafiltration of the centrifuged supernatant using an ultrafiltration membrane with a molecular weight cutoff of 10K, and concentrating the supernatant volume to 1 / 10 of the initial supernatant volume to obtain a fermentation concentrate containing recombinant batroxobin, and optionally purifying it; in, The composition of the YPD medium used was: 1% peptone, 0.5% yeast extract, 2% glucose, water, and hydrochloric acid or sodium hydroxide to adjust the pH to 6.0 ± 0.2; The composition of the fermentation medium used is: 85% phosphoric acid 13ml / L, calcium sulfate dihydrate 0.46g / L, potassium sulfate 9.1g / L, magnesium sulfate heptahydrate 7.46g / L, potassium hydroxide 2.06g / L, glycerol 30.0g / L, PTM1 solution 4.35ml / L, balance water; The composition of the PTM1 solution used was: Copper sulfate 6g / L, sodium iodide 0.088g / L, manganese sulfate 3g / L, sodium molybdate 0.2g / L, boric acid 0.02g / L, cobalt chloride 0.5g / L, zinc chloride 20g / L, ferrous sulfate 65g / L, biotin 0.2g / L, inositol 1.2g / L, calcium pantothenate 2.5g / L, balance water.

2. The method according to claim 1, wherein when the structural gene sequence of batroxobin is artificially synthesized, in order to allow the synthesized target gene to be recombined into the yeast secretory expression vector pPICZα, an Xho I restriction site is added to the 5′ end of the gene, and a termination codon TAATGA and a SacII restriction site are added to the 3′ end of the gene.

3. The method according to claim 1, wherein when the structural gene sequence of batroxobin is artificially synthesized, the synthesized target gene and pPICZα vector are double-digested with Xho I and SacII, electrophoresed and recovered, the target gene is recombined into pPICZα, transformed into Escherichia coli Top10F', and screened; Escherichia coli Top10F' transformed with pPICZα is cultured on an LB medium plate supplemented with Zeocin, a single colony is picked, cultured in a liquid medium, the plasmid is extracted, and double-digestion detection with Xho I and SacII, α-factor priming and 3'AOX1 priming PCR detection are performed, which shows that pPICZα contains the target gene and can be used to transform Pichia pastoris.

4. The method according to claim 3, wherein when the structural gene sequence of batroxobin is artificially synthesized, the LB medium with Zeocin added is composed of: 1% tryptone, 0.5% yeast extract, 0.5% sodium chloride, 1.5% agar, the pH is adjusted to 7.0, autoclaved, and 25 µg / mg of zeocin is added during plating; the liquid culture medium: 1% tryptone, 0.5% yeast extract, 0.5% sodium chloride, the pH is adjusted to 7.0, and autoclaved.

5. The method according to claim 1, wherein when Pichia pastoris is used for cloning expression screening to obtain the seed solution of highly expressed engineered bacteria, pPICZα-Bg is linearized with the DNA restriction endonuclease SacI-HF™, and a multi-copy Pichia pastoris expression kit of Gibco is used to prepare a yeast host competent state and transform it according to the method described in the kit, and the transformed cells are spread on YPDZ medium, grown at 30°C for 4 to 5 days to produce single colonies, and clones with large and full colonies are selected for expression screening, and highly expressed engineered bacteria are screened in test tubes and inoculated into shake flasks for proliferation as seed solution of engineered bacteria; the YPDZ medium is composed of: 1% yeast extract, 2% peptone, 2% glucose, 2% agar, and 200µg / ml bleomycin.

6. The method according to claim 1, wherein in step (1) the first-level seed solution is prepared: the screened high-expression engineered bacteria are inoculated into a shake flask of YPD medium at an inoculation rate of 1‰, and the shake flask is cultured at 30°C and 250rpm until the OD600 reaches 7-9, which is used as the first-level seed solution.

7. The method according to claim 1, wherein in step (2) of preparing the secondary seed solution: the primary seed solution is inoculated into a shake flask of YPD medium at a 10% inoculation rate, and the shake flask is cultured at 30°C and 250rpm until the OD600 reaches 7-9, which is used as the secondary seed solution.

8. The method according to claim 1, wherein in step (3), when the secondary seed liquid is inoculated into the fermentation medium of the fermenter, the volume ratio of the secondary seed liquid to the fermentation medium is 1:8-12.

9. The method according to claim 1, wherein in step (5), while maintaining the methanol feeding rate until the end of fermentation, samples are taken at regular intervals to detect the expression level of the recombinant batroxobin in the supernatant, and when the expression level no longer increases, it is regarded as the fermentation end point.

10. The method according to claim 1, comprising the steps of: (1) Preparation of primary seed solution: The selected high-expression engineered bacteria were inoculated at a 1‰ inoculum into a 2L shake flask containing 500 ml of YPD medium, and cultured at 30°C and 250 rpm until the OD600 reached 7-9, which was used as the primary seed solution; (2) Preparation of secondary seed solution: 10% of the primary seed solution was inoculated into three 4 L shake flasks containing 1000 ml of YPD medium, respectively, and cultured at 30°C and 250 rpm until the OD600 reached 7-9, which was used as the secondary seed solution; (3) Fermentation tank inoculation and pre-culture: 2 L of secondary seed liquid was inoculated into a 30 L fermentation tank containing 20 L of fermentation medium for fermentation. The initial culture conditions were 30 °C, pH 6.0, rotation speed 200 rpm, and air ventilation volume 1 vvm. During the fermentation process, the rotation speed and air ventilation volume were gradually increased, and the dissolved oxygen was controlled within the range of 15-25% until the rotation speed was increased to 750 rpm and the air ventilation volume was increased to 2 vvm. The culture was then continued until the carbon source was exhausted, and then glycerol fed-batch culture was performed. (4) Glycerol feeding culture: Start feeding by adding 50% glycerol containing 12 ml / L of PTM1 solution at a rate of 5 ml / h / L. Gradually increase the feeding rate to 50 ml / h / L within 30 min. During this period, adjust the rotation speed and air ventilation volume to control the dissolved oxygen in the range of 15-25%. Continue to culture for 6.5 h until the wet weight of the bacteria reaches 250-270 g / L, and the liquid feed is 23 L. Then enter the next methanol feeding; (5) Methanol feeding induction culture: the fermentation broth temperature was lowered to 25°C and the pH was adjusted to 3.

0. Methanol containing 12 ml / L of PTM1 solution was added dropwise at a rate of 3 ml / h / L. The methanol feeding rate was maintained until the end of the fermentation. Samples were taken every 5 hours to detect the expression level of the recombinant batroxobin in the supernatant. When the expression level no longer increased, it was regarded as the end point of the fermentation. During this period, the rotation speed and air ventilation volume were adjusted to control the dissolved oxygen in the range of 15-25%. After the rotation speed and air ventilation volume reached the maximum, oxygen was introduced, and the maximum ventilation volume was 0.3 vvm. After the fermentation was completed, the fermentation broth was centrifuged and 24 L of the supernatant was collected. (6) ultrafiltration of the centrifuged supernatant using an ultrafiltration membrane with a molecular weight cutoff of 10K, and concentrating the supernatant volume to 1 / 10 of the initial supernatant volume to obtain a fermentation concentrate containing recombinant batroxobin, and optionally purifying it; in, The composition of the YPD medium used was: 1% peptone, 0.5% yeast extract, 2% glucose, water, and pH adjusted to 6.0 ± 0.2 with hydrochloric acid or sodium hydroxide; The composition of the fermentation medium used: 85% phosphoric acid 13 ml / L, calcium sulfate dihydrate 0.46 g / L, potassium sulfate 9.1 g / L, magnesium sulfate heptahydrate 7.46 g / L, potassium hydroxide 2.06 g / L, glycerol 30.0 g / L, PTM1 solution 4.35 ml / L, balance water; The composition of the PTM1 solution used: copper sulfate 6 g / L, sodium iodide 0.088 g / L, manganese sulfate 3 g / L, sodium molybdate 0.2 g / L, boric acid 0.02 g / L, cobalt chloride 0.5 g / L, zinc chloride 20 g / L, ferrous sulfate 65 g / L, biotin 0.2 g / L, inositol 1.2 g / L, calcium pantothenate 2.5 g / L, and the balance water.

11. The method according to claim 1, comprising the steps of: (1) Preparation of primary seed solution: The selected high-expression engineered bacteria were inoculated at a 1‰ inoculum into a 2L shake flask containing 500 ml of YPD medium, and cultured at 30°C and 250 rpm until the OD600 reached 7-9, which was used as the primary seed solution; (2) Preparation of secondary seed solution: 10% of the primary seed solution was inoculated into three 4 L shake flasks containing 1000 ml of YPD medium, respectively, and cultured at 30°C and 250 rpm until the OD600 reached 7-9, which was used as the secondary seed solution; (3) Fermentation tank inoculation and pre-culture: 2 L of secondary seed liquid was inoculated into a 30 L fermentation tank containing 20 L of fermentation medium for fermentation. The initial culture conditions were 30 °C, pH 6.2, rotation speed 150 rpm, and air ventilation volume 1.2 vvm. During the fermentation process, the rotation speed and air ventilation volume were gradually increased, and the dissolved oxygen was controlled within the range of 15-25% until the rotation speed was increased to 700 rpm and the air ventilation volume was increased to 2.5 vvm. The culture was then continued until the carbon source was exhausted, and then glycerol fed-batch culture was performed. (4) Glycerol feeding culture: Start feeding by adding 50% glycerol containing 10 ml / L of PTM1 solution at a rate of 5 ml / h / L. Gradually increase the feeding rate to 55 ml / h / L within 30 min. During this period, adjust the rotation speed and air ventilation volume to control the dissolved oxygen in the range of 15-25%. Continue to culture for 6.3 h until the wet weight of the bacteria reaches the range of 250-270 g / L, and the liquid feed is 23 L. Then enter the next methanol feeding; (5) Methanol feeding induction culture: the fermentation broth temperature was lowered to 25°C and the pH was adjusted to 3.

2. Methanol containing 14 ml / L of PTM1 solution was added dropwise at a rate of 3.5 ml / h / L. The methanol feeding rate was maintained until the end of the fermentation. Samples were taken every 5 hours to detect the expression level of the recombinant batroxobin in the supernatant. When the expression level no longer increased, it was regarded as the end point of the fermentation. During this period, the rotation speed and air ventilation volume were adjusted to control the dissolved oxygen in the range of 15-25%. After the rotation speed and air ventilation volume reached the maximum, oxygen was introduced, and the maximum ventilation volume was 0.32 vvm. After the fermentation was completed, the fermentation broth was centrifuged and 24 L of the supernatant was collected. (6) ultrafiltration of the centrifuged supernatant using an ultrafiltration membrane with a molecular weight cutoff of 10K, and concentrating the supernatant volume to 1 / 10 of the initial supernatant volume to obtain a fermentation concentrate containing recombinant batroxobin, and optionally purifying it; in, The composition of the YPD medium used was: 1% peptone, 0.5% yeast extract, 2% glucose, water, and hydrochloric acid or sodium hydroxide to adjust the pH to 6.0 ± 0.2; The composition of the fermentation medium used: 85% phosphoric acid 13 ml / L, calcium sulfate dihydrate 0.46 g / L, potassium sulfate 9.1 g / L, magnesium sulfate heptahydrate 7.46 g / L, potassium hydroxide 2.06 g / L, glycerol 30.0 g / L, PTM1 solution 4.35 ml / L, balance water; The composition of the PTM1 solution used: copper sulfate 6 g / L, sodium iodide 0.088 g / L, manganese sulfate 3 g / L, sodium molybdate 0.2 g / L, boric acid 0.02 g / L, cobalt chloride 0.5 g / L, zinc chloride 20 g / L, ferrous sulfate 65 g / L, biotin 0.2 g / L, inositol 1.2 g / L, calcium pantothenate 2.5 g / L, and the balance water.

12. The method according to claim 1, comprising the steps of: (1) Preparation of primary seed solution: The selected high-expression engineered bacteria were inoculated at a 1‰ inoculum into a 2L shake flask containing 500 ml of YPD medium, and cultured at 30°C and 250 rpm until the OD600 reached 7-9, which was used as the primary seed solution; (2) Preparation of secondary seed solution: 10% of the primary seed solution was inoculated into three 4 L shake flasks containing 1000 ml of YPD medium, respectively, and cultured at 30°C and 250 rpm until the OD600 reached 7-9, which was used as the secondary seed solution; (3) Fermentation tank inoculation and pre-culture: 2 L of secondary seed liquid was inoculated into a 30 L fermentation tank containing 20 L of fermentation medium for fermentation. The initial culture conditions were 30 °C, pH 5.8, rotation speed 220 rpm, and air ventilation volume 0.8 vvm. During the fermentation process, the rotation speed and air ventilation volume were gradually increased, and the dissolved oxygen was controlled within the range of 15-25% until the rotation speed was increased to 650 rpm and the air ventilation volume was increased to 1.8 vvm. The culture was then continued until the carbon source was exhausted, and then glycerol fed-batch culture was performed. (4) Glycerol feeding culture: Start feeding by adding 50% glycerol containing 14 ml / L of PTM1 solution at a rate of 5 ml / h / L. Gradually increase the feeding rate to 45 ml / h / L within 30 min. During this period, adjust the rotation speed and air ventilation volume to control the dissolved oxygen in the range of 15-25%. Continue to culture for 7 h until the wet weight of the bacteria reaches the range of 250-270 g / L, and the liquid feed is 23 L. Then enter the next methanol feeding; (5) Methanol feeding induction culture: the fermentation broth temperature was lowered to 25°C and the pH was adjusted to 2.

8. Methanol containing 10 ml / L of PTM1 solution was added dropwise at a rate of 2.8 ml / h / L. The methanol feeding rate was maintained until the end of the fermentation. Samples were taken every 5 hours to detect the expression level of the recombinant batroxobin in the supernatant. When the expression level no longer increased, it was regarded as the end point of the fermentation. During this period, the rotation speed and air ventilation volume were adjusted to control the dissolved oxygen in the range of 15-25%. After the rotation speed and air ventilation volume reached the maximum, oxygen was introduced, and the maximum ventilation volume was 0.28 vvm. After the fermentation was completed, the fermentation broth was centrifuged and 24 L of the supernatant was collected. (6) ultrafiltration of the centrifuged supernatant using an ultrafiltration membrane with a molecular weight cutoff of 10K, and concentrating the supernatant volume to 1 / 10 of the initial supernatant volume to obtain a fermentation concentrate containing recombinant batroxobin, and optionally purifying it; in, The composition of the YPD medium used was: 1% peptone, 0.5% yeast extract, 2% glucose, water, and pH adjusted to 6.0 ± 0.2 with hydrochloric acid or sodium hydroxide; The composition of the fermentation medium used: 85% phosphoric acid 13 ml / L, calcium sulfate dihydrate 0.46 g / L, potassium sulfate 9.1 g / L, magnesium sulfate heptahydrate 7.46 g / L, potassium hydroxide 2.06 g / L, glycerol 30.0 g / L, PTM1 solution 4.35 ml / L, balance water; The composition of the PTM1 solution used: copper sulfate 6 g / L, sodium iodide 0.088 g / L, manganese sulfate 3 g / L, sodium molybdate 0.2 g / L, boric acid 0.02 g / L, cobalt chloride 0.5 g / L, zinc chloride 20 g / L, ferrous sulfate 65 g / L, biotin 0.2 g / L, inositol 1.2 g / L, calcium pantothenate 2.5 g / L, and the balance water.

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