阅读说明：本技术 中介蝮蛇毒纤溶酶及其制备方法和应用 (Medium Agkistrodon halys venom fibrinolytic enzyme and preparation method and application thereof ) 是由 杨章民 张扭 王璐 陈毓 刘珍珍 张腾 裴剑竹 王喆之 于 2019-10-21 设计创作，主要内容包括：一种中介蝮蛇毒纤溶酶,其基因为Gisp6,具有序列表中SEQ ID No.1所示的核苷酸序列,具有序列表中SEQ ID No.2所示的氨基酸序列；其制备方法由中介蝮蛇毒纤溶酶的纯化方法、纯度鉴定以及质谱分析、中介蝮蛇毒纤溶酶Gisp6基因的克隆三个步骤组成；中介蝮蛇毒纤溶酶在HEK293T细胞建立稳定表达细胞株中的用途,中介蝮蛇毒纤溶酶在溶解血栓药物中的用途。由于采取了最佳的盐浓度,减少了组分与填料的非特异性吸附；采用了最佳pH值,使得峰之间不重叠,排除了杂质、提高了产物的纯度。(A middle Agkistrodon halys venom fibrinolysin, whose gene is Gisp6, has the nucleotide sequence shown in SEQ ID No.1 in the sequence table, has the amino acid sequence shown in SEQ ID No.2 in the sequence table; the preparation method comprises three steps of a purification method of intermediate Agkistrodon halys venom fibrinolysin, purity identification and mass spectrometry, and cloning of intermediate Agkistrodon halys venom fibrinolysin Gisp6 gene; the use of intermediate Agkistrodon halys venom plasmin in the establishment of stable expression cell strain of HEK293T cell, and the use of intermediate Agkistrodon halys venom plasmin in thrombolytic drug. Due to the adoption of the optimal salt concentration, the nonspecific adsorption of the components and the filler is reduced; the optimum pH value is adopted, so that peaks are not overlapped, impurities are removed, and the purity of the product is improved.)

1. A medium Agkistrodon halys venom fibrinolysin, which is characterized in that: the gene is Gisp6, has a nucleotide sequence shown by SEQ ID No.1 in a sequence table and has an amino acid sequence shown by SEQ ID No.2 in the sequence table.

2. A process for preparing the intermediate Agkistrodon halys venom plasmin of claim 1, comprising the steps of:

(1) method for purifying intermediate pallas pit viper venom fibrinolysin

1) Gel filtration chromatography separation

Dissolving intermediate Agkistrodon Halys crude drug lyophilized powder in 50mM ammonium acetate buffer solution to prepare 50mg/mL sample solution, loading on gel filtration chromatographic column with Sephacry1-200HR as filler and 30-60 mM ammonium acetate buffer solution containing 0.10-0.25M NaCl and having pH of 7.3, performing gel filtration by conventional method, and collecting primary component P3 with plasmin activity;

2) anion exchange chromatography separation

Taking 45-55 mM ammonium acetate with the pH value of 8.2 as an initial buffer solution, taking the initial buffer solution containing 0.8-1.2M NaCl as an elution buffer solution, and separating a primary component P3 with the plasmin activity by passing through a HiTrap DEAE FF 16/10 anion exchange column to obtain a secondary component P3-3 with the plasmin activity;

3) cation exchange chromatography separation

Taking 45-55 mM ammonium acetate with pH of 5.6 as an initial buffer solution, taking the initial buffer solution containing 1M NaCl as an elution buffer solution, separating a secondary component P3-3 of plasmin activity by passing through a HiTrap CM FF 16/10 cation exchange column for anion exchange chromatography to obtain a refined extract component P3-3-2 with the plasmin activity, namely the intermediate agkistrodon halys venom plasmin;

4) determination of intermediate Agkistrodon Halys venom fibrinolytic enzyme activity

Detecting the activity of the intermediate Agkistrodon halys venom fibrinolytic enzyme according to a conventional enzymology method, wherein a substrate used is plasmin specificity artificial polypeptide V7127, a solvent is 100mM Tris-hydrochloric acid buffer solution with the pH value of 8.0, the reaction condition is that the reaction is carried out for 20min in a dark place at the temperature of 37 ℃, and the wavelength of a light absorption value measured by an enzyme-labeling instrument according to the operation steps is 405 nm;

(2) purity determination and mass spectrometry

Taking the refined extract component P3-3-2 for sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis, determining the purity, cutting a protein band, and performing MALDI-TOF/TOF identification to obtain peptide fragment sequence information;

(3) cloning method of intermediate Agkistrodon halys venom fibrinolysin Gisp6 gene

1) Extracting total RNA from intermediate pallas pit viper venom gland by conventional molecular biological method;

2) carrying out reverse transcription on Oligo dT serving as a primer into cDNA to obtain a template;

3) according to the information provided by the peptide sequence of the intermediate Agkistrodon halys venom plasmin, a pair of Gisp6 degenerate primers is designed, and the sequences are as follows:

an upstream primer: 5'-GCTATG [ T/G ] [ G/T ] GCTGATCA [ A/G ] AGTG [ C/T ] TAG [ C/T ] -3'

A downstream primer: 5'-GTTTTCA [ T/C/A ] [ T/G ] G [ A/G ] GGGCA [ G/A ] [ T/G ] [ T/C ] [ G/C ] [ G/A ] CATC-3'

Synthesizing the primer;

4) establishing a PCR system according to a conventional molecular cloning method, wherein primers corresponding to the sequences and the prepared cDNA are used as templates, high-fidelity DNA polymerase is used for carrying out PCR amplification to obtain a product with the length of 780bp, and the reaction conditions are as follows: pre-denaturation at 98 ℃ for 20 s; denaturation at 98 ℃ for 15 seconds, annealing at 52 ℃ for 35 seconds, extension at 72 ℃ for 1.5 minutes, and running for 30 cycles;

5) after amplification is finished, performing 2% agarose gel electrophoresis, recovering a Gisp6 gene DNA fragment with the length of 780bp, connecting the DNA fragment with a pEASY-Blunt Zero T vector, transforming competent cells by a connecting system, coating an ampicillin resistance plate, selecting clone culture, extracting plasmids, and screening positive clones by using M13F and M13R primers as conventional PCR; sequencing the obtained positive clone by using M13F or M13R primer to obtain a full-length nucleotide sequence of the intermediate Agkistrodon halys venom fibrinolytic enzyme gene Gisp6 shown as SEQ ID NO.1, and an amino acid sequence of the intermediate Agkistrodon halys venom fibrinolytic enzyme deduced by using BioEdit software is shown as SEQ ID NO. 2.

3. The method for preparing intermediate Agkistrodon halys venom plasmin according to claim 2, wherein the step 1) of said method for purifying intermediate Agkistrodon halys venom plasmin comprises: gel filtration chromatography is carried out by taking Sephacry1-200HR as a filler and 45mM ammonium acetate containing 0.18M NaCl and having pH of 7.3 as a buffer solution to obtain an initial component P3 with plasmin activity.

4. The use of the intermediate Agkistrodon halys venom plasmin of claim 1 in the establishment of stably expressing cell lines in HEK293T cells by the following method:

(1) construction of recombinant expression vectors

The intermediate Agkistrodon halys venom plasmin gene is inserted between the BamH I and EcoRI enzyme cutting sites of an expression vector pcDNA3.1(+) to obtain a recombinant expression plasmid;

(2) transfection of host cells

The HEK293 cell is taken as a host cell, and the recombinant expression plasmid is transferred into the HEK293 cell under the mediation of polyethyleneimine to realize transient expression;

(3) g418 antibiotic screening

Screening by G418 antibiotic with concentration of 600-1050 mug/mL to obtain middle Agkistrodon halys venom fibrinolytic enzyme gene integrated HEK293 cells;

the G418 antibiotic described above was purchased from Calbiochem, USA;

(4) medium Agkistrodon halys venom fibrinolysin expression detection

Collecting the above-mentioned intermediate Agkistrodon halys venom fibrinolytic enzyme gene integrated HEK293 cell, cracking and preparing sample, using 6 × His monoclonal antibody as western blot to detect the expression of intermediate Agkistrodon halys venom fibrinolytic enzyme;

(5) subcloning of engineered cells

The HEK293 cell integrated with the intermediate Agkistrodon halys venom plasmin gene is subcloned to obtain an engineering cell stably expressing the snake venom plasmin, which is named as HEK293-Gisp 6.

5. The use of an intermediate viper venom plasmin enzyme of claim 4 in expression of HEK293T in a cell line, wherein said step (3) is: g418 antibiotic with concentration of 800 mug/mL is used for screening to obtain the middle Agkistrodon halys venom fibrinolytic enzyme gene integrated HEK293 cell.

6. The use of the intermediate viper venom plasmin of claim 1 in thrombolytic drugs.

7. The use of the intermediate Agkistrodon halys venom plasmin in thrombolytic drug according to claim 6, wherein the intermediate Agkistrodon halys venom plasmin is prepared into 1000mL injection by conventional preparation method from the following raw materials and their mixture ratio:

6.5mg of fibrinolysin as intermediate Agkistrodon halys venom

Water for injection was added to 1000 mL.

Technical Field

The invention belongs to the technical field of enzyme, and particularly relates to intermediate agkistrodon halys venom fibrinolytic enzyme.

Background

Thrombi are blood clots formed by damaged blood components on the surface of vascular endothelial cells and exposed collagen fibers, and the main components of thrombi are deposited platelets and insoluble fibrin gel and red blood cells. Thrombi are generally classified into six types, mixed thrombi including head (platelets + a few fibrin), body (trabecular + fibrin network + red blood cells) and tail (fibrin network + red blood cells), and clear thrombi (microthrombus) are mainly composed of fibrin. In the blood circulation system of the human body, there are two antagonistic and interdependent physiological action systems, namely the coagulation system and the anticoagulation system, namely the fibrinolysis system, abbreviated as fibrinolysis system, which are in dynamic equilibrium in the body.

The fibrinolytic system has the main functions of removing fibrin gel blocks in thrombus deposited on the vessel wall, dissolving the thrombus and keeping the blood vessel recanalization and normal blood flow. The fibrinolytic system in the human body mainly comprises 4 components: plasminogen, plasmin, plasminogen activator and plasminogen activator inhibitor. The thrombocytic disease is a common cardiovascular and cerebrovascular disease, and with the improvement of the living standard of people in China and the arrival of the aging era, the thrombocytic disease becomes one of the diseases with the highest morbidity, disability rate and mortality in China, thrombolytic therapy is undoubtedly the most effective therapy, and the search for novel high-efficiency thrombolytic drugs is a hotspot of natural drug research.

The development of thrombolytic drugs has been advanced by first generation urokinase, tissue plasminogen activator, second generation thrombolytic drug, recombinant t-PA (rt-PA, third generation thrombolytic drug. finding thrombolytic drugs from nature is also an important direction, and successively found thrombolytic drugs include lumbrokinase, nattokinase, hirudin, etc. plasmin isolated from snake venom has two major types, namely metallo-protease (SVMP) plasmin and serine protease (SVSP) plasmin, from 1991, several metalloproteinases (alpha-chain of hydrolyzed fibrin) with plasmin activity have been found in viperidae venom, e.g., Fibrolase (trade name: Alfimeprase) from Pallas crater copperas, Atroxas of Croton ruppus rupestris, Protobothros Tokanensis (Protobothrops tokanensis), metalloproteinases PT-H2, etc., whose action is not affected by plasma serine inhibitors F, etc., and its plasmin activity is dependent on metal ions.

The venom of Agkistrodon of Viperidae contains abundant serine protease plasmin, and its active center has marked amino acid residue-serine, and is independent of metal ion. Although the plasmin has different amino acid sequences, molecular weights, isoelectric points and the like, the plasmin has similar fibrinolytic activity. The most famous are plasmin from the snake venom of the solder head and plasmin from the snake venom of the white-eyebrow pit viper (namely the black-perilla pit viper) which are prepared into plasmin injection, and the clinical use effect is good.

The intermediate Agkistrodon Halys (Gloydius intermedia) is Viperidae (Viperidae) Agkistrodon Halys of Asian, and is widely distributed in northwest and north China. Because the content of plasmin in intermediate Agkistrodon halys venom is very low, the plasmin purified by column chromatography separation technology has low purity, and the impurities in plasmin can cause side effects.

Disclosure of Invention

The technical problem to be solved by the present invention is to overcome the disadvantages of the prior art and to provide a medium for Agkistrodon halys venom plasmin.

The invention aims to solve another technical problem of providing a preparation method of intermediate Agkistrodon halys plasmin.

The invention also aims to solve the technical problem of providing a new application of the medium Agkistrodon halys plasmin.

The technical scheme for solving the technical problems is as follows: the gene of the intermediate Agkistrodon halys venom plasmin is Gisp6, has a nucleotide sequence shown as SEQ ID No.1 in a sequence table and has an amino acid sequence shown as SEQ ID No.2 in the sequence table.

The preparation method of the intermediate Agkistrodon halys venom fibrinolytic enzyme comprises the following steps:

(1) method for purifying intermediate pallas pit viper venom fibrinolysin

1) Gel filtration chromatography separation

Dissolving the intermediate Agkistrodon Halys crude drug lyophilized powder in 50mM ammonium acetate buffer solution to prepare 50mg/mL sample solution, loading the sample solution on a gel filtration chromatographic column by using Sephacry1-200HR as a filler and 30-60 mM ammonium acetate buffer solution containing 0.10-0.25M NaCl and having pH of 7.3, performing gel filtration by a conventional method, and collecting an initial component P3 with fibrinolytic enzyme activity.

2) Anion exchange chromatography separation

Taking 45-55 mM ammonium acetate with the pH value of 8.2 as an initial buffer solution, taking the initial buffer solution containing 0.8-1.2M NaCl as an elution buffer solution, and separating a primary component P3 with the plasmin activity by using a HiTrap DEAE FF 16/10 anion exchange column to obtain a secondary component P3-3 with the plasmin activity.

3) Cation exchange chromatography separation

Taking 45-55 mM ammonium acetate with pH of 5.6 as an initial buffer solution, taking the initial buffer solution containing 1M NaCl as an elution buffer solution, and separating a secondary component P3-3 of the plasmin activity by passing through a HiTrap CM FF 16/10 cation exchange column for anion exchange chromatography to obtain a refined component P3-3-2 with the plasmin activity, namely the intermediate agkistrodon halys venom plasmin.

4) Determination of intermediate Agkistrodon Halys venom fibrinolytic enzyme activity

The detection of the intermediate Agkistrodon halys venom fibrinolytic enzyme activity is carried out according to a conventional enzymology method, a used substrate is plasmin specificity artificial polypeptide V7127, a solvent is 100mM Tris-hydrochloric acid buffer solution with the pH value of 8.0, the reaction condition is that the reaction is carried out for 20min in a dark place at the temperature of 37 ℃, and the wavelength of a light absorption value measured by an enzyme-labeling instrument according to the operation steps is 405 nm.

(2) Purity determination and mass spectrometry

Taking the refined extract component P3-3-2 component for sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis, determining the purity, cutting a protein band, and performing MALDI-TOF/TOF identification to obtain peptide segment sequence information.

(3) Cloning method of intermediate Agkistrodon halys venom fibrinolysin Gisp6 gene

1) Taking intermediate pallas pit viper venom gland as a raw material, and extracting total RNA by a conventional molecular biology method.

2) And (3) carrying out reverse transcription to form cDNA by using Oligo dT as a primer to obtain a template.

3) According to the information provided by the peptide sequence of the intermediate Agkistrodon halys venom plasmin, a pair of Gisp6 degenerate primers is designed, and the sequences are as follows:

an upstream primer: 5'-GCTATG [ T/G ] [ G/T ] GCTGATCA [ A/G ] AGTG [ C/T ] TAG [ C/T ] -3'

A downstream primer: 5'-GTTTTCA [ T/C/A ] [ T/G ] G [ A/G ] GGGCA [ G/A ] [ T/G ] [ T/C ] [ G/C ] [ G/A ] CATC-3'

The above primers were synthesized.

4) Establishing a PCR system according to a conventional molecular cloning method, wherein primers corresponding to the sequences and the prepared cDNA are used as templates, high-fidelity DNA polymerase is used for carrying out PCR amplification to obtain a product with the length of 780bp, and the reaction conditions are as follows: pre-denaturation at 98 ℃ for 20 s; denaturation at 98 ℃ for 15 seconds, annealing at 52 ℃ for 35 seconds, and extension at 72 ℃ for 1.5 minutes, for a total of 30 cycles.

5) After amplification is finished, performing 2% agarose gel electrophoresis, recovering a Gisp6 gene DNA fragment with the length of 780bp, connecting the DNA fragment with a pEASY-Blunt Zero T vector, transforming competent cells by a connecting system, coating an ampicillin resistance plate, selecting clone culture, extracting plasmids, and screening positive clones by using M13F and M13R primers as conventional PCR; sequencing the obtained positive clone by using M13F or M13R primer to obtain a full-length nucleotide sequence of the intermediate Agkistrodon halys venom fibrinolytic enzyme gene Gisp6 shown as SEQ ID NO.1, and an amino acid sequence of the intermediate Agkistrodon halys venom fibrinolytic enzyme deduced by using BioEdit software is shown as SEQ ID NO. 2.

The step 1) of the purification method of the intermediate agkistrodon halys venom plasmin comprises the following steps: gel filtration chromatography is carried out by taking Sephacry1-200HR as a filler, and preferably 45mM ammonium acetate containing 0.18M NaCl and having pH of 7.3 as a buffer solution to obtain a primary component P3 with plasmin activity.

The application of the intermediate Agkistrodon halys venom plasmin in establishing a stable expression cell strain in HEK293T cells comprises the following steps:

(1) construction of recombinant expression vectors

The intermediate Agkistrodon halys venom plasmin gene is inserted between the BamH I and EcoRI cleavage sites of expression vector pcDNA3.1(+) to obtain recombinant expression plasmid.

(2) Transfection of host cells

The HEK293 cell is taken as a host cell, and the recombinant expression plasmid is transferred into the HEK293 cell under the mediation of polyethyleneimine to realize transient expression.

(3) G418 antibiotic screening

Screening by G418 antibiotic with concentration of 600-1050 mug/mL to obtain middle Agkistrodon halys venom fibrinolytic enzyme gene integrated HEK293 cells;

the G418 antibiotic described above was purchased from Calbiochem, USA.

(4) Medium Agkistrodon halys venom fibrinolysin expression detection

Collecting the above-mentioned intermediate Agkistrodon halys venom fibrinolytic enzyme gene integrated HEK293 cell, cracking and preparing sample, using 6 × His monoclonal antibody as western blot to detect the expression of intermediate Agkistrodon halys venom fibrinolytic enzyme.

(5) Subcloning of engineered cells

The HEK293 cell integrated with the intermediate Agkistrodon halys venom plasmin gene is subcloned to obtain an engineering cell stably expressing the snake venom plasmin, which is named as HEK293-Gisp 6.

The step (3) is as follows: optimally, G418 antibiotic with the concentration of 800 mug/mL is used for screening to obtain the middle Agkistrodon halys venom fibrinolysin gene integrated HEK293 cell.

The application of the medium Agkistrodon halys venom fibrinolysin in the medicine for dissolving thrombus is provided.

The application of the medium Agkistrodon halys venom fibrinolysin in dissolving thrombus is prepared into 1000mL injection by the following raw materials and the mixture ratio according to the conventional preparation method:

6.5mg of fibrinolysin as intermediate Agkistrodon halys venom

Water for injection was added to 1000 mL.

The medium agkistrodon halys venom is used as a material, gel filtration chromatography, anion exchange chromatography and cation exchange technology are sequentially adopted to separate and obtain medium agkistrodon halys venom fibrinolytic enzyme, and due to the adoption of the optimal salt concentration, the non-specific adsorption of components and fillers is reduced; the optimum pH value is adopted, so that peaks are not overlapped, impurities are removed, and the purity of the product is improved.

Taking venom gland of intermediate Agkistrodon Halys as material, extracting total RNA, reverse transcribing to prepare cDNA, designing primer based on peptide segment information provided by mass spectrum identification of intermediate Agkistrodon Halys venom plasmin, and cloning plasmin gene by RT-PCR technology to obtain complete sequence of the plasmin gene.

The invention uses pcDNA3.1(+) as an expression vector and uses HEK293 cell line as a host cell to realize the method for stably expressing the intermediate Agkistrodon halys venom fibrinolysin Gisp6 in the HEK293 cell, and lays a foundation for developing the gene engineering product into clinical drugs.

Drawings

FIG. 1 is a diagram of a process for separating P3-3 components by cation exchange chromatography.

FIG. 2 detection of plasmin activity of fractions obtained by cation exchange chromatography separation of P3-3 fraction.

FIG. 3 shows the SDS-PAGE analysis of the intermediate Agkistrodon halys venom plasmin.

FIG. 4 is a MALDI-TOF/TOF tandem mass spectrometry identification of the intermediate Agkistrodon halys venom plasmin A band.

FIG. 5 is a MALDI-TOF/TOF tandem mass spectrometry identification of the intermediate Agkistrodon halys venom plasmin B band.

FIG. 6 is an electrophoretogram of PCR amplification product of the intermediate Agkistrodon halys venom plasmin gene Gisp 6.

FIG. 7 is a PCR identification electrophoretogram of pcDNA3.1/Gisp6 positive clones.

FIG. 8 is a forward sequencing diagram of the pcDNA3.1-Gisp6 recombinant plasmid.

FIG. 9 is an optical microscopic image of G418 screening 15 days after the recombinant plasmid was transfected into HEK293 cells.

FIG. 10 shows Western blot detection of the intermediate Agkistrodon halys venom plasmin expressed in HEK293-Gisp6 cells.

FIG. 11 is a Western blot analysis of the subcloning of HEK293/Gisp6 cells to generate expression of 7 cell lines Gisp 6.

FIG. 12 is a graph showing the measurement of plasmin activity of supernatants of 7 HEK293/Gisp6 engineered cell lines.

FIG. 13 is a thrombolysis assay of supernatants from seven HEK293/Gisp6 engineered cells.

FIG. 14 is a graph comparing fibrinolytic activity of intermediate Agkistrodon halys venom plasmin and injectable plasmin.

FIG. 15 shows the electrophoretic analysis of SDS-PAGE of fibrinogen hydrolyzed by plasmin from intermediate Agkistrodon halys venom.

FIG. 16 is a graph comparing the activity of the intermediate Agkistrodon halys venom plasmin and batroxobin to hydrolyze fibrinogen.

Detailed Description

The present invention will be described in further detail with reference to the following drawings and examples, but the present invention is not limited to these examples.