CN114150007B

CN114150007B - Coding gene applicable to rabbit mammary gland specific expression deaminase and application thereof

Info

Publication number: CN114150007B
Application number: CN202111525445.8A
Authority: CN
Inventors: 成勇; 张婷; 周敏雅
Original assignee: Yangzhou University
Current assignee: Yangzhou University
Priority date: 2021-12-14
Filing date: 2021-12-14
Publication date: 2023-07-21
Anticipated expiration: 2041-12-14
Also published as: CN114150007A

Abstract

The invention relates to a coding gene suitable for rabbit mammary gland specificity expression deaminase DSPA alpha 1, which is used for preparing the deaminase DSPA alpha 1 with high expression quantity and low production cost. The invention also relates to a method for extracting and purifying deaminase rDSPA from rabbit milk, which is characterized by separating and purifying by combining anisole affinity chromatography, cation exchange chromatography and active blue affinity chromatography, and has simple operation and high product purity which can reach 98 percent.

Description

Coding gene applicable to rabbit mammary gland specific expression deaminase and application thereof

Technical Field

The invention relates to genetic engineering, in particular to a gene for encoding protein and application thereof, and more particularly relates to a method for preparing recombinant protein by using the encoding gene.

Background

Thrombotic disease is one of the major diseases that endanger human life and health, and thrombolytic drugs currently studied do not directly dissolve thrombus, but rather dissolve thrombus by activating the plasminogen system, thus depleting the amounts of fibrinogen and plasminogen upon dissolving thrombus, causing a significant decrease in clotting factors and thus bleeding tendency. Thrombolytic drugs used clinically to treat this class of diseases are represented by natural human tissue plasminogen activator (tPA), however tPA has a very short half-life in blood (3-5 minutes) which necessitates large doses of intravenous instillation in order to maintain its effective concentration in clinical treatment, thereby increasing the risk of bleeding in the circulatory system.

Deaminase (DSPA) is a plasminogen activator isolated from blood-sucking bat saliva in south america, and mainly comprises four proteins: DSPA α1, DSPA α2, DSPA β, and DSPA γ. It has been found that among them, DSPA alpha 1 has the best biochemical and pharmacological properties, and compared with other plasminogen activators, DSPA alpha 1 is most characterized by high specificity for fibrin, and compared with the current widely used tissue plasminogen activators (tPA), in the presence of fibrin, DSPA alpha 1 has an enzymatic activity increased by 102100 times, while tPA has only an increase of 550 times; under the condition that fibrin and fibrinogen exist respectively, the activity ratio of DSPA alpha 1 to DSPA alpha 1 is 12900, and tPA is only 72; clinically, DSPA alpha 1 has small dosage, little bleeding, no neurotoxicity and better medicinal value than tPA. The DSPA is used as a new thrombolytic drug under development, is suitable for old thrombus, has the characteristics of wide treatment window period (9 hours), long half-life period of 2.3 hours, capability of directly degrading fibrin, reduction of bleeding, toxic and side effects and the like, and has wide development prospect. Although recombinant DSPA can be expressed in prokaryotic cells, the expression product is inactive and needs to be renatured, thereby increasing the production links. Even though the activity is generated by renaturation, the specific activity is low. This results in increased production costs, which lead to high clinical treatment costs. In addition, the prokaryotic product has poor compatibility with human body, is easy to cause adverse reaction, reduces the safety of the medicine and increases the clinical application risk. The animal mammary gland bioreactor technology is utilized to produce medicinal recombinant protein, and has high commercial application value. The rabbit has the advantages of high lactation yield, short gestation period, multiple litter size per fetus, and the like. The use of rabbit mammary gland bioreactors to produce recombinant DSPA has not been reported so far.

Therefore, designing and developing the preparation method for prolonging the half-life, reducing the bleeding tendency of the system, improving the specific activity and reducing the price become important targets for developing novel DSPA medicaments, and the development of the novel medicaments has important practical significance for guaranteeing the life health of human beings.

The history of research on the separation and purification of target proteins by transgenic animal emulsion is short, so no mature purification technology exists at home and abroad. Although the basic principle of the recombinant protein produced by the purification mammary gland bioreactor is similar to that of the general protein, the separation and purification are carried out by utilizing the physicochemical and biological differences of different proteins, the milk components of mammals are complex, various milk proteins and globulins are contained, and the recombinant protein also contains rich fat, sugar, vitamins, minerals, fragments of animal tissues and the like. In addition, the active structure and the function of the recombinant protein are maintained in the process of extracting the recombinant protein, and the requirements of separation and purification of the target protein are improved. It is generally necessary to combine various purification techniques to isolate and purify a recombinant protein, and the selection of the scheme is made with reference to the expression system of the recombinant protein, since the expression system determines the nature of the recombinant protein and the type of the hybrid protein.

Disclosure of Invention

The invention aims to solve the technical problem of providing a coding gene suitable for rabbit mammary gland specific expression deaminase DSPA alpha 1.

Another technical problem to be solved by the present invention is to provide a method for preparing desmopressin DSPA alpha 1 using the coding gene.

The invention also provides a method for extracting and purifying the desmopressin DSPA from the rabbit milk.

In order to solve the technical problems, the invention adopts the following technical scheme:

the coding gene is suitable for rabbit mammary gland specific expression deaminase, and is characterized in that the nucleotide sequence of the gene is shown as SEQ ID NO. 1.

The gene has the following characteristics: (1) The codon of the coding sequence is compatible with the characteristics expressed in rabbit individuals; (2) The recombinant coding sequence meets the requirement of high-efficiency expression in rabbit mammary tissue specificity.

The amino acid sequence of the deaminase DSPA alpha 1 recombinant protein coded by the gene is shown as SEQ ID NO. 2.

The coded DSPA alpha 1 recombinant protein has the following characteristics: (1) The primary structure N-terminus facilitates processing of the translated polypeptide into a mature protein in the cell; (2) The recombinant DSPA primary structure has folding or chemical bond formation to form functional protein, so as to generate thrombolytic activity.

The application of the coding gene in the preparation of the desmopressin.

Further, the application is to prepare the desmopressin through a rabbit mammary gland bioreactor.

Further, the preparation method comprises the following steps:

(1) Constructing a mammary gland specific expression deaminase gene vector;

(2) Preparing a mammary gland specific expression deaminase gene rabbit;

(3) Detecting and analyzing deaminase in rabbit milk, and collecting the milk after the rabbit is bred and delivered;

(4) The rDSPA recombinant protein in the milk is extracted and purified.

Preferably, the deaminase is DSPA alpha 1.

Preferably, the method combines anisole affinity chromatography, cation exchange chromatography and active blue affinity chromatography for separation and purification.

Further, the method comprises the following steps:

(1) The rabbit milk is subjected to ultracentrifugation and 55% ammonium sulfate precipitation to primarily separate rDSPA in the rabbit milk, so as to obtain a rabbit milk pretreatment sample;

(2) Anisole affinity chromatography:

A. balancing the chromatographic column with a balancing liquid; the balance liquid is 20mmol/L glycine solution, and the pH value is 8.75;

B. loading the sample at a flow rate of 3mL/min after balancing, and washing the chromatographic column with a balancing solution with a volume of 6 times of the column volume until the ultraviolet absorption line UV280 is zeroed;

C. eluting with 3 times of column volume of eluting solution (20 mmol/L phosphate buffer solution+0.5 mol/L NaCl, pH 7.5), wherein the eluting condition is set to 0.1mol/L glycine, and the pH is 3.0;

(3) Cation exchange chromatography:

A. balancing the chromatographic column with a flow rate of 5mL/min, and balancing 200mL after the ultraviolet absorption value UV280 and the electric conductivity value are stable; the balance liquid is 50mmol/L phosphate buffer solution, and the pH value is 8.0;

B. washing the chromatographic column with 6 times column volume of equilibrium liquid (20 mmol/L glycine solution, pH 8.75) until the ultraviolet absorption line (UV 280) is zeroed, wherein the elution condition is set to 20m mol glycine+0.31 mol sodium chloride;

(4) Lan Zhu affinity chromatography:

A. balancing the balancing solution, wherein the flow rate is 3mL/min, and balancing 30mL after the ultraviolet absorption value UV280 and the electric conduction value are stable; the balance liquid is 50mmol/L phosphate buffer solution, and the pH value is 8.0;

b.20mmol/L phosphate buffer pH7.5, and loading after diluting to 500mL, wherein the flow rate is 3mL/min;

c.20mmol/L phosphate buffer pH7.5+0.15mol/L sodium chloride eluent is discarded; 20mmol/L phosphate buffer pH7.5+0.4mol/L sodium chloride eluent can be recovered and reused; collecting 20mmol/L+1.0mol/L sodium chloride;

(5) Desalting to obtain rDSPA pure product.

9. The method of extracting and purifying rdpa recombinant protein as claimed in claim 8, further comprising: the purified rDSPA was subjected to anion exchange chromatography to remove endotoxin from the purified product. The rabbit is a rabbit for expressing the deaminase gene specifically in mammary gland.

Compared with the prior art, the invention has the beneficial technical effects that:

(1) The gene of the invention can be adopted to efficiently express DSPA through a rabbit mammary gland bioreactor;

(2) Obtaining DSPA transgenic rabbits with high-efficiency specificity expression of mammary gland and biological activity, wherein fibrinolytic activity is higher than that of other expression systems (bacteria, fungi and CHO cells);

(3) The production cost is low, and the yield is enlarged simply;

(4) Post-translational modifications and correct folding of the recombinant DSPA protein can be achieved as a mature protein.

(5) The extraction and purification process of the invention has simple operation and high product purity (up to 98%).

Drawings

Fig. 1: plasmid map of pUC57+DSPA;

fig. 2: plasmid map of pcl25+rhpa;

fig. 3: pCL25/DSPA construction flow diagram;

fig. 4: restriction enzyme map of recombinant plasmid Xho I;

fig. 5: sequence analysis to identify the forward and reverse of the insert in the recombinant plasmid pCL 25/DSPA;

fig. 6: microinjection of fertilized eggs of rabbits in a prokaryotic period;

fig. 7: PCR amplification electrophoretogram of integrated DSPA gene rabbit

Fig. 8: ELASA detection graph of rDSPA in transgenic rabbit milk;

fig. 9: FAPA analysis of DSPA transgenic rabbit whey;

fig. 10: purifying an rDSPA transgenic rabbit milk eluting peak diagram by using an anisole affinity chromatographic column;

fig. 11: FAPA detects fibrinolytic activity of the sample in the process of anisole affinity chromatographic column purification;

Fig. 12: linear gradient elution peak diagram of strong cation exchange chromatographic column;

fig. 13: FAPA detection strong cation exchange chromatographic column linear gradient elution sample fibrinolysis activity;

fig. 14: performing SDS-PAGE detection on a strong cation exchange chromatographic column linear gradient elution sample;

fig. 15: purifying rDSPA transgenic rabbit milk peak diagram by using an active blue affinity chromatographic column;

fig. 16: FAPA detection of fibrinolytic Activity of sample in purification process of active blue affinity chromatography column

Fig. 17: hiTrap Capto Q column elution peak diagram;

fig. 18: endotoxin detection results.

Nucleotide and amino acid sequence specification

SEQ ID NO.1: the coding gene is suitable for rabbit mammary gland specificity expression deaminase DSPA alpha 1;

SEQ ID NO.2: the amino acid sequence of the deaminase DSPA alpha 1 of the invention;

SEQ ID NO.3: nucleotide sequence of primer DSPA-S;

SEQ ID NO.4: nucleotide sequence of primer DSPA-A.

Detailed Description

The technical scheme of the invention is further described below by combining examples.

EXAMPLE 1 construction of vector for mammary gland-specific expression of Deaminopeptidase (DSPA) Gene

1. And (3) a carrier:

pCL25+tPA is a mammary gland specific expression vector constructed in the laboratory, and is prepared by taking goat beta-casein as an expression control sequence, chicken beta-globin as an isolation element and CMV as an enhancer; this vector has been validated at the cellular and individual levels of goats and rabbits. The recombinant DSPA alpha 1cDNA sequence was synthesized by biological engineering (Shanghai) Inc., and inserted into the multiple cloning site (MSC) of pUC57 vector; plasmid pUC57 was purchased from Shanghai Ming Biotech Co., ltd; host bacteria Trans10 were purchased from Beijing full gold organisms. The pCL25 plasmid is formed by splicing PBC1 and CMV sequences, and the details are shown in the patent of the invention: "construction method of transgenic animal mammary gland specific vector with high expression level", the inventors: chenglong, patent number: ZL200610040436.9; construction of pcl25+rhpa vector reference 2016, song Shaozheng, doctor graduation paper: "transgenic rabbit mammary gland expression recombinant human plasminogen activator (rhPA) and pharmacodynamics research thereof" (China network, shuo Bo paper database).

plasmid maps of pUC57+DSPA and pCL25+rhPA are shown in FIGS. 1 and 2.

2. Material

2.1 major instruments and equipment

DNA amplification apparatus (Bio-Rad Co., USA); DNA electrophoresis apparatus and electrophoresis tank (south kyo university biotechnology development company); digital gel imaging system (Shanghai Technical Co., ltd.); a water-proof electrothermal constant temperature incubator (PYX-DHS-35X 40-B-H, shanghai medical equipment factory); an electrothermal constant temperature water tank (Shanghai Jing Hongjingzhi laboratory equipment Co., ltd.); ice maker (SANYO corporation, japan); electronic balance (Shanghai precision balance manufacturing Co., ltd.); PH electrode (Metrehler-Tolyx group Shanghai Co.); a constant temperature oscillator (IS-RDV-1,INCUBATOR SHAKER); automatic double pure water distiller (Shanghai Shenli glass Co., ltd.); low temperature high speed centrifuges (Eppendorf, germany); desk-top refrigerated high speed centrifuges (Eppendorf, germany); a vacuum extractor (Speed Vac Plus SC, U.S.); ultra clean bench (su zhou clean instrumentation plant); common refrigerator (Qingdao sea company); ultra low temperature refrigerator (SANYO corporation, japan); nucleic acid protein quantitative analyzer (One Drop) ^TM OD-1000+, gene group, shanghai Instrument waves biological instruments Co., ltd.); laboratory grade ultra pure water machine (EPED-20 TF, nanjing Yi Puyi, science and technology development Co., ltd.); ultrasonic cleaner (KQ-300 DE, kunshan ultrasonic instruments Co., ltd.).

2.2 principal drugs and reagents

Restriction enzymes such as Xho I, sal I, not I, etc., and markers such as DL2000, lambda-HindIII, etc., are available from TaKaRa company; endo-Free Plasmid Mini Kit II D6950 (endotoxin free plasmid miniprep kit II) was purchased from OMEGA, inc. of America; the DNA recovery and purification kit is purchased from QIAGEN company, ampicillin is purchased from Beijing Soy pal, and other chemical reagents are all of domestic analytical purity.

3. Method of

3.1 Synthesis of recombinant DSPA functional Gene

The NCBI is searched for a DSPA cDNA sequence, a mature peptide sequence is selected, and a coat BLG sig-peptide sequence is added to the 5' end of the mature peptide sequence to form a recombinant DSPA functional gene, and the recombinant DSPA functional gene is synthesized by the division of biological engineering (Shanghai) and connected to a pUC57 vector. After the received pUC57+DSPA glycerol bacteria were subjected to a small amount of culture to identify the activity, the activity was expressed as 50% glycerol (3): the bacterial liquid (7) is mixed in proportion and kept for standby, and meanwhile, recombinant plasmid is prepared to be constructed.

The mRNA sequence of DSPA is retrieved in NCBI, and the names and numbers are as follows:

Desmodus rotundus plasminogen activator alpha 1mRNA

GenBank:M63987.1

the sequence obtained by modifying the cDNA sequence is shown as SEQ ID NO.1.

3.2 construction of recombinant DSPA plasmids

The synthesized DSPA cDNA sequence is inserted into a breast specific expression vector (pCL 25) containing the expression elements described above and maintained in the laboratory, and the newly constructed breast specific expression vector is designated pCL25/DSPA.

3.2.1 extraction of plasmid DNA and purification of recovered DNA fragments

The experimental procedures for preparing pCL25+tPA plasmid DNA and pUC57+DSPA plasmid DNA using endotoxin-free plasmid miniprep kit II (OMEGA) were as follows:

(1) Respectively inoculating the two plasmids into 50mL LB culture solution containing AMP, and shake culturing at 37 ℃ for 12h;

(2) Collecting bacteria by centrifugation at 5,000g for 5min at room temperature;

(3) Removing LB culture Solution, adding 500. Mu.l Solution I mixed Solution containing RNase, and shaking to make it completely suspended;

(4) Transferring the Solution to a new 2mL centrifuge tube, adding 500 μl Solution II, gently reversing and mixing, and standing the mixed Solution at room temperature for 2-3 min; note: the cracking time after adding Solution II should not exceed 5min; sealing and preserving;

(5) Adding pre-chilled N3 Buffer 250 μl, and gently turning upside down the centrifuge tube for 7-8 times until white floccules are generated in the tube;

(6) Centrifuging at room temperature of 12000g for 10min, and sucking supernatant into the clean sterilization finger tube;

(7) Adding ETR Solution of 0.1×total volume of the liquid in the tube (6), turning upside down for 7-8 times, and standing in ice bath for 10min;

(8) Adjusting the constant-temperature water bath kettle to 42 ℃ in advance, immediately transferring to the water bath kettle after the ice bath in the step (7) is finished, and incubating for 5min;

(9) Centrifuging at room temperature of 12,000g for 3min, transferring the colorless transparent liquid on the upper layer into a clean sterilization finger tube, adding absolute ethanol which is 0.5 times of the total volume of the liquid in the tube, turning upside down for 7-8 times, and standing at 20deg.C for 2min;

(10) Sucking up not more than 700 mu L of the liquid in the step (9), transferring the liquid into a DNA binding column containing a 2mL collecting pipe, centrifuging for 1min at 12000g, and discarding the waste liquid in the collecting pipe;

(11) Repeating (10) until all the columns are crossed;

(12) Finally, the waste liquid is discarded, 500 μl of HBC Buffer is added, 12000g is centrifuged for 1min, and the waste liquid is discarded;

(13) Adding 700 μl of DNA washing buffer, centrifuging for 1min with 12000g, discarding the waste liquid, and repeating for 2-3 times;

(14) 12000g of air is separated for 2min;

(15) Discarding the collecting tube, inverting the DNA binding column, transferring the alcohol smell into a sterile finger tube after the alcohol smell is removed, adding 60 μl of sterilized double distilled water, standing at room temperature for 2min, and centrifuging 12000g for 1min;

(16) Storing the two collected filtrates, namely plasmids, at-20deg.C respectively;

the two plasmids were digested with XhoI restriction enzymes, respectively, and incubated overnight (about 8 h) in a 37℃water bath, the digestion system being as follows in Table 1:

TABLE 1 XhoI cleavage reaction System

The purification kit used for the recovery of the DNA fragment required for ligation was manufactured by Axygen company, and comprises the following specific steps:

(1) Preparing 0.8% agarose gel with 1×TAE electrophoresis buffer, and respectively electrophoresis two DNA enzyme cutting products;

(2) Setting the electrophoresis condition to be constant pressure of 90V, observing whether the strip is askew, adjusting at any time, and ending electrophoresis after 3-4 h;

(3) Sucking up the liquid on the surface of the undyed gel by using a paper towel, cutting off a strip-shaped gel block with the left side of the gel being about 1cm wide, dyeing for 20s by using EB, decolorizing for 5min, finding the strip of the target DNA under an ultraviolet lamp, marking, and cutting off agarose gel containing the target DNA in the undyed gel by taking the strip as a reference;

(4) Calculating gel mass, firstly peeling the finger tube, weighing gel mass not more than 300mg, and calculating the volume of the added reagent according to the specification, wherein the following steps are as follows: 1mg = 1 μl;

(5) Adding the DE-A buffer solution with the volume of 3 times of the gel amount in the step (4), and after being turned upside down vigorously, placing the mixture in a water bath kettle at 70 ℃ for melting, and turning upside down for 7-8 times every 2 minutes until all gel blocks in the pipe are melted, wherein the liquid is uniformly transparent pink;

(6) Adding 0.5 times of the volume of the DE-A buffer solution of the step (5) into the DE-B buffer solution, and reversely and uniformly mixing until the liquid is uniformly transparent yellow;

(7) Sucking the yellow liquid in the step (6), transferring the yellow liquid into a DNA preparation tube containing a 2mL collecting tube, centrifuging for 1min at 12,000g, discarding the waste liquid in the collecting tube, and repeating until all the liquid in the step (6) passes through the column;

(8) Adding 500 mu L W1 buffer solution into the tube, centrifuging for 0.5min at 12,000g, and pouring out waste liquid in the collecting tube;

(9) Adding 750 mu L W2 buffer solution into the tube, centrifuging for 0.5min at 12,000g, pouring out waste liquid in the collecting tube, and repeating for 3 times;

(10) Air-separating for 2min at 12,000 g;

(11) Discarding the collecting tube, inverting the DNA preparation column, transferring the alcohol smell into a sterile finger tube after the alcohol smell is removed, adding 40 μl of sterilized double distilled water heated to 50deg.C in advance, standing at room temperature for 10min, centrifuging 12000g for 1min, and preserving at-20deg.C for use.

3.2.2 ligation of recombinant DSPA with pCL25 vector

The pCL25/DSPA plasmid was prepared using T4 DNA ligase manufactured by Promega, inc., as follows:

(1) The purified DSPA fragment and the mammary gland specific expression vector pCL25 fragment were calculated separately according to the following formula, ng required for ligation, and converted to volume;

(1) The concentration of the purified DSPA fragment was 191.3 ng/. Mu.L and the concentration of the purified pCL25 vector portion was 148.9 ng/. Mu.L as determined by One Drop. According to the specifications and experimental practical conditions of the T4 DNA ligase, the reaction system is adjusted as follows (Table 2):

TABLE 2 T4 DNA ligase reaction System

The reaction system is placed in a refrigerator at 4 ℃ for overnight connection or reacted for 4-18h at 15 ℃.

3.2.3 transformation of recombinant pCL25/DSPA plasmid

The transformation of the recombinant pCL25/DSPA plasmid into Trans 10 competent cells was as follows:

(1) Slowly sucking 50 mu L of Trans 10 competent cells melted on the ice bath, placing the cells into a 1.5mL centrifuge tube, lightly adding 10 mu L of the connecting product, gently mixing, and standing in the ice bath for 30min;

(2) The water bath kettle is accurately adjusted to 42 ℃ before the experiment, the water bath heat shock is carried out for 45s after the confirmation by a thermometer, then the centrifuge tube is quickly transferred into an ice-water mixture, the ice bath is kept stand for 2min, and the centrifuge tube is prevented from being shaken in the transfer process;

(3) Adding 500 mu L of sterile LB liquid medium without Ampicillin (AMP) into a centrifuge tube, gently mixing, placing on a shaking table at 37 ℃ and 200rpm/min, and culturing for 1h to recover cells;

(4) According to the experimental requirements, centrifuging at 5000rpm/min for 5min, discarding 300 mu L of supernatant, re-suspending cells, then sucking 100 mu L of transformed competent cells, adding the mixture onto an AMP-containing LB solid agar medium, uniformly spreading, placing the medium in a constant temperature incubator at 37 ℃, inverting the LB medium after 20min, culturing overnight at 37 ℃ (12 h), taking out the supernatant and placing in a refrigerator at 4 ℃ the morning for preparation of cloning and amplification at night.

3.2.4 Enzyme digestion identification and sequence analysis of pCL25/DSPA plasmid

The LB solid medium placed in a refrigerator at 4℃was removed, and individual colonies were picked up and inoculated into 7mL of LB liquid medium (containing AMP at a final concentration of 50. Mu.g/mL), 48 individual colonies were picked up at a time, labeled and placed on a shaker at 37℃at 200rpm/min for overnight culture. 2ml of plasmids of bacterial liquid are extracted from each tube by SDS alkaline lysis after 12-14h of shaking, and simultaneously 32 μl of ribonucleobase A (10 mg/ml) is added to 1568 μl of DW and kept at 4 ℃ for later use; each pCL25/DSPA plasmid is dissolved by 30 mu l of DW containing RNAse, mixed evenly and reacted for 1 hour at 37 ℃,2 mu l of plasmid extract is taken and reacted for 2 hours by using an Xho I enzyme digestion system (the enzyme digestion reaction system is shown in Table 3), 5 mu l of reaction liquid is taken and electrophoresed, and plasmids with only 1398bp and 16341bp bands are screened (namely recombinant plasmids inserted with target genes are obtained); and (3) sending the screened sample to Shanghai biological engineering Limited company for sequencing, and identifying the forward and reverse directions of DSPA DNA insertion and the mutation condition. And comparing the theoretical sequence with the actual sequencing result through MegAlign software, finally obtaining the pCL25/DSPA plasmid and strain which are connected correctly, and storing the pCL25/DSPA plasmid and strain at the temperature of-20 ℃ for standby. The construction flow of the pCL25/DSPA mammary gland specific expression vector is shown in figure 3.

TABLE 3 XhoI cleavage reaction System

4. Design requirements and identification analysis

4.1 Enzyme digestion identification and sequence analysis result of pCL25/DSPA plasmid

The study uses Xho I enzyme to cut plasmids of 48 bacterial liquid samples, 23 enzyme cutting products are randomly selected for agarose gel electrophoresis, and 9 recombinant plasmids meeting the conditions are obtained through screening, and the electrophoresis result is shown in figure 4; meanwhile, 7 samples with obvious bands are selected for sequencing by a worker, and 3 pCL25/DSPA recombinant plasmids which are connected in forward direction and have no mutation are screened by sequence analysis, wherein the sequences of S6, S25 and S27 in the tables 4 and the theoretical sequence are consistent, and the sequence is shown in figure 5.

TABLE 4 recombinant pCL25/DSPA plasmid ligation data

EXAMPLE 2 preparation of mammary gland-specific expression DSPA Gene Rabbit

1. Material

1.1 laboratory animals

The animals selected in the experiment are all common New Zealand white rabbits, and are purchased from the Gandensae canal species rabbit farm in Yangzhou, jiangsu province. All experimental rabbits were purchased for 14 days later, wherein female rabbits were 5-12 months old, male rabbits were 6-12 months old, and the body weight was 2.5-3.5 kg/animal. Feeding conditions: cage-raising and equipped with automatic flushing system; an indoor air conditioner is arranged, the annual temperature is controlled at 18-25 ℃, and ventilation is good; the relative humidity is 40% -45%; light treatment is carried out for 12 hours every day; the pellet feed is fed for 1 time in the morning and at night, and drinking water is free. All experimental procedures referred to in this study were strictly in accordance with standard protocols and ethical ethics for experimental animals.

1.2 major instrumentation

Stereoscopic microscope (Xiamen Megaodi; TMS, NIKON, japan); fluorescence inversion microscopes and micromanipulators (Leica, germany; eppendorf TransferMan NK 2; IX-71, olympus, japan); a needle puller (MODEL P-97, INSTRUMENT, USA); needle milling instrument (BV-10Micropipette SUTTER;Beveler SUTTER); CO ₂ Incubator (MCO-18M ThermoForma); DNA amplification apparatus (S1000 TM Thermal Cycler BiO-RAD company; PTC-200, gene company, USA); single sheetHuman double-sided decontamination work table (su zhou decontamination facilities limited); DNA electrophoresis apparatus, electrophoresis tank (Nanjing university student biotechnology development company); digital gel imager (GIS-1000, shanghai Technical Co., ltd.); ice machine (SIM-F124, japan three ocean); a water-proof electrothermal constant temperature incubator (GSP-9080 MBE, shanghai, china); an electronic balance (FA 1604, shanghai precision balance); PH detector (HA 405-K2, metretolide Shanghai Co.); an electric thermostatic drying oven (MICRO-4 HYBAID); table-top high-speed cryocentrifuge (Biofuge, germany; eppendorf, germany); a low-speed table centrifuge (model 80-2, shanghai medical instruments Co., ltd.); vertical electrophoresis System (POWER-P, sanpeng technology development Co., ltd.); PVDF film (Pall corporation, usa); enzyme label instrument (Shenzhen Rayto Co., ltd.); DNA concentrator (Speed Vac Plus SC, USA); ultrapure water meter (Nanjing Yi Puyi to technology development Co.); portable pressure steam sterilizing pot (Shanghai Boqing Ultrafiltration medical equipment factory); thermostatic water bath (Beijing Long wind instruments & meters; DK-600, shanghai Jing Hongjingzhu laboratory equipment Co., ltd.); pipettes (Eppendorf, germany); common refrigerator (Qingdao sea company); ultra low temperature refrigerator (SANYO corporation, japan); vacuum freeze-dryer (FDU-2100,EYELA,TOKYO RIKAKIKAI CO, LTD, japan); vertical constant temperature oscillator (IS-RDV 1, south kyo chang instrument equipment, liability company); nucleic acid protein quantitative analyzer (One Drop) ^TM OD-1000+, gene group, shanghai Instrument waves biological instruments Co., ltd.); fluorescent quantitative PCR instrument (ABI StepOne) ^TM Plus)。

1.3 preparation of Main reagents and solutions

Restriction enzymes Not I and Sal I (Takara Shuzo Co., ltd., takara) and a plasmid endotoxin-free extraction kit (OMEGA, USA); DNA gel purification recovery kit (OMEGA, USA); FSH (Ningbo Sansheng pharmaceutical Co., ltd.); HCG (department of reproduction in the northhospital, jiangsu province); liu Mianning injection (Jilin Hua animal health products Co., ltd.); m2, M16 medium (Sigma, usa); normal saline (Sichuan Korea pharmaceutical Co., ltd.); absolute ethanol (Shanghai Su chemical company, inc.); phenol, proteinase K (Beijing Soy Bao technology Co., ltd.); trichloromethane (national pharmaceutical group chemical agents limited); PCR detection primers were synthesized by Shanghai Biotechnology Co., ltd, other solid reagents involved were purchased from Sigma, USA; the relevant solution formulations are shown in table 5.

Table 5 relevant reagent formulation table

Table 2-1 The preparation table of related reagents

2. Method of

2.1 preparation of Gene fragments for microinjection

The transgenic fragment of about 17.74kb in size (as in FIG. 6) was recovered by the OMEGA gel recovery kit by double digestion of the pCL25/DSPA vector with Sal1 and Not1 (digestion system as in Table 6 below), and diluted to 2.5ng/uL with TE for microinjection.

2.2 superovulation and synchronous estrus treatment of donor and recipient rabbits

2-3 female rabbits which are not in oestrus and are 5-12 months old are selected as donors in each group of operations, and the selected female rabbits are characterized in that the female parts of the female rabbits are not swollen, and the color of the female rabbits is white and is more close to the tragic white. Selected donors were marked with a Marker pen by writing a cage number in the ear. Donor rabbits were subjected to intramuscular injection of FSH and marginal intravenous injection of HCG using a fsh+hcg combined injection protocol, wherein each donor female rabbit had a total FSH injection of 60IU and a total HCG injection of 100IU.

Day 6 of the last FSH: 30pm, 4-5 receptors are selected for each group of operations, and the selected subjects are characterized in that female rabbits have swelling and flushing of the pudendum, obvious wettability and special smell, and often have climbing phenomenon when placed on the ground together with other female rabbits. The selected recipient was marked with a Marker pen by writing a cage number in the ear. The acceptor female rabbits and the donor female rabbits are simultaneously injected with 100IU of HCG (hydrogen chloride) at the ear margin, the donor female rabbits are prepared for mating and breeding, the acceptor female rabbits are put back into the cage according to the marks, and the trough is taken down for starvation treatment.

2.3 collecting fertilized eggs

The fertilized eggs are recovered from the umbrella part of the conventional oviduct after hCG is injected into a donor rabbit for 18-20 hours, picked up under a stereoscopic microscope, counted and transferred into an M16 culture solution, and placed into a culture box with the temperature of 38 ℃ and the humidity of 5% CO2 for culture.

2.4 microinjection of fertilized eggs from rabbits

The console was preheated in advance, the fertilized eggs were transferred into M2 drops of autoclaved siliconized glass, and a hold needle and an injection needle were prepared simultaneously, wherein the outer diameter of the hold needle was about 80 μm and the inner diameter was about 25 μm, and the injection needle was prepared according to the setting procedure of the needle puller. Taking out the split-packed DSPA injection fragments, centrifuging for 2 minutes at 12000r/min, transferring into an injection needle, and starting injection after each item is ready, wherein the injection part is cell nucleus (male procaryote) as shown in figure 7; after microinjection is finished, the reconstructed eggs are washed by M16 for 8-10 times, transferred into a new M16 square cup, placed in a constant temperature incubator for culturing for 30min, and prepared for embryo transfer operation.

2.5 embryo transfer

Conventional embryo transplantation requires observing the survival rate of microinjected eggs before transplantation, and removing dead eggs and eggs with bad conditions. Oviduct and ovary, observing whether the ovary has ovulation point or follicular, and transplanting. The embryo is transferred into the oviduct, and fertilized eggs are injected into the ampulla of the oviduct. In general, the female rabbits can be naturally born after pregnant for about 30 days, and the female rabbits are regularly nursing after delivery and suckling.

2.6 extraction of genomic DNA from birth Rabbit

Phenol-chloroform extraction:

(1) The next day after birth of the young rabbits, numbering the young rabbits, shearing a small tail tissue by using sterilized ophthalmic scissors, putting the small tail tissue into a 2mL sterilizing tube for shearing, adding 750 μl of tissue lysate and 7.5 μl of proteinase k into each tube, sealing by using a sealing film, and putting the small tail tissue into a hybridization furnace at 55 ℃ for rotary digestion until the tissue is digested to be transparent and clear;

(2) Taking out digested tissue fluid, tearing off a sealing film, adding 750 mu l of phenol into a tube, reversing, gently mixing, centrifuging for 8min at 12000r/min, and taking the upper clear mucus layer into a 2ml sterilizing tube;

(3) Adding 750 μl of 1:1 phenol-chloroform mixture into the tube, mixing gently, centrifuging for 8min at 12000r/min, and collecting the supernatant clear mucus in 2mL sterilizing tube;

(4) Adding 750 μl chloroform into the tube, mixing gently, centrifuging for 8min at 12000r/min, and collecting the supernatant clear mucus in a 2mL sterilizing tube;

(5) 750 μl of pre-chilled isopropanol is added into the tube, and the mixture is inverted and mixed for many times to precipitate DNA;

(6) Taking clean sterilization finger tubes, adding 1ml of 70% ethanol into each tube, picking white DNA clusters by using a sterilization gun head, placing the white DNA clusters into the tubes, centrifuging for 3min at 12000r/min, removing the supernatant, and repeatedly washing for one time;

(7) Removing supernatant, centrifuging at 12000r/min for 1min, removing residual liquid with gun head, air drying at room temperature without alcohol smell, adding sterilized double distilled water according to size of DNA block, dissolving at 37deg.C for 2 hr or 4 deg.C overnight, and preserving at-20deg.C.

2.7 PCR integration detection

The primer is designed by taking a mammary gland specific expression vector pCL25/DSPA as a template, whether the birth rabbit is transferred into an exogenous gene or not is detected by a PCR method, and if a target band appears in an electrophoresis result, the PCR product is sent to a sequencing step to further verify the detection result. (1) primer design and synthesis: a pair of primers was designed using Primer5.0 software with the construction vector pCL25/DSPA as template, the upstream primer on CMV and the downstream primer on DSPA coding region. The size of the amplified product of the primer is 722bp, and the amplified product is synthesized by Shanghai Biotechnology Co., ltd. The primers are shown in Table 6:

TABLE 6 primer information

Table6 The information of Prime

(2) Exogenous gene integration detection is carried out on the genomic DNA of the newborn rabbits by a PCR method, and sterilized double distilled water is respectively set as a blank control, normal genomic DNA of the newborn rabbits is set as a negative control, and microinjection fragments are set as positive controls. The PCR reaction system is shown in Table 7:

TABLE 7 PCR reaction System

Table 7 The reaction System of PCR

The PCR amplification conditions were: pre-denaturation at 94℃for 5min; denaturation at 94℃for 30s, annealing at 55℃for 30s, elongation at 72℃for 50s, and repeating for 33 cycles; incubating at 72 ℃ for 7min; preserving at 4 ℃.

(3) Detecting the PCR product by 1% agarose gel electrophoresis, dyeing for 2min by using EB after electrophoresis is finished, decolorizing for 10min, placing the obtained product in a gel imager for observing the result, if the genomic DNA of the newborn rabbit can be amplified into a band with the size of about 722bp by PCR reaction, initially proving that the newborn rabbit integrates the exogenous gene CMV+DSPA gene, and sending the PCR product to sequencing to eliminate the non-integrated newborn rabbit.

(4) Performing key culture on the DSPA gene obtained through PCR and sequence analysis, marking the earmarks after weaning, numbering, and feeding for maturation for later experiments.

3. Technical Effect determination and DSPA expression detection

3.1 exogenous Gene integration detection in birth Rabbit

The obtained DSPA transgenic rabbits are sampled, integrated and detected after numbering. The results are shown in FIG. 7, wherein D4, D7, D9 show integration bands, D13 is positive control M: DL-2000Marker.

3.2 detection of DSPA and Activity in transgenic Rabbit milk

3.2.1 ELISA detection of transgenic Rabbit whey

(1) Coating: 100. Mu.L of whey and 100. Mu.L of coating solution were added to each well of the ELISA strip overnight at 4 ℃; at the same time, alteplase was added as positive control, normal rabbit whey as negative control, and PBS as blank control.

(2) Washing: the coating was discarded, washed 3 times with PBS-T for 5min each, and then patted dry.

(3) Closing: 200. Mu.L of blocking solution was added to each well, and the mixture was subjected to a water bath at 37℃for 2 hours.

(4) Washing: the blocking solution was discarded, washed 3 times with PBS-T for 5min each, and then patted dry.

(5) Adding an antibody: 100. Mu.L of antibody diluted in a 1:2000 ratio with antibody dilution was added to each well, and the mixture was subjected to a 37℃water bath for 2 hours.

(6) Washing: the primary antibody was discarded, washed 3 times with PBS-T for 5min each, and then patted dry.

(7) Adding enzyme-labeled secondary antibodies: 100 μl of goat anti-mouse IgG-HRP diluted 1:2000 with antibody dilution was added to each well, and the mixture was incubated in a 37℃water bath for 2h.

(8) Washing: the enzyme-labeled secondary antibody solution is discarded, washed for 3 times by PBS-T for 5min each time, and then is beaten to dryness.

(9) Color development: 50. Mu.L of a color development solution was added to each well, and incubated at 37℃for 20min in the absence of light.

(10) And (3) terminating: 50 mu L of 2M H are added to each well ₂ SO ₄ The reaction was stopped by the stop solution.

(11) And (3) result judgment: eye-observation color change and simultaneously measuring OD by using enzyme-labeled instrument ₄₅₀ And (5) taking a picture and recording.

Results referring to fig. 8, all milk samples were diluted 5-fold with PBS. A1-A8: positive standards (2000, 1000, 500, 250, 125, 62.5, 31.25,0 μg/mL); B1-B8, C1-C6 are respectively D20, A1, A2, A3, C1, C12, C13, C15, C16, C17, C20, C22, C26 and C28 transgenic rabbit milk; C7-C8 wild rabbit milk.

3.2.2 Western Blot detection of transgenic Rabbit whey

(1) The vertical electrophoresis tank is correctly installed.

(2) 12% of separation gel was prepared: taking a 10mL centrifuge tube sterilized in advance, sequentially adding 3.5mL sterilized redistilled water, 4mLA solution, 2.5mL B solution, 50 μL 10% (NH 4) ₂ S ₂ O ₈ And 10 μl TEMED; after being quickly and fully and uniformly mixed, the mixture is added into a glass plate with a gap of 1.5mm until the mixture is stopped at a position which is about 1cm away from the lower edge of the comb teeth to be inserted; 1mL of sterilized redistilled water is added to isolate the mixture from air and play a role in leveling the rubber surface. Standing at room temperature, removing the redistilled water after solidification, and sucking the residual water.

(3) 4% of concentrated gum was prepared: taking a 10mL centrifuge tube sterilized in advance, sequentially adding 2.3mL sterilized redistilled water, 0.67mL A solution, 1.0mL C solution, 30 μL 10% (NH 4) ₂ S ₂ O ₈ And 5 μl TEMED; adding the mixture to 12% of separating gel after rapid and full mixing until the upper edge of the glass plate stops; slightly inserting comb teeth, and standing at room temperature After the comb is solidified, the comb teeth are gently pulled off.

(4) Boiling and adding: mixing 30 μl sample and 6 μl 6×electrophoresis loading Buffer, and boiling in boiling water for 7-8min to allow protein in the mixture to be denatured; taking out, centrifuging with a small centrifuge for 5min, removing impurities, sequentially adding samples into gel holes, and adding 30-40 μl of sample into each hole, wherein the sample adding amount is generally 5-50 μg; wherein normal rabbit whey is used as a negative control group, alteplase is used as a positive control group, and a proper protein Marker is added as a reference standard.

(5) Electrophoresis: and (3) carrying out electrophoresis by using two voltages, namely firstly, finishing the migration of the bromophenol blue dye to the tail end of the concentrated gel under the constant voltage of 70V, and then finishing the migration of the bromophenol blue dye to the bottom of the separation gel under the constant voltage of 120V, and turning off the power supply.

(6) After SDS-PAGE electrophoresis, the gel was gently removed and washed 3-5 times with redistilled water.

(7) The PVDF film of 0.45 μm was cut according to the size of the gel, and immersed in absolute methanol for 45s.

(8) Soaking gel, PVDF membrane, filter paper, etc. in transfer buffer solution, and balancing for 10-20min.

(9) The placing sequence on the clip is as follows: the method comprises the steps of lightly removing bubbles between layers of a filter paper pad, filter paper, a PVDF film, gel, filter paper and a filter paper pad, closing a clamp (particularly important is that bubbles between the PVDF film and the gel are removed and marked), enabling the gel to be close to a cathode side, enabling the PVDF film to be close to an anode side, ensuring that the gel and the PVDF film are completely immersed in transfer printing liquid, covering a cover, and starting a power supply.

(10) And (3) transferring the PVDF film with the 300mA constant current film for 1.5-2 hours, taking out the PVDF film, observing whether a clear Marker exists, if so, carrying out subsequent operation, and if not or slightly, repeating the experiment after finding out the condition.

(11) Washing with sterilized redistilled water for 5-10min for 3 times. PVDF membrane was fully immersed in 10mL of blocking solution at 4℃overnight or 37℃for 2h.

(12) The membrane was removed and washed 3 times with TBS-T for 5-10min each, immersed in 10mL of Mouse-Anti-human T-PA antibody diluted in a 1:000 ratio with antibody dilution, and incubated for 1.5h in a 37℃hybridization oven.

(13) The membrane was removed and washed 3 times with TBS-T for 5-10min each. The membrane was immersed in 10mL of the coat-Anti Mouse IgG/HRP diluted 1:1000 with antibody dilution and incubated for 1.5h in a hybridization oven at 37 ℃.

(14) Taking out the membrane, washing with TBS-T for 3 times, 5-10min each time, and pre-cooling the instrument to obtain the final product.

(15) ECL color development: preparing a developing solution in a darkroom, and sucking a proper amount of A solution B solution 1:1, putting the film into a developing solution, developing for 1min, adjusting the exposure time, and photographing and recording.

3.2.3 FAPA fibrinolytic activity assay

And adopting FAPA and FAPA heating methods to perform preliminary exploration on the mechanism of dissolving fibrin in the DSPA transgenic rabbit mammary gland expression product. Wherein, if the mammary gland expression product can generate transparent dissolving ring through FAPA, the mammary gland expression product has in vitro fibrinolytic activity; if the expression product produced a transparent loop on the FAPA heated plate and the alteplase group did not produce a soluble loop, it was shown that the expression product could directly degrade fibrin.

(1) Weighing: 0.15g of agarose was weighed into a Erlenmeyer flask, 0.015g of fibrinogen was placed into a 2mL centrifuge tube, 15U of thrombin was removed at-20℃and appropriate amounts of physiological saline (about 20mL, considering heat evaporation) were added, respectively.

(2) Heating: microwave heating the triangular flask to prepare 1.0% agarose gel solution; simultaneously, fibrinogen and thrombin are put into a constant-temperature water bath kettle at 37 ℃ for dissolution and preheating.

(3) Preparing a solution: pouring the gel sugar solution into a 50mL centrifuge tube, cooling the solution to 40 ℃, sequentially adding fibrinogen and thrombin to make the final concentration of the fibrinogen and thrombin be 1mg/mL and 1U/mL respectively, gently blowing with a gun head, pouring the mixture into a colorless glass culture dish sterilized under high pressure after fully mixing, and ensuring the uniform thickness of gel during pouring.

* When fibrinogen is added to the agarose gel solution, if flocs are found to be present, this indicates a failure in preparation and need to be reconstituted.

(4) Preparing a fibrin heating plate method: based on FAPA, the preparation method is placed in a constant temperature water bath kettle with the temperature of 70 ℃ and heated for 40min, so as to inactivate the plasminogen in the flat plate.

(5) Punching and sealing the bottom: and (3) solidifying for 2-3 hours at room temperature, reasonably dispersing and punching according to the number of sample adding samples, sealing the bottom by using a heated screw after punching, and preparing sample adding by using each sample adding hole with the diameter of about 7 mm.

(6) Sample adding: respectively adding a sample group to be detected, a negative control group and a positive control group which are 50 mu L/hole; culturing in a constant temperature incubator at 37 ℃ for 12 hours; wherein, the alteplase drug is a positive control group and a normal rabbit whey negative control.

(7) And (3) result judgment: first, it is observed whether or not a fibrinolytic transparent ring is produced, and then the diameter (d) of the transparent ring is measured. If only the sample to be tested and the positive control generate transparent circles and the negative control is unchanged, the sample to be tested has fibrinolysis function, and the larger the area of the transparent circles is, the higher the thrombolytic activity is, and the record is photographed. The results are shown in fig. 9, where 1: normal male rabbit milk sample; 2: normal female rabbit milk negative control group; 3: a transgenic rabbit No. D1 milk sample; 4: a transgenic rabbit No. D2D milk sample; 5: pharmaceutical alteplase drug (concentration 20. Mu.g/mL).

EXAMPLE 3 isolation and purification of DSPA from Rabbit milk

1.1 laboratory apparatus

The main experimental instruments and equipment are shown in Table 8.

Table 8 main experimental instrument and equipment

Table 8 The main experimental instruments and equipment

1.2 Experimental reagent and consumables

The experimental reagents and consumables used in this example are shown in Table 9.

Table 9 main experimental reagents and consumables

Table 9 The main experimental material reagents and consumables

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1.3 preparation of commonly used reagents

CNBr-activated Bestarose 4FF-IgG ligand conjugate reagent related reagents are shown in Table 10:

TABLE 10 preparation of CNBr-activated Bestarose 4FF-IgG ligand coupling related reagents Table

Table 10 CNBr-activated Bestarose 4FF-IgG ligand conjugation-related reagents

Protein purification assay-related reagents were formulated as in table 11:

TABLE 11 preparation of protein purification-related Agents Table

Table 11 The preparation table of protein purification related reagents

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2.1 pretreatment of Rabbit milk

After the rDSPA transgenic rabbits were bred and delivered, the milk was collected manually and stored in a-80℃refrigerator. 50mL of transgenic rabbit milk stored in a refrigerator at-80 ℃ is quickly melted at 37 ℃ before the rabbit milk is purified, and after the transgenic rabbit milk is melted, the transgenic rabbit milk is centrifuged at 4 ℃ and 35000g/min for 30min. The milk after centrifugation is divided into three layers from top to bottom: milk fat, whey and casein precipitate (fig. 3-1). Carefully aspirate the whey for later use, this step is mainly to separate the whey and remove the casein.

3.2 precipitation of Rabbit whey proteins with ammonium sulfate

At 0 ℃, saturated ammonium sulfate with the concentration of 55% ammonium sulfate is suitable for salting out, rDSPA in rabbit whey is almost completely precipitated, and partial impurity proteins can be removed by removing supernatant.

3.3 purification of rDSPA in transgenic Rabbit milk by anisole affinity chromatography

Opening upAfter the full-automatic protein purifier and the sterile double distilled water washing apparatus, benzamidine Bestarose 4FF (20.0X12.6 cm) was attached to the purifier. The chromatographic column is equilibrated with equilibration solution (20 mmol/L phosphate buffer+0.5 mol/L NaCl, pH 7.5), the flow rate is set to 10mL/min, the upper limit of the column is 0.35MPa, and 200mL is equilibrated after the ultraviolet absorption value (UV 280) and the electric conductivity value are stable. A sample of the rabbit milk precipitated by saturated ammonium sulfate was diluted to 2L with 20m mol/L phosphate buffer (pH 7.5), mixed with a solution of 20m mol/L phosphate buffer+3 mol/L NaCl (pH 7.5) at a ratio of 5:1, loaded at a flow rate of 8mL/min, and the penetration was collected for detection. After the loading was completed, the column was rinsed with 3 column volumes of equilibration solution (20 mmol/L phosphate buffer+0.5 mol/L NaCl, pH 7.5) until the ultraviolet absorption line (UV 280) was zeroed, and 200mL was equilibrated again. Eluting with eluent (0.1 mol/L glycine, pH 3.0), collecting eluent, immediately adding alkaline neutralization solution (1 mol/L Tris-HCl, pH 9.0) into the eluent, and adjusting pH of the solution to about 7.5. The purification Peak graph is shown in FIG. 10 (wherein Peak 1: breakthrough Peak; peak 2: elution Peak). The detection and identification results of the fibrinolytic activity of the collected liquid FAPA are shown in the figure 11 (1-5: anisole affinity chromatography eluent; 6-9: anisole affinity chromatography regenerated liquid; 10: alteplase (20 mug/mL), 11: rDSPA transgenic rabbit milk; 12: anisole affinity chromatography sample loading liquid; 13: anisole affinity chromatography penetrating liquid; 14: negative rabbit whey; 15: blank; 16: PB), and the rDSPA can be extracted from rabbit whey ammonium sulfate precipitate by using anisole affinity chromatography.

3.4 Strong cation exchange chromatography purification of rDSPA in transgenic Rabbit milk

Opening upAfter the full-automatic protein purification apparatus was cleaned with sterile double distilled water, a strong cation exchange chromatography column (Capto S ImpAct column,20ml, ge medical life sciences, sweden) was connected to the purification apparatus. The chromatographic column is equilibrated with an equilibration solution (20 mmol/L glycine solution, pH 8.75), the flow rate is set at 7mL/min, the upper limit of the column is 0.35MPa, and after the ultraviolet absorption value (UV 280) and the conductivity value are stable, 90mL is equilibrated. The rabbit milk pretreatment sample (pH 8.75) and 20mmol/L glycine solution (pH 8.75) were mixed at a ratio of 1:3 and loaded at a flow rate of 3mL/min, and the penetration was collected for detection. After the loading was completed, the column was washed with 6 column volumes of equilibration solution (20 mmol/L glycine solution, pH 8.75) until the UV absorption line (UV 280) was zeroed. The elution conditions were set at 20mM glycine+0.31M sodium chloride. The strong cation exchange chromatography elution peaks are shown in FIG. 12, rDSPA is mainly in Peak 2. The activity detection is shown in figure 13 (1: strong cation exchange chromatography loading liquid; 2: strong cation exchange chromatography penetrating liquid; 3-10: strong cation exchange chromatography eluent; 11: strong cation exchange chromatography regenerating liquid; 12: negative rabbit whey; 13: alteplase (20 mug/mL; 14: PB), and the separation to obtain DSPA protein electrophoresis diagram is shown in figure 14, and the obvious improvement of rDSPA purity by applying strong cation exchange is verified. / >

3.5 purification of rDSPA in transgenic Rabbit milk by active blue affinity chromatography

After the protein purifier was started up and washed with sterile ultra-pure water, blue Sepharose 6Fast Flow (5.0X1.2 cm, GE medical life sciences, sweden) was connected to the purifier. Equilibrated with 20mmol/L phosphate buffer (pH 7.5) at a flow rate of 3mL/min, and equilibrated again at 30mL after the UV absorbance (UV 280) and conductivity values were stabilized. The pre-treated sample of rabbit milk was diluted to 500mL with 20mmol/L phosphate buffer (pH 7.5), loaded at a flow rate of 3mL/min, and the permeate was collected for subsequent testing. And after the sample loading is finished, continuing to rinse the sample by using a balancing solution with the volume of 3 times of the column until the ultraviolet absorption (UV 280) returns to zero and the conductivity value is stable. 20mM PB+0.15M sodium chloride, discarded; 20mM PB+0.4M sodium chloride, recoverable multiplexing; 20mM PB+1.0M sodium chloride, collecting, and desalting to obtain pure product. The purification peak diagram is shown in FIG. 15. The collected sample solutions of the tubes are used for FAPA fibrinolysis activity detection, and obvious transparent fibrinolysis rings are found in the active blue affinity chromatography eluent (see figure 16, wherein 1 is active blue affinity chromatography sample loading liquid, 2 is active blue affinity chromatography penetrating liquid, 3 is active blue affinity chromatography eluting peak 1 eluent, 4 is active blue affinity chromatography eluting peak 3 eluent, 5,6 is active blue affinity chromatography eluting peak 4 eluent, 7,8 is active blue affinity chromatography eluting peak 5 eluent, 9,10 is active blue affinity chromatography regenerating liquid, 11 is negative rabbit whey, 12:PB and 13 is alteplase (20 mug/mL), and rDSPA can be purified by the active blue affinity chromatography column.

3.6 purification of the product to remove endotoxins

Opening upAfter the full-automatic protein purification apparatus was cleaned with sterile double distilled water, an anion exchange chromatography column (HiTrap Capto Q column,1ml, ge medical life sciences, sweden) was connected to the purification apparatus. The chromatographic column is equilibrated with an equilibration solution (20 mmol/L Tris-HCl, pH 8.0), the flow rate is set to 1mL/min, the upper limit of the column pressure is 0.35MPa, and the volume of the column is equilibrated for 3-5 times after the ultraviolet absorption value (UV 280) and the electric conductivity value are stable. The desalted sample of 3.5 was loaded at a flow rate of 1mL/min. The penetration was collected in endotoxin-free tubes. After the loading was completed, the column was washed with equilibration solution (20 mmol/L Tris-HCl, pH 8.0) until the UV absorption line (UV 280) was zeroed. Then eluted with an eluent (20 mmol/L Tris-HCl+1mol/L NaCl, pH 8.0). The peak diagram is shown in fig. 17.

The endotoxin detection results of the purified product are shown in FIG. 18, A: DSPA after endotoxin removal; b: negative control (endotoxin free water); c: positive control (endotoxin); both the purified sample and the negative control were in a flowable liquid state with no gel solids formed, while the positive control was in a non-flowable gel solid state. Thereby determining that the endotoxin content in the purified sample after removal of endotoxin is less than 0.015EU/mL.

It is apparent that the above examples are merely illustrative of the technical solution and are not limiting of the embodiments of the present invention. Other variations or modifications of the above teachings will be apparent to those of ordinary skill in the art. It is not necessary here nor is it exhaustive of all embodiments. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention should be included in the scope of protection claimed by the present invention.

Sequence listing

<110> university of Yangzhou

<120> coding gene for rabbit mammary gland specific expression deaminase and application thereof

<160> 4

<170> SIPOSequenceListing 1.0

<210> 1

<211> 1398

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 1

ctcgaggcag ccatgaagtg cctcctgctt gccctgggcc tggccctcgc ctgtggcatc 60

caggccgcct acggtgtggc ttgcaaggac gaaattaccc agatgacata ccggcgacaa 120

gagtcgtggc tgcgccccga ggtcagaagc aagcgggtgg aacactgcca gtgcgataga 180

ggccaggccc ggtgccacac cgtgcccgtc aacagttgca gtgaaccaag gtgcttcaat 240

ggggggacat gctggcaggc tgtatatttc tcagactttg tctgtcagtg ccctgcagga 300

tatacgggga aacggtgtga agtagatacc cgtgccacct gctatgaggg ccagggtgtc 360

acctacaggg gcacatggag cacagcagaa agtagggttg agtgtatcaa ctggaacagc 420

agccttctga cccggaggac ctacaatggg cggatgccag atgccttcaa cctgggcctt 480

gggaatcaca attactgcag aaacccaaat ggcgccccaa aaccttggtg ctatgtcatc 540

aaggcaggga agttcacctc ggagtcctgt agcgtgcctg tctgctccaa ggccacctgt 600

ggcctgagaa agtacaagga gccacagctt cacagtacag gcggcctctt cacagacatc 660

acctctcatc catggcaggc tgccatcttt gcccagaaca gaaggtcatc aggcgaaagg 720

ttcctgtgtg gggggatact gatcagttcc tgctgggtcc tgactgctgc ccactgcttc 780

caggagagct atcttcctga ccagcttaag gtggttttgg gcaggacata ccgggtgaaa 840

cctggagagg aagagcagac atttaaagtc aaaaaataca tcgtccataa ggaatttgat 900

gacgacactt acaacaatga cattgcactg ctgcagctga aatcggactc accacagtgt 960

gcccaagaga gtgacagtgt ccgcgccatt tgtctccctg aagccaacct gcagctgccc 1020

gactggacag aatgtgagct gtctggctac ggcaagcata agtcatcttc tcctttctat 1080

tctgagcagc tgaaggaagg gcatgtcagg ctgtacccct ccagccgctg cgcacccaag 1140

tttctgttta acaaaaccgt cacaaacaac atgctgtgtg ctggagacac ccggagcgga 1200

gagatctatc caaatgtgca cgatgcctgc cagggtgact caggaggccc cttggtgtgt 1260

atgaatgaca accacatgac tttgcttggc atcatcagtt ggggtgttgg ctgtggggag 1320

aaagacgttc caggtgtata caccaaggtt actaattacc taggctggat tcgagacaac 1380

atgcacctgt gactcgag 1398

<210> 2

<211> 459

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 2

Met Lys Cys Leu Leu Leu Ala Leu Gly Leu Ala Leu Ala Cys Gly Ile

1 5 10 15

Gln Ala Ala Tyr Gly Val Ala Cys Lys Asp Glu Ile Thr Gln Met Thr

20 25 30

Tyr Arg Arg Gln Glu Ser Trp Leu Arg Pro Glu Val Arg Ser Lys Arg

35 40 45

Val Glu His Cys Gln Cys Asp Arg Gly Gln Ala Arg Cys His Thr Val

50 55 60

Pro Val Asn Ser Cys Ser Glu Pro Arg Cys Phe Asn Gly Gly Thr Cys

65 70 75 80

Trp Gln Ala Val Tyr Phe Ser Asp Phe Val Cys Gln Cys Pro Ala Gly

85 90 95

Tyr Thr Gly Lys Arg Cys Glu Val Asp Thr Arg Ala Thr Cys Tyr Glu

100 105 110

Gly Gln Gly Val Thr Tyr Arg Gly Thr Trp Ser Thr Ala Glu Ser Arg

115 120 125

Val Glu Cys Ile Asn Trp Asn Ser Ser Leu Leu Thr Arg Arg Thr Tyr

130 135 140

Asn Gly Arg Met Pro Asp Ala Phe Asn Leu Gly Leu Gly Asn His Asn

145 150 155 160

Tyr Cys Arg Asn Pro Asn Gly Ala Pro Lys Pro Trp Cys Tyr Val Ile

165 170 175

Lys Ala Gly Lys Phe Thr Ser Glu Ser Cys Ser Val Pro Val Cys Ser

180 185 190

Lys Ala Thr Cys Gly Leu Arg Lys Tyr Lys Glu Pro Gln Leu His Ser

195 200 205

Thr Gly Gly Leu Phe Thr Asp Ile Thr Ser His Pro Trp Gln Ala Ala

210 215 220

Ile Phe Ala Gln Asn Arg Arg Ser Ser Gly Glu Arg Phe Leu Cys Gly

225 230 235 240

Gly Ile Leu Ile Ser Ser Cys Trp Val Leu Thr Ala Ala His Cys Phe

245 250 255

Gln Glu Ser Tyr Leu Pro Asp Gln Leu Lys Val Val Leu Gly Arg Thr

260 265 270

Tyr Arg Val Lys Pro Gly Glu Glu Glu Gln Thr Phe Lys Val Lys Lys

275 280 285

Tyr Ile Val His Lys Glu Phe Asp Asp Asp Thr Tyr Asn Asn Asp Ile

290 295 300

Ala Leu Leu Gln Leu Lys Ser Asp Ser Pro Gln Cys Ala Gln Glu Ser

305 310 315 320

Asp Ser Val Arg Ala Ile Cys Leu Pro Glu Ala Asn Leu Gln Leu Pro

325 330 335

Asp Trp Thr Glu Cys Glu Leu Ser Gly Tyr Gly Lys His Lys Ser Ser

340 345 350

Ser Pro Phe Tyr Ser Glu Gln Leu Lys Glu Gly His Val Arg Leu Tyr

355 360 365

Pro Ser Ser Arg Cys Ala Pro Lys Phe Leu Phe Asn Lys Thr Val Thr

370 375 380

Asn Asn Met Leu Cys Ala Gly Asp Thr Arg Ser Gly Glu Ile Tyr Pro

385 390 395 400

Asn Val His Asp Ala Cys Gln Gly Asp Ser Gly Gly Pro Leu Val Cys

405 410 415

Met Asn Asp Asn His Met Thr Leu Leu Gly Ile Ile Ser Trp Gly Val

420 425 430

Gly Cys Gly Glu Lys Asp Val Pro Gly Val Tyr Thr Lys Val Thr Asn

435 440 445

Tyr Leu Gly Trp Ile Arg Asp Asn Met His Leu

450 455

<210> 3

<211> 19

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 3

tcccatagta acgccaata 19

<210> 4

<211> 18

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 4

agggcactga cagacaaa 18

Claims

1. The coding gene suitable for rabbit mammary gland specific expression deaminase is characterized in that the nucleotide sequence of the gene is shown as SEQ ID NO. 1.

2. A desmopressin encoded by the gene of claim 1, wherein the amino acid sequence of the desmopressin is shown in SEQ ID No. 2.

3. Use of the coding gene of claim 1 for the preparation of desmopressin.

4. Use according to claim 3, characterized in that the desmopressin is prepared by means of a rabbit mammary gland bioreactor.

5. The use according to claim 4, wherein the method of preparation is as follows:

(1) Constructing a mammary gland specific expression deaminase gene vector;

(2) Preparing a mammary gland specific expression deaminase gene rabbit;

(3) Identifying deaminase in rabbit milk and analyzing fibrinolytic activity, and collecting the milk after rabbit hybridization and delivery;

(4) The deaminase recombinant protein in the milk is extracted and purified.

6. A method for extracting and purifying a deaminase recombinant protein from rabbit milk, wherein the rabbit is a rabbit for expressing the deaminase gene of claim 1 specifically on the mammary gland, and the method is characterized in that anisole affinity chromatography, cation exchange chromatography and active blue affinity chromatography are combined for separation and purification.

7. The method for extracting and purifying a desmopressin recombinant protein as claimed in claim 6, wherein the method comprises the steps of:

(1) Preliminary separation of deaminase in the rabbit milk by ultracentrifugation and 55% ammonium sulfate precipitation of the rabbit milk;

(2) Anisole affinity chromatography:

C. the leaching condition is 20mmol/L phosphate buffer solution plus 0.5 mol/L NaCl, pH7.5; the elution conditions were set to 0.1 mol/L glycine, pH3.0;

(3) Cation exchange chromatography:

A. balancing the chromatographic column with a flow rate of 5mL/min, and balancing 200 mL after the ultraviolet absorption value UV280 and the electric conductivity value are stable; the balance liquid is 50mmol/L phosphate buffer solution, and the pH value is 8.0;

B. the leaching conditions are set to 20mmol/L glycine solution, and the pH value is 8.75; the elution conditions were set to 20m mol glycine+0.31 mol sodium chloride;

(4) Lan Zhu affinity chromatography:

A. balancing the balancing solution, wherein the flow rate is 3mL/min, and balancing for 30 mL after the ultraviolet absorption value UV280 and the electric conductivity value are stable; the balance liquid is 50mmol/L phosphate buffer solution, and the pH value is 8.0;

c.20mmol/L phosphate buffer pH7.5+0.15 mol/L sodium chloride eluent is discarded; 20 The mmol/L phosphate buffer solution pH7.5+0.4mol/L sodium chloride eluent can be recycled and reused; collecting 20 mmol/L+1.0mol/L sodium chloride;

(5) Desalting to obtain deaminase pure product.

8. The method of extracting and purifying a desmopressin recombinant protein as defined in claim 7, wherein the method further comprises: the purified desmopressin is subjected to anion exchange chromatography to remove endotoxin in the purified product.