CN100445390C - A highly active, low antigenic staphylokinase derivative and its preparation method - Google Patents

Abstract

The invention belongs to the field of mutation or genetic engineering, and discloses a preparation method and application of SAK derivatives with enhanced thrombolytic activity and reduced immunogenicity. Studies have found that after the deletion of the first 10 amino acids of SAK, its thrombolytic activity can basically remain unchanged, and the segments where amino acids 35, 74, 82, 130, 135 and 136 are located have multiple B lymphocyte epitopes, so The first 10 amino acids of SAK were deleted by fragment deletion and point mutation methods to construct SAK mutants with N-terminal deletion and modification of the above sites, and a lysine was added to the C-terminus of SAK molecule to construct SAK derivatives. The thrombolytic activity of the mutant was significantly improved. Due to the shielding of some antigenic epitopes in the SAK molecule, the antigenicity of the mutants was significantly reduced. The mutant of the present invention is suitable for expression and preparation in prokaryotic cells. Because of its high thrombolytic activity and low antigenicity, it is expected to be developed into a new drug for treating cardiovascular and cerebrovascular diseases.

Description

A highly active, low antigenic staphylokinase derivative and its preparation method

technical field

The invention belongs to the field of mutation or genetic engineering.

Background technique

Cardiovascular and cerebrovascular diseases are currently the number one killer that endangers people's health, and acute myocardial infarction is the main cause of death of patients. Although there are some clinically available thrombolytic drugs, there are still many problems to be solved in terms of thrombolytic effect, specificity, side effects and drug price. Staphylokinase (Staphylokinase, SAK) is a protein produced by lysogenic Staphylococcus aureus. Its gene encodes a protein of 163 amino acids, which is processed to form a mature protein consisting of 136 amino acids with a molecular weight of about 15kDa , no disulfide bridges. SAK is an elliptical molecule whose crystal structure has been resolved. It can specifically combine with plasminogen to form a complex to activate plasminogen into plasmin, thereby specifically degrading fibrin and dissolving blood clots. Strictly speaking, staphylokinase is not an enzyme, but it can form a 1:1 stoichiometric complex with plasmin to activate other plasminogen molecules, and staphylokinase is highly selective for fibrin. Clinical studies have shown that SAK is the only new thrombolytic drug comparable to tPA in terms of thrombolytic effect and specificity. However, because it is a foreign protein, its antigenicity will cause some allergic reactions in clinical use, especially when it is used repeatedly, its antigenicity has a great impact on the curative effect.

Contents of the invention

The purpose of the present invention is to study the recombinant staphylokinase (vSAK), and discover the influence of its antigenicity on the thrombolytic effect. Therefore, the systematic analysis and elucidation of its antigenicity and its directional modification by means of in vitro recombination are the key to reduce the antigenicity of SAK, improve the curative effect and expand the scope of its clinical application.

We used bioinformatics and gene deletion mutation technology to analyze the antigen distribution characteristics of SAK molecules, and used deletion and amino acid substitution methods to modify wild-type SAK molecules to minimize the size of SAK molecules and shield SAK molecules. At the same time, modify the C-terminus of ASK to construct SAK mutants with reduced molecular weight and reduced antigenicity and greatly improved thrombolytic activity, and use gene in vitro recombination technology to construct SAK mutants that highly express SAK mutants Engineering bacteria. After molecular modification of SAK was carried out, the proportion of soluble expression decreased. Therefore, how to balance the number of modified amino acids and select those amino acids for modification is the key to the successful construction of the mutant. On the basis of achieving high-efficiency soluble expression of recombinant proteins, we use chromatography techniques such as ion exchange and molecular sieves to purify the expressed recombinant proteins, and the purity of the prepared recombinant proteins can reach more than 95%.

Advantages of the present invention:

The activity assay results showed that the thrombolytic activity of the mutant was increased by about 3 times compared with the wild type. In addition, the preliminary detection of its antigenicity by ELISA method shows that the SAK mutant we constructed has lower antigenic activity, and also provides a better safety guarantee for the clinical use of SAK. Due to its high activity and low antigenicity, it is possible to transform from a single rescue drug to a multi-purpose thrombolytic drug that combines first aid, recovery and preventive treatment, and its application range is greatly expanded. The research of this new drug must be There will be huge economic benefits.

The SAK derivative sequence constructed by the present invention is:

AAA GGC GAT GAC GCG AGT TAT TTT GAA CCA ACA GGC

Lys Gly Asp Asp Ala Ser Tyr Phe Glu Pro Pro Thr Gly

CCG TAT TTG ATG GTA AAT GTG ACT GGA GTT GAT GGT GCA GGA AAT GAA TTG CTA TCC CCT CGT TAT

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

GTC GAG TTT CCT ATT AAA CCT GGG ACT ACA CTT ACA AAA GAA AAA ATT GAA TAC TAT GTC GAA TGG GCA

Val Glu Phe Pro Ile Lys Pro Gly Thr Thr Leu Thr Lys Glu Lys Ile Glu Tyr Tyr Val Glu Trp Ala

TTA GAT GCG ACA GCA TAT CAA GAG TTT AGA GTA GTT GAA TTA GCG CCA AGC GCA AAG ATC GAA GTC ACT

Leu Asp Ala Thr Ala Tyr Gln Glu Phe Arg Val Val Glu Leu Ala Pro Ser Ala Lys Ile Glu Val Thr

TAT TAT GAT AAG AAT AAG AAA AAA GAA GAA ACG AAG TCT TTC CCT ATA ACA GAA AAA GGT TTT GTT GTC

Tyr Tyr Asp Lys Asn Lys Lys Lys Glu Glu Thr Lys Ser Phe Pro Ile Thr Glu Lys Gly Phe Val Val

CCA GAT TTA TCA GAG CAT ATT AAA AAC CCT GGA TTC AAC TTA ATT ACA GCG GTT GTT ATA GAA CGT GCG

Pro Asp Leu Ser Glu His Ile Lys Asn Pro Gly Phe Asn Leu Ile Thr Ala Val Val Ile Glu Arg Ala

\\\

Lys

Note: The first 10 amino acids of SAK are missing; 35-position lysine lys AAA is changed to alanine Ala GCA; 74-position lysine lys AAA is changed to glutamine Gln CAA; 82-position aspartic acid Asp GAT is changed Alanine Ala GCG; lysine 130 Lys AAG to alanine Ala GCG; 135 lysine Lys AAG to arginine ArgCGT; 136 lysine Lys AAA to alanine Ala GCG ; Lysine Lys AAA was inserted at position 137.

The preparation method of SAK derivative body:

1. Experimental materials and equipment

1.1 Strains: Staphlococcus aureus (Staphlococcus aureus 1.1476) was purchased from the National Culture Collection Center, and E.coli DH5, BL21, etc. were preserved strains by the Institute of Basic Medical Sciences, Academy of Military Medical Sciences.

1.2 Plasmid: T vector was purchased from Promega Company in the United States. The pBV220 expression vector is 3.66kb in size. It is a temperature-sensitive high-efficiency expression vector, which contains a P _R P _L tandem promoter, cIts857 temperature-sensitive regulatory gene and P _R P _L It exists so that the vector can transform any recipient bacteria. The SD sequence is followed by multiple cloning sites, which provides convenience for the operation of the gene and is preserved for the Institute of Basic Medical Sciences, Academy of Military Medical Sciences. pT-SAK-M is preserved by the Institute of Basic Medical Sciences, Academy of Military Medical Sciences.

1.3 Enzymes and biochemical reagents: BamH I, EcoR I, Taq enzyme and T4 DNA ligase, small and large plasmid kits, DNA and protein molecular weight standards were purchased from Promega, USA.

1.4 Experimental equipment: The PCR instrument is a product of PE Company in the United States. Electrophoresis apparatus and chromatography apparatus are products of BIO-RAD Company. The ultra-clean workbench and shaker are domestically produced.

2. Preparation process

2.1 Construction and identification of SAK mutants

We used the N-terminal deletion of 6 amino acid SAK mutants as a template, and removed the first 10 amino acids in the SAK molecule by PCR method to reduce the size of the SAK derivative molecule. In addition, some B lymphocyte epitope segments are modified, mainly including: amino acids 35, 74, 82, 130, 135 and 136, and on this basis, a lysine is added after the modified amino acid 136 (137). The nucleotide sequences at these sites were changed by synthetic primer sequences, and then the expected mutant genes were obtained by PCR and annealing reaction (Fig. 1).

The constructed mutants were identified by double enzyme digestion and PCR (Fig. 2). Then carry out nucleic acid sequence analysis (Fig. 3)

2.2 Construction and identification of SAK mutant expression vector

The expression vector pBV220 and the SAK derivative fragment spliced by PCR were cut with EcoRI and BamHI, ligated with T4 DNA ligase, transformed into host bacteria, and positive clones were screened by enzyme digestion. Construction of expression vector pBV220-SAK-M1

2.3 Expression of recombinant SAK mutant protein

The cloning strain containing the recombinant plasmid was inoculated in LB medium containing ampicillin (Ap ⁺ ), cultured with shaking at 30°C overnight, and then inoculated in LB medium at a concentration of 5% the next day, and cultured with shaking at 30°C for 2 hours to When the OD ₆₀₀ is between 0.4 and 0.6, quickly move the culture to a water bath at 42°C, culture with shaking, and induce expression for 5 hours. At the same time, use the BL21 strain containing an empty vector as a control. After the induction, centrifuge at 12,000 rpm for 20 seconds, collect To induce expression, add 80 μl TE buffer to suspend the cells, then add an equal volume of 2X sample loading buffer to mix, boil at 100°C for 5 minutes, and then perform SDS-PAGE detection. The results showed that the engineered bacteria we constructed could express target protein accounting for 56% of the total protein of the bacteria after being induced.

2.4 Purification of recombinant SAK mutant protein

The induced expression bacteria were washed twice with PB buffer (50mM PB pH7.6, 2mM EDTA), and then suspended with 2-5 times the volume of PBE buffer, placed in an ice bath, and the bacteria were broken by an ultrasonic breaker. After the crushing is completed, pour the cell crushing solution into a centrifuge tube, centrifuge at 12000 rpm for 20 minutes at 4°C, and take the supernatant for later use. Using the principle of ion-exchange negative adsorption, the crushed supernatant was passed through an anion-exchange column (Q Sephrose FF) to allow the rSAK derivative protein to pass through, the bacterial protein was adsorbed on the column, and the passing liquid was collected. After purification, a part of impurity proteins are removed, so that the purity of rSAK is improved, thereby achieving the effect of preliminary purification.

The protein solution purified by anion exchange was adjusted to pH 6.2, and then purified by cation exchange (SP Sephrose FF) to further improve the purity of rSAK derivative protein. Then, Sephacryl S-200 produced by Amersham-Phamacia was selected as the gel filtration medium, the cation exchange eluate was loaded, and the elution peaks were collected. Purity analysis was carried out by SDS-PAGE electrophoresis (figure), and the purity of the recombinant protein was >95%.

Description of drawings

Figure 1 is the result of PCR amplification of the SAK mutant gene

1. Quantum standard, 2. SAK mutant gene

Fig. 2 is the identification diagram of recombinant SAK mutant plasmid

1. Quantity standards, 2. pBV220EcoRI/BamHI digestion

3. EcoRI/BamHI digestion of recombinant plasmids, 4. PCR identification results of recombinant plasmids

Figure 3 is the nucleic acid sequence analysis map of the constructed SAK derivative

Figure 4 is the identification map of the recombinant expression vector pBV220-SAK-M

1. Quantity standards, 2. Empty vector control 3. pBV220-SAK-M1 double enzyme digestion

Figure 5 is a graph showing the expression results of foreign genes in recombinant expression vectors

1. Protein molecular weight standard 2. pBV220-SAK-M1/BL21 3. Supernatant after sonication

Figure 6 is a diagram of the purification results of recombinant SAK derivative protein

1. Protein molecular weight standard 2. Whole bacteria expressed 3. Supernatant after sonication:

4, 5. Purified rSAK derivative protein

Figure 7 is an activity assay diagram of the purified product

A2, A3, A4: Test samples, B1-5, C1-5: Dissolving circles of reference products,

D2, D3, D4: Duplicate loading of test samples A2, A3, A4

Detailed ways

Activity assay of recombinant SAK mutants

Weigh 125mg of agarose (Biorad electrophoresis grade), add 23ml of normal saline, boil to dissolve, equilibrate in a water bath at 60°C, add 14μl of thrombin (100IU/ml), 280μl of plasminogen (0.5mg/ml), add as you go Shake well, add 2.2ml of human fibrinogen (6mg/ml), shake constantly, pour it into a flat plate (diameter 8cm) immediately after cloudiness, place it horizontally in a refrigerator at 4°C for at least half an hour, and wait until it is fully solidified before use.

Dilution of standards and samples to be tested: standard dilutions are performed as follows:

Pipe No. 1 2 3 4 5

Normal saline (μl) 500 500 500 500

Reference solution (μl) 1000 500 500 500 500

Reference product activity (AU/ml) 250 125 62.5 31.25 15.625

The sample to be tested was diluted to a concentration of 2 μg/ml according to the labeled amount, ready for use.

Punching, spotting:

Punch holes (diameter 2mm) in the formed fibrin plate, spot 6 μl per hole, duplicate wells for each sample and standard, and place in a wet box (add a small amount of water in the lunch box to maintain a certain humidity) at 25°C Place it for 16h.

Result calculation and verification report:

Measure the diameter of the dissolving ring twice vertically and horizontally on the spotting plate under the black background, take the logarithm (x) of the activity of each dilution as the abscissa, and take the logarithm of the average of the diameter of the dissolving ring (the value obtained for four times) is the ordinate (y), and the results are analyzed using biostatistical software. Use the regression analysis method in the statistical software to make a standard curve, and obtain the a and b in y=a+bx and the linear regression coefficient r value, and the activity of the sample can be obtained according to the diameter of the dissolved ring of the sample.

Results: After calculation, the specific thrombolytic activity was 1.03×10 ⁵ AU/mg. Compared with the wild type (4.12×10 ⁴ AU/mg), it increased by about 3 times.

Antigenic Analysis of Recombinant SAK Mutants

The SAK polyclonal antibody was prepared by the laboratory itself, and the mice were immunized by subcutaneous injection in the peritoneal cavity, and the SAK antibody was obtained after preliminary purification. We conducted a preliminary analysis of the antigenic changes of the recombinant protein, respectively coated 96-well plates with wild-type SAK and constructed SAK derivatives, washed 3 times after blocking, added SAK mouse polyclonal antibodies, incubated at 37°C for 1 hour, and washed 5 times, add horseradish enzyme-labeled goat anti-mouse antibody, incubate for 1 hour, wash 5 times, add substrate to develop color, use 2M H ₂ SO ₄ to terminate the reaction, make 8 duplicate holes on each plate, and compare the results at 490nm OD value.

OD (SAK wild type) = 1.23 ± 0.32

OD (SAK derivative)＝0.82±0.16

It can be seen that the antigenicity of the derivatives has been reduced, and animal experiments on the dynamic changes of antibodies are in progress.

Claims (2)

1, a kind of high reactivity, low antigen glucokinase derivant is characterized in that: the aminoacid sequence of described staphylokinase redundant organism is:

Lys?Gly?Asp?Asp?Ala?Ser?Tyr?Phe?Glu?Pro?Thr?Gly

Pro?Tyr?Leu?Met?Val?Asn?Val?Thr?Gly?Val?Asp?Gly?Ala?Gly?Asn?Glu?Leu?Leu?Ser?Pro?Arg?Tyr

Val?Glu?Phe?Pro?Ile?Lys?Pro?Gly?Thr?Thr?Leu?Thr?Lys?Glu?Lys?Ile?Glu?Tyr?Tyr?Val?Glu?Trp?Ala

Leu?Asp?Ala?Thr?Ala?Tyr?Gln?Glu?Phe?Arg?Val?Val?Glu?Leu?Ala?Pro?Ser?Ala?Lys?Ile?Glu?Val?Thr

Tyr?Tyr?Asp?Lys?Asn?Lys?Lys?Lys?Glu?Glu?Thr?Lys?Ser?Phe?Pro?Ile?Thr?Glu?Lys?Gly?Phe?Val?Val

Pro?Asp?Leu?Ser?Glu?His?Ile?Lys?Asn?Pro?Gly?Phe?Asn?Leu?Ile?Thr?Ala?Val?Val?Ile?Glu?Arg?Ala

Lys。

2, the preparation method of the described staphylokinase redundant organism of a kind of claim 1 is characterized in that:

A, experiment material and equipment

A bacterial strain: streptococcus aureus Staphlococcus aureus 1.1476 is available from national DSMZ, and E.coliDH5, BL21 are that Institute of Basic Medical Sciences, A Cademy of Military Medical Sciences, preserves bacterial classification;

The b plasmid: the T carrier is available from U.S. Promega company, and pBV220 expression vector size is 3.66kb, is a kind of temperature sensitive type efficient expression vector, contains P _RP _LPlaced in-line promotor, temperature sensitive regulatory gene of cIts857 and P _RP _LExistence, make this carrier can transform any recipient bacterium, the back of SD sequence for the operation of gene is provided convenience, is that Institute of Basic Medical Sciences, A Cademy of Military Medical Sciences, preserves immediately following a plurality of cloning sites;

C enzyme and biochemical reagents: BamH I, EcoR I, Taq enzyme and T4 dna ligase, carry for a short time, big upgrading grain test kit, DNA and molecular weight of albumen standard be available from U.S. Promega company;

The d experiment equipment: the PCR instrument is a U.S. PE company product, and electrophoresis apparatus, chromatograph are BIO-RAD company product, and Bechtop, shaking table are homemade;

B, preparation process

The structure and the evaluation of a SAK mutant

With 6 amino acid SAK mutant of N end disappearance is template, by PCR method preceding 10 amino acids in the SAK molecule are got rid of, reduced SAK redundant organism bulk of molecule, in addition following bone-marrow-derived lymphocyte epi-position section is modified, mainly comprise: 35th, 74,82,130,135 and 136 amino acids, and after the 136th amino acids after the modification, add a Methionin 137 on this basis, by the nucleotide sequence in these sites of synthetic primer sequence fixed point change, obtain the mutant gene of expection then by PCR and annealing reaction;

Utilize double digestion and PCR method that the mutant that makes up is identified, carry out nucleic acid sequence analysis then;

The structure and the evaluation of b SAK mutant expression vector

The SAK redundant organism fragment of utilizing EcoRI and BamHI double digestion expression vector pBV220 and utilizing PCR to splice utilizes the T4DNA ligase enzyme to connect, and transforms host bacteria, utilizes enzyme to cut the evaluation and screening positive colony, construction of expression vector pBV220-SAK-M1;

The expression of c reorganization SAK mutant protein

The clone strain that will contain recombinant plasmid is inoculated in and contains penbritin Ap ⁺The LB substratum in, 30 ℃ of concussion overnight incubation, be inoculated in the LB substratum with 5% concentration next day again, 30 ℃ of concussions are cultivated 2 hours to OD ₆₀₀Between 0.4-0.6, culture is moved to rapidly in 42 ℃ of water-baths, concussion is cultivated, abduction delivering 5 hours, simultaneously in contrast with the BL21 bacterial strain that contains empty carrier, after inducing end, with 12000rpm centrifugal 20 seconds, collect the thalline of abduction delivering, add 80 μ l TE damping fluid suspension thalline, add isopyknic 2X load sample damping fluid mixing again, 100 ℃ are boiled processing 5 minutes, carry out SDS-PAGE then and detect, the result shows, the engineering bacteria that makes up, after inducing, the target protein of expression accounts for 56% of bacterial protein;

The purifying of d reorganization SAK mutant protein

With the abduction delivering bacterium with PB damping fluid 50mM PB pH7.6, twice of 2mM EDTA washing thalline, PBE damping fluid with 2-5 times of volume suspends again, place ice bath, utilize the broken bacterium of Ultrasonic Cell Disruptor, behind broken the end, bacterial cell disruption liquid is fallen in centrifuge tube, 12000rpm is centrifugal 20 minutes under 4 ℃ of conditions, and it is standby to get supernatant; Utilize the negative adsorption principle of ion-exchange, supernatant after the fragmentation is passed anion-exchange column Q Sephrose FF, rSAK redundant organism albumen is passed, tropina is adsorbed on the post, liquid is passed in collection, through behind the purifying, removes a part of foreign protein, the purity of rSAK is improved, thereby reaches the effect of preliminary purification;

Will be through the protein solution of anionresin purifying, transferring pH is 6.2, passes through the purifying of cationic exchange SP Sephrose FF then, and the proteic purity of rSAK redundant organism is further improved;

Then, the Sephacryl S-200 that selects Amersham-Phamacia company to produce is a gel filter medium, with sample on the cationic exchange elutriant, collects elution peak, utilizes the SDS-PAGE electrophoresis to carry out purity check, the purity of recombinant protein＞95%.

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