CN102199208B - Universal medicine delivery system and preparation method thereof - Google Patents

Abstract

The invention discloses a universal medicine delivery system and a preparation method thereof, and provides a method for upgrading a protein or polypeptide medicine. The method comprises the following step that: a vitamin H (VH) gene of a human serum albumin (HSA) antibody capable of specifically combining albumin and the gene of the protein or polypeptide medicine are subjected to fusion expression so as to form a fusion protein. The method comprises the following steps of: cloning the nucleotide sequence for coding the protein or polypeptide into a BL21/pETa(32+)-HSA gene engineering expression vector; and expressing the fusion protein containing a VH segment of an antibody generated by a hybridoma cell line with the collection number of CCTCC(China Center For Type Culture Collection)-C2010111.

Description

A kind of general drug-loading system and preparation method

Technical field

The invention belongs to biomedicine field, especially relate to the medicine-feeding technology field.Utilize drug-loading system of the present invention the protein or the polypeptide drugs that emerge existing or future at present can be upgraded, solve the shorter difficult problem of transformation period in vivo that current protein or polypeptide drugs face.Preparation method of the present invention is easy, is suitable for scale operation, is suitable for preparation all polypeptide protein class medicines at present.

Background technology

Pharmaceutical grade protein successfully is widely used in clinical treatment for a long time, and the numerous protein medicine provides effective and unique result for the treatment of for some diseases.Yet, due to the singularity of the protein structure of pharmaceutical grade protein own, people's physical efficiency is very effectively degraded and is removed the pharmaceutical grade protein entered in body.Different pharmaceutical grade proteins is due to the difference of its molecular size and structure, and their speed removed that is degraded in human body is different ^[1].Therefore, the pharmaceutical grade protein transformation period in vivo is limited by the removing degradation effect of body to this protein.Due to pharmaceutical grade protein in vivo the transformation period short, in order to reach effective result for the treatment of, often adopt high dosage or repeatedly, long-time medication, can increase its toxic side effect so on the one hand, also to patient's medication, bring great inconvenience simultaneously.

In order to extend the pharmaceutical grade protein transformation period in vivo, thereby strengthen the result for the treatment of of its medicine, people wish to delay the degraded scavenging(action) of body to pharmaceutical grade protein.At first there is the people itself to be modified the pharmaceutical grade protein molecule, thereby change identification and the proteases for decomposing effect of body protein degraded scavenge system to pharmaceutical grade protein ^[2-3].For example, in order to improve the pharmacokinetics of IFN-α, reduce medicine frequency, some drugmaker has prepared semisynthetic alpha-interferon medicine together with polyoxyethylene glycol (PEG) the molecule covalent attachment of alpha-interferon and about 12KD or 30KD, is referred to as Peg-Intron (Pegglated IFN).Although this Peg-Intron can be brought into play the antiviral biologic activity of alpha-interferon, but and the significant prolongation alpha-interferon transformation period in vivo.But this pegylation can obviously reduce the specific biological specific activity of alpha-interferon.Simultaneously, the effect of its Increased Plasma Half-life is also relevant with composition and the binding site of the peg molecule of its combination ^[4-5].

Albumin is the main protein in blood plasma, and it can be with water molecules, ion (as Ca ²⁺, Na ⁺and K ⁺) combination, also can be combined with lipid acid, hormone and medicine.Its major function maintains plasma colloid osmotic pressure exactly.Albumin in vivo the transformation period longer, simultaneously it can be combined with some drug molecules, therefore, it is a kind of desirable pharmaceutical carrier molecule.It can provide provide protection to drug molecule, avoids being disposed by body degraded too early, like this can the prolong drug transformation period in vivo.For example, the people such as Andersen merges the albumin function land of staphylococcal protein G (albumin binding domain, ABD) form a fusion rotein with the Fc acceptor.This fusion rotein can with blood in the albumin specific binding, and the FC acceptor in vivo transformation period greatly extend.By human epidermal growth factor receptor 2 (HER ₂) make together fusion rotein with ABD identical effect is also arranged ^[6].Therefore, by biological or chemical medicine prolong drug transformation period in vivo significantly together with albumin bound, thereby reach the enhancing medication effect, and the adaptedness that improves drug dose and patient's medication.

In the present invention, we utilize the characteristics of anti-human albuminous monoclonal antibody and albumin specific binding, and the specificity albumin bound functional zone (domains) of protein drug and this antibody are merged and make fusion rotein.Like this this fusion rotein specificity guiding (targetting) is combined with human albumin, thereby reaches the result for the treatment of that extends the transformation period of protein drug in human body and improve protein drug.In this invention we found a strain can stablize, efficiently, secretion specifically carries out the hybridoma cell strain (preserving number is CCTCC-C 2010111) of the antibody of combination with human albumin.We obtain its DNA encoding sequence through round pcr, then through Protocols in Molecular Biology construction expression HSA antibody V _hprokaryotic expression engineering bacteria BL21/pETa (32+)-HSA (its preserving number is CCTCC-M 2010288).We using this expression vector as the drug-loading system of albumen and polypeptide drug, at first select to carry out amalgamation and expression with the encoding sequence of α 1-Interferon, rabbit, obtain and express HSA antibody V _hwith the fusion rotein BL21/pETa (32+) of IFN-IFN-Fab (HSA), be called for short IFN-Fab (HSA).Adopt the biochemical separation purification method of our uniqueness to obtain highly purified IFN-Fab (HSA) fusion rotein from the intestinal bacteria lysate.Experiment in vitro proves that this fusion rotein has identical antivirus action with IFN-α 1.This fusion rotein is expelled to interior its transformation period of rat body than alone IFN-α 1 prolongation 6-12 doubly.IFN-α 1 injects the transformation period that can not extend IFN-α 1 together with VH antibody function district egg white mixture separately.This illustrates IFN-Fab (HSA) fusion rotein in the antiviral biologic activity with IFN-α 1 simultaneously, its transformation period significant prolongation in vivo, thus its clinical treatment effect itself has obvious advantage than IFN-α 1.

Summary of the invention

The invention provides a kind of novel universal drug-loading system and preparation method thereof.

Know-why of the present invention:

The present invention utilize a kind of can with blood plasma in the normal human in the genetic engineering antibody V of albumin bound _has carrier, produce a kind of general drug-loading system.By genetic engineering means, current existing some protein or polypeptide drugs are carried out to amalgamation and expression, for the upgrading of protein and peptide drugs, when guaranteeing medicine effect, can the prolong drug transformation period in vivo.

Technical scheme of the present invention:

On the one hand, the invention provides the antibody V for human serum albumin _hfragment, is characterized in that described fragment is the V of the antibody that produces of hybridoma cell strain that preserving number is CCTCC-C 2010111 _hfragment.Preserving number is CCTCC-C 2010111, and depositary institution is Chinese Typical Representative culture collection center, preservation address Wuhan, China university, and preservation date on November 10th, 2010, Classification And Nomenclature is hybridoma cell strain 1C1.

On the other hand, the invention provides V _hfragment, is characterized in that the aminoacid sequence of described fragment is by nucleotide sequence coded shown in SEQ ID NO:4.

Again on the one hand, the invention provides the method for upgrading protein or polypeptide drugs, it is characterized in that can with the antibody V of albumin specific binding _hcarry out amalgamation and expression with described protein or polypeptide drugs, thereby form fusion rotein, preferably, described can with the albumin specific binding be the V of the antibody that produces of the hybridoma cell strain of CCTCC-C 2010111 _hfragment, preserving number is CCTCC-C 2010111, depositary institution is Chinese Typical Representative culture collection center, preservation address Wuhan, China university, preservation date on November 10th, 2010, Classification And Nomenclature is hybridoma cell strain 1C1, more preferably, described can be with the VH fragment of albumin specific binding by nucleotide sequence coded shown in SEQ ID NO:4; Wherein said protein or polypeptide drug can be Interferon, rabbit or other.

On the other hand, the invention provides the fusion rotein obtained according to aforesaid method.

On the other hand, the invention provides the gene engineering expression carrier of expressed fusion protein, be characterised in that the V that described fusion rotein comprises AHS's albumin HSA antibody that hybridoma cell strain that preserving number is CCTCC-C 2010111 produces _hfragment, preserving number is CCTCC-C 2010111, depositary institution is Chinese Typical Representative culture collection center, preservation address Wuhan, China university, preservation date on November 10th, 2010, Classification And Nomenclature is hybridoma cell strain 1C1, described carrier is BL21/pETa (32+)-HSA gene engineering expression carrier (its preserving number is CCTCC-M 2010288), preserving number is CCTCC-M 2010288, depositary institution is Chinese Typical Representative culture collection center, preservation address Wuhan, China university, preservation date on November 10th, 2010, Classification And Nomenclature is Escherichia coli BL21/pETa (32+)-HAS.

On the other hand, the present invention also provides a kind of method of producing the fusion rotein medicine of protein or polypeptide, it is characterized in that described method comprises is cloned into the nucleotide sequence of code for said proteins or polypeptide in said gene engineering expression vector, and expressed fusion protein.

At first prepare the monoclonal antibody for human serum albumin, then by genetic engineering means, increase and obtain the V for human serum albumin antibody _hgene order, be cloned into plasmid pETa (32+) expression vector by this gene order, is converted into e. coli bl21 and expresses bacterium, by screening, obtains the energy specific secretion for people HSA antibody V _he. coli bl21/the pETa of albumen (32+)-HSA, confirm this antibody V by competition inhibition test, IP etc. _halbumen energy specificity is combined with HSA; Existing some protein or polypeptide drugs and BL21/pETa (32+)-HSA carry out amalgamation and expression at present, this amalgamation and expression albumen of purifying, carry out animal experiment, the protein of the protein of relatively transforming by this novel universal drug-loading system or polypeptide drugs and not transformation or polypeptide drugs are drug effect and transformation period length in vivo.

The preparation method carries out according to following steps:

1. the preparation specificity is for the hybridoma cell strain (its preserving number is CCTCC-C 2010111) of HSA;

2. utilize the RT-PCR technology to take HSA hybridoma cell strain RNA as the antibody V of template acquisition specificity for HSA _hgene order, be cloned into the pMD-18T carrier, after order-checking is identified, is cloned into PETa (32+) expression vector again;

3. express bacterium through being converted into e. coli bl21, by screening, obtain the energy specific secretion for people HSA antibody V _he. coli bl21/pETa (32+)-HSA (English name: Escherichia coli BL21/pETa (32+)-HSA)) gene engineering expression bacterium (its preserving number is CCTCC-M 2010288);

4. normal fermentation amplification, after IPTG induces, collect the thalline supernatant through ultrasonication.Carry out chromatography through the HIS affinity column again, obtain the energy specificity for HSA antibody V _halbumen;

5. confirm this antibody V by competition inhibition test, IP etc. _halbumen energy specificity is combined with HSA; We are by the e. coli bl21/pETa (32+) of success foundation-HSA (Escherichia coliBL21/pETa (32+)-HSA)) genetically engineered medicine carrying expression system, carry out amalgamation and expression by genetic engineering technique and current existing some protein or polypeptide drugs;

6. the protein that the experimentation on animals test is relatively transformed by this novel universal drug-loading system or the protein of polypeptide drugs and not transformation or polypeptide drugs are drug effect and transformation period length in vivo.

Concrete Technology Roadmap is referring to Fig. 1.

Main innovate point shows following two aspects:

(1) by genetic engineering means can with the anti-human albumin antibody V of albumin specific binding _hsegment, as carrier and protein and peptide medicine amalgamation and expression, does not only affect the drug effect of medicine, and its transformation period extends approximately 12 times in vivo than conventional protein/polypeptide medicine simultaneously;

(2) this carrier is a kind of universal support, can be for the upgrading of current all proteins polypeptide drug.

The accompanying drawing explanation

Fig. 1 is concrete route map of the present invention.

Fig. 2 is preservation cell strain 1C1 (preserving number: CCTCC-C 2010111) chromosome map.

Fig. 3 .Western-blot normal people Peripheral Blood through the SDS-PAGE electrophoresis, be transferred to pvdf membrane, 1%Casein sealing; Hybridoma cell strain (preserving number: the CCTCC-M 2010111) ascites that adds the HSA diluted at 1: 5000 of purifying, 37 ℃, 2 hours; The goat anti-mouse igg of the Biotion mark of dilution in 1: 1000,37 ℃, 1 hour; 1: 1000 the dilution Streptavidin37 ℃, 1 hour; The DAB colour developing.

Fig. 4 is RT-PCR figure, and a band appears in visible 368bp place.

Fig. 5 and Fig. 6 are respectively sequence and the order-checking peak figure that the order-checking step described in embodiment 2 obtains.

Fig. 7 be purifying pMD-18T for vector plasmid SacI, HindIII (selecting M buffer) carry out double digestion, connect into pET32a (+) after Purified in electrophoresis, in being converted into bacillus coli DH 5 alpha, extracting plasmid and carry out agarose electrophoresis figure.

Fig. 8 is the Insert Fragment sequence chart of inserting pETa (32+) expression vector.

Fig. 9 is the abduction delivering figure (SDS-PAGE) of BL21/pETa (32+)-HSA genetically engineered medicine carrying expression system albumen.Swimming lane from left to right is respectively: Marker, 0.1mol/L supernatant, 0.1mol/L precipitation, 0.2mol/L supernatant, 0.2mol/L precipitation, 0.5mol/L supernatant, 0.5mol/L precipitation, 1mol/L supernatant, 1mol/L precipitation, do not induce supernatant liquor and induced precipitation not.

Figure 10 is HIS affinitive layer purification schematic diagram.

Figure 11 is that BL21/pETa (32+)-HSA genetically engineered medicine carrying expression system albumen-specificity is for the SDS-PAGE electrophorogram before and after HSA antibody VH purifying protein purifying.Before swimming lane from left to right is respectively Marker, purifying, after purifying.

Figure 12. the Western-bolt figure of immunoprecipitation test.Swimming lane from left to right is respectively Marker, GST-HSA-V _h, GST.Visible GST-HSA-V in figure _hthe colour developing band of the about 6800da of a part amount appears in swimming lane; The GST swimming lane does not occur.

Figure 13 is HSA antibody V _hthe expressing protein competition suppresses graphic representation.

Figure 14 is the DNA sequence dna that inserts the IFNA1 of fusion expression vector.

Figure 15 is amalgamation and expression, the Purification and Characterization result of BL21/pETa (32+)-IFN-Fab (HSA) protein and peptide class medicine, the about 57kd of expressing protein molecular weight.Before swimming lane from left to right is respectively Marker, purifying, after purifying.

Figure 16 is HSA-IFN fusion rotein pharmacokinetics comparison diagram.

Embodiment

Embodiment 1 is for the preparation of human serum albumin monoclonal antibody specific ^[7-9]

1) immune mouse flow process

Commercialization human serum albumin (HSA) the 1 μ l (200 μ g) that to get concentration be 20%, 0.01mol/L, pH7.2PBS 500 μ l, freund adjuvant 500 μ l, magnetic stirring apparatus fully mixes emulsification, makes into water in oil chyle shape (put dropping on the water surface to be difficult for spreading and be droplet-shaped and show to reach water in oil state).Concrete operations are as follows:

A. initial immunity: after getting the emulsification of HSA Freund Freund's complete adjuvant, in mouse four limbs and the subcutaneous multi-point injection mouse 100 μ g/0.5ml/ in back only;

B. immunity for the second time: the two weeks post doses in interval are the same, the subcutaneous multi-point injection mouse of Freund Freunds incomplete adjuvant;

C. immunity for the third time: two Zhou Houtong are immunity for the second time, again subcutaneous multi-point injection mouse.(rear blood sampling in about a week is surveyed it and is tired);

D. booster immunization: detect serum titer and be greater than 1: 10000, get 100 μ g/0.5ml and do not add the direct abdominal injection of adjuvant;

2) preparation of immune spleen cell

After booster immunization 3 days, aseptic extracting spleen cell, prepare splenocyte suspension, and concrete operations are as follows:

A. dislocation method is put to death mouse;

B. mouse is put in 75% alcohol after soaking disinfection, cuts off its belly with sterile scissors and take out spleen, be placed in the aseptic plate stainless steel sift containing a small amount of nutrient solution (RPMI-1640) online, count after grinding to form cell suspension with the syringe nook closing member;

C. collect splenocyte suspension on ice bath, the centrifugal supernatant that goes;

D. will precipitate with fresh RPMI-1640 nutrient solution recentrifuge washing;

E. add fresh RPMI-1640 nutrient solution, the counting splenocyte.

3) myeloma cell's preparation

Be stored in-196 ℃ of liquid nitrogen containers, about one week recovery myeloma cell SP2/0 before merging, breed, go down to posterity.The take the logarithm vegetative period during fusion myeloma cell of (cultivating 15h～20h), with suction pipe piping and druming collecting cell suspension, washing counting.

4) preparation of feeder cell

Concrete operations are as follows:

A. mouse, 75% alcohol-pickled sterilization 10min are put to death in dislocation;

B. with operating scissors, mouse web portion is cut off to an osculum, strip off skin, expose abdominal cavity;

C. with syringe, 4～5ml serum-free RPMI-1640 nutrient solution is injected to abdominal cavity, by light finger, rub the massage belly, still use this syringe pumpback abdominal cavity liquid, move into centrifuge tube;

D.1000r/min centrifugal 10min, remove supernatant;

E. with the RPMI-1640 nutrient solution suspendible containing 20% calf serum (NCS), adjust cell count 4 * 10 ⁵/ ml;

F. with the 1ml suction pipe, cell is added to 96 well culture plates, every hole 100 μ l, put into 37 ℃, 5%CO ₂incubator is cultivated, what the gained feeder cell can be for cytogamy and cloning.

5) cytogamy flow process

The basic step of cytogamy be by the myeloma cell with after immune spleen cell mixes, under the effect of PEG, make two kinds of cells merge each other, the cell after merging is suitably diluted, split in the culture plate containing feeder cell and cultivate.Concrete operations are as follows:

A. the myeloma cell through anticipating is mixed in the ratio of 1: 5 with immune spleen cell, the full nutrient solution that toos many or too much for use in the 50ml plastic centrifuge tube is washed 1 time, 1200rpm/min, 8min;

B. abandon supernatant, with the dropper residual liquid that exhausts, in order to avoid affect the concentration of PEG.At the bottom of the attack centrifuge tube, make cell precipitation loosening slightly gently;

C. by 50%PEG (molecular weight 4000) 0.8ml be incubated in 37 ℃ of water-baths, slowly splash into centrifuge tube, add in 1min, shake while dripping;

D. the incomplete nutrient solution that adds preheating, added respectively 1ml every 2 minutes, 2ml, and 3ml, 4ml, 5ml and 10ml, stop the PEG effect.

E. centrifugal, 800rpm/min, 6min.

F. abandon supernatant, first with about 6ml containing the RPMI1640 substratum (Gibco, the U.S.) of 20% calf serum suspendible cell gently;

G. after merging with aseptic straw, cell suspension adds and contains 96 orifice plates of having inoculated in advance feeder cell, 100 μ l/ holes, 37 ℃ of lower 5%CO ₂incubator is cultivated.

6) HAT screening hybridoma

Generally, after merging 24 hours, add xanthoglobulin-methotrexate-thymidine (HAT, Sigma, the U.S.) and select nutrient solution.Used time adds 50ml to contain in the complete culture solution of 20% calf serum 1ml.Because splenocyte can only be survived several days under culture condition in vitro, do not affect Growth of Hybridoma Cell; And the myeloma cell is due to can't the purine biosynthesis ring when folic acid inhibitor aminopterin-induced syndrome exists and thymus pyrimidine methyl and can not synthetic DNA, simultaneously again because it is the defective type of HGPRT-and TK-, can't utilize HGPRT and TK in nutrient solution to carry out synthetic DNA, so the myeloma cell can't be survived in this nutrient solution.Last like this can in the HAT nutrient solution, survive just only had hybridoma, because it has the karyomit(e) of two kinds of parental cells, can produce the HGPRT that original splenocyte has, obtained again the ability of long-term survival and reproduction in cell culture medium from the myeloma cell.

When HAT selects the nutrient solution maintain after one week, use the HT nutrient solution instead, then maintain one week, use general nutrient solution instead.

By selectivity, cultivate in the hybrid cell line obtained, only minority can be secreted for immunogenic specific antibody.Generally at the bottom of hybridoma is covered with hole, during 1/5 area, can start the anti-HSA antibody of detection specificity, filter out the hybridoma cell line of needed hypersecretion.

7) cloning of hybridoma

Fused cell is clonal growth, and after limiting dilution, every hole adds 1 cell.Detect and filter out high antibody secretory pit according to the ELISA method, by the capable cloning again of cell in hole.The principle of cloning is should carry out as early as possible cloning for the hybridization clone who detects anti-HSA antibody positive, even the hybridoma of cloning also needs regular cloning again.Concrete operations are as follows:

A. prepare feeder cell suspension (preparing with before merging);

B. collect the counting of positive porocyte, and regulate cell count to 10 cell/ml, every hole adds cell suspension 100 μ l;

C., after cultivating 4～5 days, add complete culture solution 200 μ l/ holes;

D. 8th～9 days the time, naked eyes visible cell clone, carry out anti-HSA antibody test in time.

8) preparation of anti-HSA monoclonal antibody ascites

The anti-HSA hybridoma cell strain of the positive filtered out 1C ₁should carry out early a large amount of preparations of anti-HSA monoclonal antibody.What this experiment adopted is mice celiac inoculation, at the anti-HSA hybridoma of mouse Inoculation, and preparation ascites.Concrete operations are as follows:

A. the abdominal injection whiteruss is in the BALB/C mouse, and 0.5ml/ only;

B.1, after～2 weeks, with method, above-mentioned mouse is carried out to abdominal injection 1 * 10 ⁶individual hybridoma;

C. inoculating cell can produce ascites after 7～10 days, and now mouse web portion obviously swells, and hand touches fluctuation, with No. 16 syringe needles, gathered ascites;

D. by the 4 ℃ of centrifugal 30min of low temperature 1500r/min of ascites that collect, collect supernatant.

9) purifying of anti-HSA monoclonal antibody

The concentration of the anti-HSA antibody of ascites specificity is than the height in antiserum(antisera), and purification effect is good.What this experiment adopted is the saturated ammonium sulphate method, and concrete operations are as follows:

A. prepare saturated ammonium sulphate solution (SAS): by 767g (NH ₄) ₂sO ₄slowly be added to while stirring in 1 liter of distilled water.Be transferred to pH 7.4 with ammoniacal liquor;

B. precipitate: by physiological saline equal-volume dilution for mouse ascites, slowly the limit edged stirs and adds isopyknic SAS in supernatant liquor, and final concentration is 1: 1 (v/v); Put 4 ℃ and spend the night, protein is fully precipitated, take out next day, in 4 ℃ of frozen centrifugations, abandons supernatant, and throw out redissolves to 2 times of ascites volumes with physiological saline, and slowly the limit edged stirs the SAS that adds 1 times of volume again; So, repeatedly once; Throw out physiological saline, redissolve, and in 4 ℃ of frozen centrifugations, collects supernatant;

C. dialysis: the supernatant of getting after above-mentioned processing is transferred in dialysis tubing, with 0.01mol/L, pH7.4PBS dialysis (4 ℃), during change dialysis buffer liquid several, thoroughly to remove ammonium sulfate, Na Shi reagent detects without NH ₄ ⁺;

D. the dialyzate of collecting is centrifugal, collect supernatant liquor concentrated with PEG-20000, in the bottle of packing into, with protective material, 4 ℃ of Refrigerator stores.

10) the anti-HSA antibody of Protein-G affinitive layer purification (summary)

11) the tiring of antibody, Chromosome Identification are shown in that (Fig. 2), specificity identification be shown in (Fig. 3)

Embodiment 2 is for the antibody V of human serum albumin _hthe clone of gene order, evaluation and expression

By laboratory Protocols in Molecular Biology routine, the hybridoma cell strain RNA of AHS's albumin (HSA) of obtaining of take in embodiment 1 is template, 5 '-GT GGC GGA GGA TCC GAG GTG MAG CTK SWS GAG TC-3 ' (SEQ ID NO:1) is upstream primer, 5 '-TGA GGA GAC KGT GAS HRW GGT CCC-3 ' (SEQ ID NO:2) is downstream primer, by RT-PCR (seeing Fig. 4) amplification, obtains the V of specificity for the HSA monoclonal antibody _hgene order, be cloned into the pMD-18T carrier, identify and (see Fig. 5 or SEQ ID NO:3 through order-checking, and Fig. 6) and double digestion identify after (seeing Fig. 7), again aim sequence (see Fig. 8, or SEQ ID NO:4) is inserted between the SacI, HindIII restriction enzyme site of pETa (32+) expression vector; Be converted into bacillus coli DH 5 alpha and carry out plasmid identification, purifying, amplification; The positive plasmid that evaluation is obtained is converted in e. coli bl21 again, routine is selected etc. and finally to be set up e. coli bl21/pETa (32+)-HSA (English name: Escherichia coli BL21/pETa (32+)-HSA) express engineering bacteria, i.e. BL21/pETa (32+)-HSA genetically engineered medicine carrying expression system.

The abduction delivering of embodiment 3BL21/pETa (32+)-HSA genetically engineered medicine carrying expression system albumen

Concrete implementation step:

1) will finally set up e. coli bl21/pETa (32+)-HSA (English name: Escherichia coli BL21/pETa (32+)-HSA) express engineering bacteria, i.e. BL21/pETa (32+)-HSA genetically engineered medicine carrying expression system engineering bacteria.Routine is seeded to 5ml LB substratum, and 37 ℃, 200rpm shaking culture activation 6 hours;

2) will activate bacterium, transferred species is to the 500ml LB substratum containing 100 μ g/ml penbritins, and 37 ℃, the 200rpm shaking culture, to OD600 to 0.6, adds 0.5mmol/L IPTG, and 28 ℃, 200rpm shaking culture, abduction delivering 4 hours;

3) cultivate and finish, the collecting cell precipitation, physiological saline washing 3 times, add 10ml to contain 1 μ g/mlAprotinin, Leupeptin, 100 μ g/ml PMSF cell pyrolysis liquids;

4) ice-bath ultrasonic fragmentation, ultrasonic 5s, stop 10s, 40 times;

5) 4 ℃, 10000RPM centrifugation;

6) collect supernatant, standby;

7) identify and confirm through SDS-PAGE (seeing Fig. 9), express good.

The purifying of embodiment 4BL21/pETa (32+)-HSA genetically engineered medicine carrying expression system albumen

Get embodiment 3 expressing proteins, first use 0.02mol/L, pH 7.4～7.6PBS+0.5mol/LNaCl balance, after 0.22 μ m aperture syringe needle filter filters, utilize HIS affinity column (Chelating) to carry out purifying; Detailed process schema shown in Figure 10.Obtain the energy specificity for HSA antibody VH purifying protein, referring to Figure 11.

The checking of embodiment 5BL21/pETa (32+)-HSA genetically engineered medicine carrying expression system and HSA specific binding

1. utilize the specificity of purifying for HSA antibody V _halbumen has the gst fusion protein characteristic, carries out the immunoprecipitation test, specific embodiments:

1) get 20 μ l 0.01mol/L, PH7.2, PBS damping fluid centrifuge washing three times for GST-Beads;

2) abandon supernatant, with 1ml 0.01mol/L, PH7.2, PBS damping fluid suspended sediment and be divided into two parts, a copy of it adds specificity for HSA antibody VH purifying protein 10 μ l, and another part adds GST 10 μ l;

3) mix (slowly) with the vibrator vibration, 4 ℃ are spent the night;

4) use PBS-Tween20 centrifuge washing three times;

5) 1%Casein was in 37 ℃ of sealings 1 hour;

6) use PBS-Tween20 centrifuge washing three times;

7) 0.5ml 0.01mol/L, PH7.2, PBS damping fluid for throw out are suspended, add 10 μ l normal human serums simultaneously;

8) 37 ℃ of vibrator vibrations mix 2 hours;

9) use PBS-Tween 20 centrifuge washing three times;

10) add 50 μ l 1 * SDS-PAGE Buffer respectively in throw out;

11) water-bath is boiled 5 minutes;

12) conventional loading, carry out Western-bolt, the results are shown in Figure 12.Can see in figure that the colour developing band of the about 6800da of a part amount appears in the GST-HSA-VH swimming lane; The GST swimming lane does not occur.Show for the antibody VH albumen of HSA can with the HSA specific binding.

2. utilize the ELISA inhibition test that is at war with

1) for HSA, 0.05mol/L, pH 9.6 carbonate buffer solution dilutions are 100ng/ml, add in elisa plate 100 μ l/ holes;

2) putting 4 ℃ of wet boxes spends the night;

3) abandon supernatant next day, pat dry at every turn, PBS-Tween20 washing three times;

4) add 1%Casein, 37 ℃, 1 hour;

5) abandon supernatant, pat dry;

6) add gradient dilution VH expressing protein 50 μ l (concentration is respectively: 1,0.5,0.25 ... μ g/ml); The 1C that simultaneously adds dilution in 1: 2500 ₁ascites 50 μ l;

7) 37 ℃, 1 hour;

8) abandon supernatant, PBS-Tween20 washing three times, pat dry;

9) add the HRP-sheep anti-mouse igg 100 μ l of dilution in 1: 5000;

10) 37 ℃, 1 hour;

11) abandon supernatant, PBS-Tween20 washing three times, pat dry;

12) TMB colour developing, measure the OD450 value, and draw and suppress curve, the results are shown in Figure 13.

Amalgamation and expression, the Purification and Characterization of embodiment 6BL21/pETa (32+)-IFN-Fab (HSA) (being called for short IFN-Fab (HSA)) genetically engineered medicine carrying expression system and protein and peptide class medicine

1) express bacterium by amplification DH5 α/pETa (32+)-HSA and DH5 α/pETa (32+)-IFN, extract respectively plasmid.Carry out double digestion through HindIII and EcoR I respectively, through glue, reclaim and obtain DH5 α/pETa (32+)-HSA expression vector and IFN gene fragment (insertion sequence is shown in Figure 14 or SEQ ID NO:5).Obtain DH5 α/pETa (32+)-IFN-Fab (HSA) fusion expression vector after connecting conversion.Extract expression plasmid through amplification again, be converted into e. coli bl21 acquisition BL21/pETa (32+)-IFN-Fab (HSA) and express bacterium.Detailed process, with embodiment 3, is carried out amalgamation and expression, the Purification and Characterization (seeing Figure 15) of protein and peptide class medicine.

2) get the BL21/pETa (32+) of purifying-IFN-Fab (HSA) amalgamation and expression albumen and detect its antiviral activity with the cytopathy political reform, concrete grammar is with reference to 2010 editions " Products in China evaluation rules ", appendix XC, interferon biological activity assay method (cytopathic-effect inhibition assay) ^[10].The antiviral specific activity of result>=8.5 * 10 ⁸iU/mg.

The animal experiment of embodiment 7BL21/pETa (32+)-IFN-Fab (HSA) (being called for short IFN-Fab (HSA)) medicine carrying expression system-protein and peptide fusion rotein medicine

1) get BL21/pETa (32+) after purifying-IFN-Fab (HSA) amalgamation and expression albumen and IFNA1 protein drug abdominal cavity and inject respectively the about 20g kunming mice of body weight, every group each five, every injection 10 μ g;

2) and injection rear 15,30,60,90,120,180,360,720,1440,2160,2800 minute tail vein blood front respectively at injection, separation of serum ,-20 ℃ save backup;

3) detect respectively IFN content in serum with the ELISA test kit;

4) get and respectively organize each time point IFN content average making pharmacokinetic curve (seeing Figure 16);

5) peak reaching time of blood concentration is approximately 1.5 hours as a result, and eliminating the transformation period is IFN A1 approximately 3 hours, and BL21/pETa (32+)-IFN-Fab (HSA) amalgamation and expression albumen elimination transformation period is IFN A1 approximately 36 hours;

6) in sum, the animal experiment effect relatively finds that BL21/pETa (32+)-IFN-Fab (HSA) medicine carrying expression system-protein and peptide fusion rotein medicine is than approximately 12 times of IFN A1 protein drug Increased Plasma Half-lifes.

Reference:

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Claims (1)

1. for the antibody V of human serum albumin _hfragment, is characterized in that described fragment is the V of the antibody that produces of hybridoma cell strain that preserving number is CCTCC-C 2010111 _hfragment;

The aminoacid sequence of described fragment is by nucleotide sequence coded shown in SEQ ID NO:4.

2. V claimed in claim 1 encodes _hthe Nucleotide of fragment, the sequence that it is characterized in that described Nucleotide is sequence shown in SEQ ID NO:4.

3. the method for modifying protein or polypeptide drug, it is characterized in that can with small peptide and described protein or the polypeptide drug amalgamation and expression of albumin specific binding, thereby the formation fusion rotein, wherein said can be the V of claim 1 with the small peptide of albumin specific binding _hfragment, described protein or polypeptide drug are α 1-Interferon, rabbit.

4. method according to claim 3, is characterized in that described modification has extended the transformation period of described protein or polypeptide drug.

5. the fusion rotein obtained according to the method for claim 3 or 4.

6. the gene engineering expression carrier of expressed fusion protein, be characterised in that the V that described fusion rotein comprises claim 1 or 2 _hfragment and α 1-Interferon, rabbit, described carrier is the BL21/pETa (32+)-HSA gene engineering expression carrier of the preserving number gene engineering expression bacterium secretion that is CCTCC-M 2010288.

7. a method of producing the fusion rotein medicine of protein or polypeptide, is characterized in that described method comprises the nucleotide sequence of code for said proteins or polypeptide is cloned in the gene engineering expression carrier of claim 6, and express the V that comprises claim 1 _hthe fusion rotein of fragment, wherein said protein or polypeptide are α 1-Interferon, rabbit.

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