CN1119415C - Clone and expression of anti-CA125 bifunctional genetically engineered antibody - Google Patents
Clone and expression of anti-CA125 bifunctional genetically engineered antibody Download PDFInfo
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Abstract
The present invention relates to clone and expression of an anti-CA125 bifunctional gene engineering antibody, which belongs to a new guide anti-cancer medicine. The present invention also provides a nucleotide sequence of a molecule guide medicine of the anti-CA125 bifunctional gene engineering antibody, the nucleotide sequence constructs gene fragments of a heavy-chain variable region of anti-CA125 immunoglobulin and a light-chain variable region into a single-chain antibody which is directly fused with human source Tuftsin genes, and the double-function gene engineering antibody expressed by the genes is composed of 259 amino acids. The antibody has double function of combining CA125 antigenic specificity, and inhibiting and killing cancer cells by regulating the immunologic mechanism, and can be widely used for treatment of tumor relevant to CA125. The present invention also provides a recombinant expression carrier of the nucleotide sequence and pichia pastoris containing the carrier.
Description
The present invention relates to a kind of cloning and expression of anti-CA 125 bifunctional genetically engineered antibody, is a kind of novel guiding immunity anticancer medicine.It belongs to the application of biotechnology in field of medicaments.
Biotechnology is one of frontline technology of life science, and each state of the world today all is considered as biotech medicine product the sunrise industry of a kind of high-tech and the next century, occupies an important position in the science and technology innovation.Last decade, biotechnology is in the application of field of medicaments, for pharmaceutical industry brings a new revolution, particularly aspect the development of new cancer therapy drug, the immunity and the targeted drug that utilize biotechnology to develop, becoming the main developing direction of cancer therapy, and will progressively replace toxic side effect than the great tradition chemotherapeutics.
The targeted therapy of cancer is to grow up on the basis of hybridoma technology, utilizes cell engineering manufacture order clonal antibody to be applied to clinically obtain certain progress as the carrier of targeted drug, but does not reach the desired ideal effect of people.Because mostly prepared monoclonal antibody is mouse source property at present; use repeatedly and can produce anti-mouse antibody (HAMA); and as carrier crosslinked medicine or toxin mainly be to connect with chemical chain; usually can medicine or toxin be lost and can't arrive target site and body is had side effects; and cause penetration power poor owing to molecular weight is excessive; these shortcomings make monoclonal antibody be subjected to certain limitation as the targeted drug carrier in clinical application; and there is the problem of building the strain difficulty in humanized antibody; simultaneously; because the cell engineering technological process is very complicated, production cost is higher.The problems referred to above have also limited monoclonal antibody medicine application and development clinically to a certain extent.In order to overcome the above-mentioned shortcoming of monoclonal antibody, the research with antibody transformation or modification is being carried out in the present many laboratories of developed country in the world, in the hope of obtaining the guiding cancer therapy drug of small-sized efficient low toxicity, this field is to be the forward position core of cancer therapy drug research at present.
The objective of the invention is to utilize genetic engineering technique to develop a kind of novel guiding cancer therapy drug, by technique means such as utilization molecular biology, immunochemistry, biological chemistry and biological fermentations, but make up the genetic engineering bacterium that a kind of secretion type expression has the target protein of antitumour activity at molecular level.This project bacterium produces a kind of fusion rotein by expressing, and this albumen has specific recognition cancer cells surface antigen CA125 and kills and wounds the double activity of cancer cells by the adjusting body immune system.The approach of research is: prepare the antigenic monoclonal antibody of anti-CA 125 by hybridoma technology, from this antibody, clone variable region gene, after the sequence checking is errorless, with 15 amino acid whose peptides variable region gene is coupled together, be built into single-chain antibody (ScFv), again single-chain antibody (ScFv) gene is combined with endogenous immune peptide Tuftsin gene, be incorporated in the plasmid vector, change among the yeast expression system Pichia pastoris, but make up the engineering bacteria of secreting, expressing fused protein, and it is carried out CHARACTERISTICS IDENTIFICATION, simultaneously expression product is carried out separation and purification and active the detection.
1. prepare the antigenic monoclonal antibody of anti-CA 125 with hybridoma method.
2. with PCR method clonal antibody variable region of light chain and heavy chain variable region gene.
3. the antibody variable gene of having cloned is carried out sequencing analysis.
4. make up single-chain antibody (ScFv).
5. the design of difunctional ScFv-tuftsin molecule:
Select expression vector; Clone ScFv gene; Clone tuftsin gene.
6. the structure of expression vector
7. conversion and the selection of recombinant expression plasmid in yeast expression system Pichia pastoris.
8. the expression of recombinant fusion protein and active detection.
Accompanying drawing of the present invention has:
Fig. 1 a, technical process and operation scheme that the b anti-CA 125 bifunctional genetically engineered antibody makes up
The structure of Fig. 2 plasmid vector and characteristic
The building process of Fig. 3 plasmid vector
Fig. 4 yeast is expressed the electrophoresis result of BACF (ScFv-Tuftsin) fusion rotein in different substratum
Fig. 5 utilizes the colour developing of immunization method hybridization protein
Fig. 6 variable region of heavy chain Nucleotide and deduced amino acid thereof
Fig. 7 variable region of light chain Nucleotide and deduced amino acid thereof
Fig. 8 single-chain antibody (ScFv) structure
Fig. 9 a, the complete nucleotide and the deduced amino acid thereof of the difunctional anticancer factor of b gene recombination (ScFv-tuftsin)
The amino acid of the difunctional anticancer factor of Figure 10 gene recombination (ScFv-tuftsin) is formed
The present invention is achieved in that
We begin to carry out the design of medicine on molecular level, this medicine is made of two funtion parts, and a part is the guiding identification division, and another part is the effector part.For the guiding identification division, what we selected is at the antigenic antibody of CA125, clones the V of variable region from this antibody
HAnd V
LGene connects to V with the two with a small peptide (Linker)
L-Linker-V
HGene, i.e. recombinant single chain antibody (SingleChain Fv, ScFv) gene.In the design of anti-cancer function, by comparative experiments with consult relevant bibliographical information, we have selected the natural tetrapeptide of a kind of humanized: tuftsin (Tuftsin).Tuftsin is a kind of immunomodulator efficiently, and it can activate intravital scavenger cell and strengthen its phagolysis, and then kills cancer cells by regulating immunity system, and has no side effect.Through more than 40 times experiment repeatedly, we successfully clone the Tuftsin gene, and single-chain antibody gene (ScFv) and Tuftsin gene are coupled together, clone out with gene recombination technology, be connected into plasmid vector, be transferred in the yeast expression system, its expression product is a fusion rotein.
Anti-CA 125 bifunctional gene antibody of the present invention will be elaborated as follows in conjunction with the accompanying drawings:
Fig. 1 represents technical process and the operation scheme that anti-CA 125 bifunctional genetically engineered antibody makes up
CA125 carries out immunity to the mouse body with the ovarian cancer cell surface antigen, merges the hybridoma of preparing anti-CA 125 external with the myeloma cell, extracts mRNA from hybridoma, and reverse transcription becomes cDNA, with PCR primer V
HIFOR, V
HIBACK, V
LIFOR, V
LIBACK, use the pcr amplification technology and obtain complete variable region of heavy chain (V
H) and variable region of light chain (V
L) gene segment.By 15 amino acid whose small peptides with V
HAnd V
LConnect and compose V
L-Linker-V
H(ScFv) gene, in intestinal bacteria, expressed single-chain antibody contains the function of the identification cancer cells surface antigen of parental antibody by plasmid integration.Using gene engineering means clone ScFv and Tuftsin gene are connected ScFv with the Tuftsin gene, be configured to the ScFv-Tuftsin dna segment of a reorganization.This segment is incorporated among the expression plasmid pPIC-9, and this plasmid contains the AOX1 strong promoter, and goal gene is efficiently expressed.This plasmid is transformed in the yeast Pichia pastoris cell, becomes the secretor type yeast expression system.Through biological fermentation, and in the presence of inductor methyl alcohol, genetic engineering bacterium is expressed and is produced desired protein, i.e. ScFv-Tuftsin.This protein secreting in the yeast culture base, the product that after separation and purification, obtains be " the difunctional anticancer factor of gene recombination " (Recombinant Bi-functional Anticancer Factor, BACF).
Fig. 2 represents the structure and the characteristic of plasmid vector
It is pPIC-9 that the ScFv-Tuftsin recombination is transformed into Pichia plasmid vector that pastoris utilizes, and this plasmid has following feature: have Amp and HIS4 selective marker; 8023bp; Has high secretion α-factor secretion inducement signal; Be controlled by AOX1 by cloned genes and start the factor; Have multiple clone site (polylinker site).
Fig. 3 represents the building process of plasmid vector
Recombinant plasmid pPIC-ScFv-Tuftsin makes up like this: after the antibody variable region dna sequence dna of anti-CA 125 uses specific PCR primer amplification, insert in the carrier pPIC-9 plasmid in the Pichia pastoris secretion type expression system, be called pPIC-ScFv, then with the Tuftsin amino-acid sequence, N-SerGlyGlyThrLysProArg-c, directly insert in the pPIC-ScFv plasmid after the coded DNA base sequence is cut by restriction endonuclease (EcoRI and EagI) enzyme, this plasmid is called as pPIC-ScFv-Tuftsin.It contains the controlled AOX1 of the being formed on promotor of difunctional guiding gene (the N-end has the excreted factor signal).Amp that this plasmid has and HIS4 gene are used for selecting intestinal bacteria and yeast.
The selection of expression system
The expression system that is used for recombinant DNA at present all has its limitation and strong point, can determine best protein expression system and working condition by the expression level of estimating target protein.In order to express and help the separation and purification of target protein matter fast, we have selected the efficient secretion type expression of yeast Pichia pastoris system for use according to experimental result, and this system successfully is used to express many eukaryotic genes that are difficult at escherichia coli expression.At first, to in the plasmid of external structure, be transformed among the Pichia pastoris cell strain GS115, select the positive colony result by the culture medium flat plate that Histidine lacks, the DNA of common 5 micrograms can obtain about 200 positive bacterium colonies that transform, these positive bacterium colonies need be selected containing on the culture medium flat plate of glucose, containing the bacterium point of not growing on the culture medium flat plate of methyl alcohol is final positive bacteria, and common second kind of selection positive findings is about 5%.
Fig. 4 represents that yeast expresses the electrophoresis result of BACF (ScFv-Tuftsin) fusion rotein in different substratum.
1.ScFv shown in the figure is in the BMGY nutrient solution; 2.ScFv-tuftsin in the BYGY nutrient solution; 3.ScFv-tuftsin in Buffer 3 nutrient solutions; 4.ScFv-tuftsin concentrate 10 times in Buffer 3 nutrient solutions; M is a molecular weight standard.
Separate later final positive bacteria and drop on the BMGY inoculation of medium, 30 ℃ of wave and culture two days, vibrational frequency was 300 rev/mins.Two days later, check OD
600, when light absorption value was between 10-20, centrifugal collection thalline changed among 200 ml methanol induced liquid BMMY or the Buffer3,30 ℃ of wave and culture two to five days.Needed protein secreting is cultivated in the induced liquid at this.We adopt purity, molecular weight and the expression amount of conventional SDS-PAGE electrophoresis detection target protein matter.Usually the expression amount of BACF (ScFv-Tuftsin) is at least the 100-150 mg/litre, and this proteinic molecular weight size is 33-35kDa.
Fig. 5 represents to utilize the colour developing of immunization method hybridization protein
1 to 5 is the electrophoresis result of BACF in different selection secretion nutrient solutions, and wherein 1 is BMGY, and 3 is that Buffer 3,6 is negative contrast.
BACF (ScFv-Tuftsin) Expression of Fusion Protein and purifying
A main advantage of biotechnology be exactly can be promptly for the preparation of recombinant protein provides the scheme of simplifying, like this, the best way is to simplify to cultivate composition, for example, the Buffer3 that we mentioned.Protein purification mainly is to use traditional ion exchange column method separation and purification, its main advantage is simple and direct, quick, can large volume preparation, and this method can be got rid of non-affinity albumen such as the white protein that might occur in the nutrient solution, transferrin, proteolytic enzyme.
The recombinant protein of being reported is at present expressed all in Pichia and can be kept its biological activity.With the external immunological method of competitiveness (RIA), we can detect the combination activity of BACF (ScFv-tuftsin).Method is as follows: the CA125 of 800 units is coated in (200 microlitres, concentration are the CA125 of 4000 units per ml) on the solid phase, adds 5 microgram I
125The reaction that is at war with of the monoclonal antibody of the anti-CA 125 of mark (100 milliliters, concentration is the antibody of 50 mcg/ml) and BACF (ScFv-tuftsin) sample.It is active that resulting difference demonstrates the distinctive biological combination of BACF (ScFv-tuftsin) after this competing reaction.
Fig. 6 represents variable region of heavy chain Nucleotide and deduced amino acid thereof
Fig. 7 represents variable region of light chain Nucleotide and deduced amino acid thereof
Fig. 8 represents single-chain antibody (ScFv) structure
Fig. 9 a, b represent the complete nucleotide and the deduced amino acid thereof of the difunctional anticancer factor of gene recombination (SeFv-tuftsin)
Figure 10 represents the amino acid composition of the difunctional anticancer factor of gene recombination (ScFv-tuftsin)
The cloning and expression of anti-CA 125 bifunctional genetically engineered antibody of the present invention is characterized in that the structure of engineering bacteria and complete aminoacid sequence:
1. this sequence is that variable region of heavy chain and chain variable region gene fragment with the anti-CA 125 immunoglobulin (Ig) is built into single-chain antibody, single-chain antibody gene and people source Tuftsin gene are directly merged, the expressed bifunctional genetically engineered antibody of this gene is made up of 259 amino acid again; This antibody has the dual-use function that combines and pass through to regulate immunologic mechanism cancer inhibitting and killing cell with the CA125 antigen-specific, and it can be widely used in the targeted therapy of the tumour relevant with CA125; The present invention also provides the recombinant expression vector that above-mentioned nucleotide sequence is arranged, and the Bi Qi yeast Pichia pastoris that contains this carrier.
2. bacterial strain and plasmid: bacterial strain: Pichia pastoris Gs115 (His4-Mut+).Plasmid: pPIC-9, this plasmid have Amp and HIS4 selective marker; 8023bp; Has high secretion α-factor secretion inducement signal; Goal gene is controlled by contained AOX1 and starts the factor; Have multiple clone site (polylinker site).Blank bacterial strain and plasmid are available from American I nvitrogene company.Promotor: AOX1, methanol evoked.
3. obtaining of anti-CA 125 hybridoma: adopt traditional cell-fusion techniques.CA125 forms through separation and purification by Proliferation of Human Ovarian Cell Ovcar-3 secretion.With CA125 mouse is carried out immunity, splenocyte after the immunity is separated, be blended in hybridoma SP2/O, choose hybridoma cell strain by the ELISA method with high-affinity antibody, be cultured to logarithmic phase, secreted anti-CA 125 monoclonal antibody is the IgGIK type.
4. the extraction and purification of the total RNA of hybridoma and cDNA are synthetic: adopt Guanidinium hydrochloride-phenol-chlorine method (Guanidium thiocyanate-phenol-chloroform) to extract total RNA, be cDNA by the mRNA that obtains through reverse transcription.
5 usefulness PCR method clonal antibody variable region genes: antibody variable region contains heavy, light two chains, so variable region gene has two, i.e. V
HAnd V
LGene, the PCR primer that extracts the VH gene is V
HIFORAnd V
HIBACK
V
HIFOR：5’-TGA?GGA?GAC?GGT?GAC?CGT?CCC?TTG?GCC?CCA?G-3’
V
HIBACK：5’-CAG?GTC/G?A/CAA/G?CTG?CAG?C/GAG?TCA/T?GG-3’
Extract V
LThe PCR primer of gene is V
LIFORAnd V
LIBACK
V
LIFOR：5’GTT?TGA?TCT?CCA?GCT?TGG?TCC?C-3’
V
LIBACK：5’-GAC?ATT?CAG?CTG?ACC?CAG?TCT?CCA-3’
In the sequence of these PCR primers, V
LIFORContain the BglII point of contact, V
LIBACKContain the PvuII point of contact.Use the Touchtown program and carry out the PCR reaction in 40 cycles, extract reaction solution after the reaction and be splined on 1% DNA sepharose, extract the PCR product from agarose gel electrophoresis, the PCR-DNA that obtains contains 350bp.
6. clone PCR products: because the dna segment that the PCR product obtains is for flat terminal, therefore can directly be cloned on the position, EcoRV point of contact of plasmid vector (pBluescriptIIKs), this carrier is 1961bp, contains the EcoRV point of contact that is positioned at 715bp and 2 and lays respectively at 529bp and 977bp PvuII point of contact.Clone's step is:
A. connect: plasmid and insertion segment ratio are 1: 3, carry out ligation with the T4-DNA ligase enzyme.
B. transform: the bacillus coli DH 5 alpha CaCl of 0.1M
2Be treated to competent cell, 20ul connects liquid and adds 100ul DH5 α cell, handles back overnight incubation under 37 ℃ of conditions.
C. select: adopt the restriction enzyme enzyme process, select positive colony.
7. variable region gene sequence analysis
A. extract plasmid DNA: V
HAnd V
LDna segment is called pBKS-V after inserting carrier
HAnd pBKS-V
L, what we adopted is the plasmid extracting method of Qiagen company.
B. sequence measurement: the main experimental program that adopts " United States Biochemical " to be provided, used primer is T
3And T
7(Stratagene), with the dna sequence dna that records Genework software processes, do contrast with known Data Base Gene Bank and obtain V accurately
HAnd V
LSequence, V
HSequence see Fig. 7, V
LSequence see Fig. 8.
7. the structure of single-chain antibody ScFv
With V
HAnd V
LBe combined into single chain molecule, should consider V
HAnd V
LDirection, our result of study shows V
L-Linker-V
HMore stable and easy expression, to consider simultaneously Linker length and the order, we adopt 15 amino acid (SerGlyGly), 5 sequences.The construction step of ScFv is as follows: adopt the two-step pcr mode with V
HAnd V
LBe combined into ScFv, and have the EcoRV point of contact, have the EcoRI point of contact at 3 ' end at 5 ' end.The used term harmonization of PCRI and PCRII was 30 cycles, and the phase is 94 ℃ of 1min weekly; 50-55 ℃, min; 72 ℃, 1min.Through extracting the PCR segment from glue after the PCRI, with the segment of two PCRI 1: 1 mixed after, as the template of PCRII.PCRII then uses V
L-EcoRV and V
H-EcoRI carries out pcr amplification, and the strand ScFv gene of composition is 752bp, and the restriction enzyme site of EcoRV and EcoRI is contained at two ends.Obtain the V of complete and accurate after the order-checking
L-Linker-V
HThe nucleotide sequence (see figure 8).
8. the structure of the experimental design of difunctional ScFv-tuftsin and expression vector
With the process of bifunctional antibody (ScFv-tuftsin) gene clone in the yeast secreted expression carrier, be divided into two steps, at first with ScFv gene clone (Invitrogene) in the pPIC-9 plasmid, required restriction enzyme site is SnaBI/EcoRV and EcoRI, and then with the tuftsin gene clone at the COOH of pPIC-9-ScFv end, required restriction enzyme site is EcoRI and EagI, simultaneously, we consider that needing certain space sequence (spacer) between ScFv and tuftsin gene makes ScFv and tuftsin function the other side that is independent of each other separately.
A. the selection of expression vector
We select is expression vector available from Invitrogene company, plasmid pPIC-9, and its collection of illustrative plates and feature are seen Fig. 2.
B. clone the ScFv gene:
Behind the ScFv dna fragmentation usefulness EcoRV that contains 752bp and EcoRI while enzymolysis that have built, be cloned into SnaBI and the EcoRI site of a pPIC-9, change bacillus coli DH 5 alpha over to, select with penbritin, the positive colony that obtains is pPIC-9-ScFv.
C. clone the Tuftsin gene:
When design tuftsin gene, need EcoRI and EagI restriction enzyme site be installed at tuftsin gene 5 ' and 3 ' end.Also to make up negative contrast molecule simultaneously.Oligonucleotide (Oligo) design of primers of synthetic tuftsin gene is as follows:
People (H-tuftsin): 5 '-AAT TCT GGA GGT GGT ACC AAG CCT AGG TAG
GA CCT?CCA?CCA?TGG?TTC?GGA?TCC?ATC?GCC?GG-5’
Mouse (m-tuftsin): 5 '-AAT TCT GGA GGT GGT ACC CAG CCT AGG TAG C
GA CCT?CCA?CCA?TGG?GTC?GGA?TCC?ATC?GCC?GG-5’
Negative contrast (G4C): 5 '-AAT TCA GCT GGA GGT GGT GGA TGT GC
GT?CGA?CCT?CCA?CCA?CCT?ACA?CGC?CGG-5’
Wherein contain the KpnI site in the H-tuftsin sequence, contain the KpnI site in the M-tuftsin sequence, contain the PvuII site in the G4C sequence.
With three groups of oligo marks good after, carry out respectively 5 ' end phosphorylation reaction (Phosphorylation).After reaction stops, with ethanol sedimentation oligo DNA.
C. the structure of expression vector:
The carrier DNA of 1pmol (pPIC-ScFv) digests simultaneously with EcoRI and EagI, and the Oligo DNA with three groups of 1pmol carries out ligation respectively.After the ligation, the pPIC9 plasmid that will contain required ScFv-tuftsin is transformed into intestinal bacteria E.coli DH5 α, and formed plasmid is called after respectively:
pDL-6(pPIC9-ScFv-m-tuftsin)
pDL-10(pPIC9-ScFv-G4c)
PDL-11 (pPIC9-ScFv-H-tuftsin) extracts three kinds of plasmid DNA respectively, and it is as follows to do the restriction endonuclease detected result:
Enzyme point of contact segment
pDL-6 kpnI 4 612bp，635bp，970bp，6633bp
pDL-10 PvaII 3 245bp，2900bp，5700bp
pDL-11 KpnI 4 612bp，635bp，970bp，6633bp
Simultaneously, be that primer carries out sequential analysis with 5 '-AOX1 and 3 '-AOX1, prove that further dna sequence dna is errorless, sequencing primer is:
5’-AOX1：5’-GAC?TGG?TTC?CAA?TTG?ACA?AGC-3’
3’-AOX1：5’-GCA?AAT?GGC?ATT?CTG?ACA?TCC-3’
Sequencing result is seen Fig. 9.According to the interpretation of result of order-checking, this sequence is made up of 259 amino acid, and the amino acid composition of BACF (ScFv-tuftsin) sees Figure 10 for details.
10. the conversion of expression plasmid pPIC-ScFv-H-tuftsin in yeast Pichia pastoris is GS115 (His4-Mut+) with selecting yeast strain Pichia pastoris.The method that electricity consumption transforms is transformed into recombinant plasmid pPIC-ScFv-H-tuftsin in the yeast strain GS115 cell.
A. the preparation of linearization plasmid: behind BglII digestion reorganization grain, ethanol sedimentation can obtain linearization plasmid.
B. electricity transforms: GS115 is cultured to OD in the 500mlYPD substratum
600Be about 1.3-1.5, centrifugal collection thalline, cell after the processing is dissolved in the 1ml Sorbitol liquid, add the plasmid DNA (5ug) of 5ul, put into the electric conversion tube of 0.2um, carry out electricity and transform (1800V pulse) with BglII digestion, the Sorbitol that adds 1ml then, the mixing cell is got 200ul and is coated with the RDB flat board, cultivates two days for 30 ℃.
C. the selection of transformant: will be inoculated in respectively on MM and the MD culture medium flat plate at the bacterium colony that generates on the RDB flat board, and cultivate two days in 30 ℃.Usually, we inoculate about 100 bacterium colonies, generally can obtain a 5-25 His+Mut-bacterium colony.Bacterium overnight incubation (30 ℃ 200rpm), are preserved bacterial classification respectively then in the YPD nutrient solution with these His+Mut-types.
11.ScFv-tuftsin fused protein is expressed and activation analysis
Screen later bacterial classification and in the MGY nutrient solution, inoculate, cultivated 48 hours, check OD at 30 ℃
600, should be between 10-20.Centrifugal then collection thalline, with thalline with among 200ml methanol induction liquid BMMY or the Buffer3 30 ℃ cultivated 2-5 days, promotor AOX1 starts goal gene under methanol induction, with required target protein secretion in inducing culture liquid.Through the SDS-PAGE electrophoretic analysis, Yi Tiao district band (Fig. 4,5) is arranged about molecular weight 35KD, expression amount reaches 100-150mg/L.Carry out biological activity determination behind the purifying, detect ScFv-tuftsin and the antigenic activity that combines with external immune competition law (RIA), the result shows that ScFv-tuftsin has specificity identical with parental antibody and avidity.
With at present use in the world more monoclonal antibody class targeted therapy medicine and compare, this product has molecular weight Little, stability is strong, is difficult for producing anti-mouse antibody, and is strong with the antigen compatibility, and effector is combined the jail with carrier Admittedly wait remarkable advantage. In addition, this product is gene engineering product on preparation technology, and the monoclonal antibody series products is The cell engineering product, technical process is greatly simplified than monoclonal antibody series products. With some laboratories are being just in the world at present Like product in research compares, and the advantage of this product shows:
(1) this product is that secreting type efficiently expresses in saccharomycete Pichia pastoris, and expression and purity are all Higher, destination protein directly is secreted in the culture medium and is automatically folded into native conformation, need not renaturation, simultaneously Reduced cost. And mostly other like product is to express in Ecoli, and resulting fusion often Form with inclusion body exists, and biologically active must just be arranged after renaturation.
That (2) this product single-chain antibody connects is endogenous immunomodulator tuftsin, even because in the body Environmental activity and single-chain antibody disconnect, and also can not cause any toxic and side effect to body. And other like product Mostly what connect is cytotoxin, in case disconnect with single-chain antibody, easily produces toxic and side effect, in the use right The requirement of dosage is very strict.
Claims (11)
1. anti-CA 125 bifunctional genetically engineered antibody, this antibody is made of 259 amino acid, has the aminoacid sequence shown in Fig. 9 a and the 9b.
2. the preparation method of the anti-CA 125 bifunctional genetically engineered antibody of claim 1, the cloning and expression that it is characterized in that anti-CA 125 bifunctional genetically engineered antibody obtains having the anti-CA 125 bifunctional genetically engineered antibody shown in Fig. 9 a and the 9b by the gene recombination structure.Prepare the antigenic monoclonal antibody of anti-CA 125 by hybridoma technology, from this antibody, clone variable region gene with PCR method, with one by 15 amino acid (SerGlyGly)
5The small peptide of forming will couple together, make up single-chain antibody ScFv, again single-chain antibody gene is combined with endogenous immune peptide Tuftsin gene, be incorporated in the plasmid vector, change among the yeast expression system Pichia pastoris, but the engineering bacteria of structure secreting, expressing ScFv-tuftsin fusion rotein carries out CHARACTERISTICS IDENTIFICATION simultaneously, engineering bacteria obtains the anti-CA 125 bifunctional genetically engineered antibody fusion rotein by secreting, expressing, and the fusion rotein ScFv-tuftsin of its expression is carried out the activity detection.
3. according to the preparation method of the anti-CA 125 bifunctional genetically engineered antibody of claim 2, it is characterized in that the yeast expression system bacterial strain is Pichia pastoris GS115 His4-Mut+; Plasmid is pPIC-9 shown in Figure 2, and this plasmid has Amp and HIS4 selective marker.
4. according to the preparation method of the anti-CA 125 bifunctional genetically engineered antibody of claim 2, it is characterized in that obtaining of anti-CA 125 hybridoma: mouse is carried out immunity with CA125 antigen, splenocyte after the immunity is blended in the SP2/O hybridoma, choose the hybridoma cell strain with high affinity antibody and cultivate, resulting anti-CA 125 monoclonal antibody is the IgG1K type.
5. according to the preparation method of the anti-CA 125 bifunctional genetically engineered antibody of claim 2, it is characterized in that: extract the total RNA of hybridoma,, be cDNA through reverse transcription by the mRNA that obtains through behind checking and the purifying with PCR method clonal antibody variable region gene.Design the variable region gene that the PCR primer is used to extract antibody again, variable region of heavy chain and variable region of light chain are contained in the variable region of antibody, so variable region gene has two, i.e. V
HAnd V
LGene extracts V
HThe PCR primer of gene is V
HIFORAnd V
HIBACK: V
HIFOR: 5 '-TGA GGA GAC GGT GAC CGT CCC TTG GCC CCA G-3 ' V
HIBACK: 5 '-CAG GTC/G A/CAA/G CTG CAG C/GAG TCA/T GG-3 ' extracts V
LThe PCR primer of gene is V
LIFORAnd V
LIBACKV
LIFOR: 5 '-GTT TGA TCT CCA GCT TGG TCC C-3 ' V
LIBACK: 5 '-GAC ATT CAG CTG ACC CAG TCT CCA-3 ' in the sequence of these PCR primers, V
LIFORContain the BglII point of contact, V
LIBACKContain the PvuII point of contact.Use the Touchtown program and carry out the PCR reaction in 40 cycles, extract reaction solution after the reaction and be splined on 1% DNA sepharose, extract the PCR product from agarose gel electrophoresis, the PCR-DNA that obtains contains 350bp.
6. according to the preparation method of the anti-CA 125 bifunctional genetically engineered antibody of claim 2, it is characterized in that clone PCR products: because the dna segment that the PCR product obtains is for flat terminal, therefore can directly be cloned on the position, EcoRV point of contact of plasmid vector pBluescriptIIKs, this carrier is 1961bp, contains the Eco-RV point of contact that is positioned at 715bp and 2 and lays respectively at 529bp and 977bp PvuII point of contact.Clone's step is:
A. connect: plasmid and insertion segment ratio are 1: 3, carry out ligation with the T4-DNA ligase enzyme.
B. transform: bacillus coli DH 5 alpha is treated to competent cell with the CaCl2 of 0.1M, and 20ul connects liquid and adds 100ul DH5 α cell, handles the back and cultivated liquid under 37 ℃ of conditions.
C. select: adopt the restriction enzyme enzyme process, select positive colony.
7. according to the preparation method of the anti-CA 125 bifunctional genetically engineered antibody of claim 2, it is characterized in that becoming district's gene sequencing:
A. extract plasmid DNA: V
HAnd V
LDna segment is called pBKS-V after inserting carrier
HAnd pBKS-V
L
B. sequence measurement mainly adopts the experimental program that " United States Biochemical " provided, used primer is T3 and T7, with the dna sequence dna that records Genework software processes, do contrast with known Data BaseGene Bank and obtain V shown in Figure 6 accurately
HSequence and V shown in Figure 7
LSequence.
8. according to the preparation method of the anti-CA 125 bifunctional genetically engineered antibody of claim 2, it is characterized in that the structure of single-chain antibody ScFv: with V
HAnd V
LBe combined into single chain molecule, should consider V
HAnd V
LDirection, our result of study shows V
L-Linker-V
HMore stable and easy expression, to consider simultaneously Linker length and the order, we adopt 15 amino acid (SerGlyGly)
5Sequence.The construction step of ScFv is as follows: adopt the two-step pcr mode with V
HAnd V
LBe combined into ScFv, and have the EcoRV point of contact, have the EcoRI point of contact at 3 ' end at 5 ' end.The used term harmonization of PCRI and PCRII was 30 cycles, and the phase is 94 ℃ of 1min weekly; 50-55 ℃; 72 ℃, 1min.Through extracting the PCR segment from glue after the PCRI, with the segment of two PCRI 1: 1 mixed after, as the template of PCRII.PCRII then carries out pcr amplification with VL-EcoRV and VH-EcoRI, and the strand ScFv gene of composition is 752bp, and the restriction enzyme site of EcoRV and EcoRI is contained at two ends.Obtain the V shown in Figure 8 of complete and accurate after the order-checking
L-Linker-V
HNucleotide sequence.
9. according to the preparation method of the anti-CA 125 bifunctional genetically engineered antibody of claim 2, it is characterized in that the experimental design of difunctional ScFv-tuftsin and the structure of expression vector: with the process of bifunctional antibody ScFv-tuftsin gene clone in the yeast secreted expression carrier, be divided into two steps, at first with the ScFv gene clone in the pPIC-9 plasmid, required restriction enzyme site is SnaBI/EcoRV and EcoRI, and then with the tuftsin gene clone at the COOH of pPIC-9-ScFv end, required restriction enzyme site is EcoRI and EagI, simultaneously, we consider that needing the certain space sequence between ScFv and tuftsin gene makes ScFv and tuftsin function the other side that is independent of each other separately.
A. we select is plasmid pPIC-9 shown in Figure 2 in the selection of expression vector.
B. clone the ScFv gene: after the ScFv dna fragmentation that contains 752bp that will build is used EcoRV and EcoRI enzymolysis, be cloned into SnaBI and the EcoRI site of plasmid pPIC-9, change bacillus coli DH 5 over to
A selects with penbritin, and the positive colony that obtains is pPIC-9-ScFv;
C. clone the Tuftsin gene: when design tuftsin gene, need EcoRI and EagI restriction enzyme site be installed at tuftsin gene 5 ' and 3 ' end.Also to make up negative contrast molecule simultaneously.The Oligonucleolide primers design of synthetic tuftsin gene is as follows:
People H-tuftsin:5 '-AAT TCT GGA GGT GGT ACC AAG CCT AGG TAG
GA CCT?CCA?CCA?TGG?TTC?GGA?TCC?ATC?GCC?GG-5’
Mouse m-tuftsin:5 '-AAT TCT GGA GGT GGT ACC CAG CCT AGG TAG C
GA CCT?CCA?CCA?TGG?GTC?GGA?TCC?ATC?GCC?GG-5’
Negative contrast G4C:5 '-AAT TCA GCT GGA GGT GGT GGA TGT GC
GT CGA?CCT?CCA?CCA?CCT?ACA?CGC?CGG-5’
Wherein contain the KpnI site in the H-tuftsin sequence, contain the KpnI site in the M-tuftsin sequence, contain the PvuII site in the G4C sequence.With three groups of oligo marks good after, carry out respectively 5 ' end phosphorylation reaction.After reaction stops, with ethanol sedimentation oligo DNA.
D. the carrier DNA of the structure of expression vector: 1pmol: pPIC-ScFv digests simultaneously with EcoRI and EagI, and the Oligo DNA with three groups of 1pmol carries out ligation respectively.After the ligation, the pPIC9 plasmid that will contain required ScFv-tuftsin is transformed into intestinal bacteria E.coli DH5 α, and formed plasmid is called after respectively:
pDL-6：pPIC9-ScFv-m-tuftsin；
pDL-10：pPIC0-ScFv-G4c；
pDL-11：pPIC9-ScFv-H-tuftsin。
Extract three kinds of plasmid DNA respectively, and it is as follows to do the restriction endonuclease detected result:
Enzyme point of contact segment
pDL-6 kpnI 4 612bp，635bp，970bp，6633bp
pDL-10 PvaII 3 245bp，2900bp，5700bp
pDL-11 KpnI 4 612bp，635bp，970bp，6633bp
Simultaneously, be that primer carries out sequential analysis with 5 '-AOX1 and 3 '-AOX1, prove that further dna sequence dna is errorless, sequencing primer is:
5’-AOX1：5’-GAC?TGG?TTC?CAA?TTG?ACA?AGC-3’
3’-AOX1：5’-GCA?AAT?GGC?ATT?CTG?ACA?TCC-3’
Obtain sequencing result shown in Figure 9.According to the interpretation of result of order-checking, this sequence is made up of 259 amino acid, and the amino acid with ScFv-tuftsin shown in Figure 10 is formed.
10. according to the preparation method of the anti-CA 125 bifunctional genetically engineered antibody of claim 2, it is characterized in that conversion and the selection of expression plasmid pPIC-ScFv-H-tuftsin in yeast Pichia pastoris: yeast strain Pichia pastoris is GS115 His4-Mut+.The method that electricity consumption transforms is transformed into recombinant plasmid pPIC-ScFv-H-tuftsin in the yeast strain GS115 cell:
A. the preparation of linearization plasmid: behind BglII digestion reorganization grain, ethanol sedimentation can obtain linearization plasmid;
B. electricity transforms: GS115 is cultured to OD in the 500mlYPD substratum
600Be about 1.3-1.5, centrifugal collection thalline, cell after the processing is dissolved in the 1ml Sorbitol liquid, add the plasmid DNA of 5ul, put into the electric conversion tube of 0.2um, carry out electricity and transform with BglII digestion, the Sorbitol that adds 1ml then, the mixing cell is got 200ul and is coated with the RDB flat board, cultivates two days for 30 ℃;
C. the selection of transformant: will be inoculated in respectively on MM and the MD culture medium flat plate at the bacterium colony that generates on the RDB flat board, and cultivate two days in 30 ℃.Usually, we inoculate about 100 bacterium colonies, generally can obtain a 5-25 His+Mut-bacterium colony.Bacterium overnight incubation (30 ℃ 200rpm), are preserved bacterial classification respectively then in the YPD nutrient solution with these His+Mut-types.
11., it is characterized in that the ScFv-tuftsin fused protein expresses and activation analysis: screen later bacterial classification and in the MGY nutrient solution, inoculate, cultivated 48 hours, inspection OD at 30 ℃ according to the preparation method of the anti-CA 125 bifunctional genetically engineered antibody of claim 2
600, between 10-20.Centrifugal then collection thalline, with thalline with among 200ml methanol induction liquid BMMY or the Buffer3 30 ℃ cultivated 2-5 days, promotor AOXI starts goal gene under methanol induction, with required target protein secretion in inducing culture liquid.Through the SDS-PAGE electrophoretic analysis, a Fig. 4, district's band shown in Figure 5 are arranged about molecular weight 35KD, expression amount reaches 100-150mg/L.Carry out biological activity determination behind the purifying, detect ScFv-tuftsin and the antigenic activity that combines with external immune competition law RIA, the result shows that ScFv-tuftsin has specificity identical with parental antibody and avidity.
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| JP2011523853A (en) | 2008-06-03 | 2011-08-25 | アボット・ラボラトリーズ | Dual variable domain immunoglobulins and uses thereof |
| RU2010153578A (en) | 2008-06-03 | 2012-07-20 | Эбботт Лэборетриз (Us) | IMMUNOGLOBULINS WITH DOUBLE VARIABLE DOMAINS AND THEIR APPLICATION |
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| BR112013002578A2 (en) | 2010-08-03 | 2019-05-14 | Abbvie Inc. | double variable domain immunoglobins and their uses |
| MX2013002270A (en) | 2010-08-26 | 2013-05-14 | Abbvie Inc | Dual variable domain immunoglobulins and uses thereof. |
| CN104159920A (en) | 2011-12-30 | 2014-11-19 | 艾伯维公司 | Dual specific binding proteins directed against il-13 and/or il-17 |
| TW202210507A (en) | 2012-11-01 | 2022-03-16 | 美商艾伯維有限公司 | Anti-vegf/dll4 dual variable domain immunoglobulins and uses thereof |
| EP2970459A2 (en) | 2013-03-15 | 2016-01-20 | AbbVie Inc. | Dual specific binding proteins directed against il-1beta and il-17 |
| CN103983791A (en) * | 2014-05-29 | 2014-08-13 | 深圳市柏明胜医疗器械有限公司 | Human CA125 protein quantum dot labeled double-sandwiched immunoassay detection kit |
| WO2016094881A2 (en) | 2014-12-11 | 2016-06-16 | Abbvie Inc. | Lrp-8 binding proteins |
| TW201710286A (en) | 2015-06-15 | 2017-03-16 | 艾伯維有限公司 | Binding proteins against VEGF, PDGF, and/or their receptors |
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