CN106222194A

CN106222194A - The plant source recombinant humanized shellfish of a kind of optimization cuts down preparation method and the medical applications of monoclonal antibody

Info

Publication number: CN106222194A
Application number: CN201610630689.5A
Authority: CN
Inventors: 于为常; 陈磊
Original assignee: Shenzhen Mekes Fish Biological Technology Development Co Ltd
Current assignee: Shenzhen Mekes Fish Biological Technology Development Co Ltd
Priority date: 2016-08-03
Filing date: 2016-08-03
Publication date: 2016-12-14

Abstract

The invention belongs to biomedicine technical field, the plant source recombinant humanized shellfish being specifically related to a kind of optimization cuts down preparation method and the medical applications of monoclonal antibody, the present invention passes through heavy chain and the light chain fusion protein of expressing recombinant antibody Bevacizumab in plant, owing to adding 2A sequence in fusion protein, the heavy chain and the light chain that make antibody obtain the expression close to 1:1 ratio, the assembling of complete antibody is played obvious facilitation by this ratio, our result indicate that the antibody production applying this system expression is higher.Simultaneously because the heavy chain of recombinant antibodies and light chain equal proportion be assembled into more stable tetramer structure, and decrease the unassembled polypeptide that comparison is degradable, thus obtain purer consistent monoclonal antibody.The shellfish that new method is developed is cut down monoclonal antibody and is used for medicine by the present invention, is used for treating breast carcinoma, is used for treating pulmonary carcinoma, glioblastoma, renal carcinoma, cervical cancer, ovarian cancer, colon cancer and rectal cancer.

Description

A kind of plant source recombinant humanized shellfish of optimization cut down monoclonal antibody preparation method and Medical applications

Technical field

The invention belongs to biomedicine technical field, the plant source recombinant humanized shellfish being specifically related to a kind of optimization cuts down Dan Ke The preparation method of grand antibody and medical applications.

Background technology

Shellfish cuts down monoclonal antibody, is called for short (bevacizumab), its trade name Arastin (Avastin), is the people of restructuring Source monoclonal antibody, for the treatment of cancer well selling medicine (http://www.avastin.com/patient) of Roche Holding Ag. On February 26th, 2004 obtain FDA approval, be first, the U.S. get the Green Light listing suppression tumor-blood-vessel growth medicine, its The sales volume of 2009 reaches 5,900,000,000 dollars.Bevacizumab is produced by Chinese hamster ovary cell expression system, molecular weight It is about 149,000 dalton.Bevacizumab is the medicine that a kind of line artery generates, by suppression VEGF The hindrance blocks blood supply to tumor, suppression tumor spreads in vivo, strengthens chemotherapy effect.It is used for treating mammary gland in approval Before cancer, this medicine also is used for treating pulmonary carcinoma, glioblastoma, renal carcinoma, cervical cancer, ovary by the approval of pencil office of the U.S. Cancer, colon cancer and rectal cancer, and be approved for treating breast carcinoma in Europe.The mechanism of action of bevacizumab is: angiogenesis is The process (Wanget al., 2004) of neovascularization.Tumor need to set up independent blood supply so that its diameter is more than 1-2mm, because of This angiogenesis is a significant process in malignant growth.VEGF is the key regulatory person of tumor-blood-vessel growth, and It it is a kind of angiogenesis factor being expressed in whole tumor life cycle.The continuous expression of VEGF, and VEGF and endothelium The genetic stability (observed result based on preclinical study) of cell, can make " directly and persistently targeting VEGF " to become A kind of important antitumor strategy (Presta et al., 1997；Ferrara et al.,2005).

Along with the development of molecular biology, at the beginning of the eighties, scientists starts with technique for gene engineering to develop antibody, And gradually formed novel interdisciplinary technology------antibody biotechnology (Antibody biotechnology).It It is with DNA recombinant technique as means, after being transformed artificially by antibody gene produced by animal lymph cell, proceeds to eucaryon or former Nucleus is expressed, produces and there is immunocompetent antibody or its functional fragment.United States Medicine biologist in 1989 Hiatt etc. (Hiatt etal., 1989) have cloned heavy chain of antibody and light chain gene from mice, and respectively with agriculture bacillus mediated Method proceeds to Nicotiana tabacum L., by sexual hybridization and Screening and Identification, gives expression to the merit assembled by heavy chain and light chain in the same plant of offspring Energy property antibody, amount of antibody is up to the 1.3% of tobacco leaf total protein.His result of study is that human use's foreign cell expresses antibody Successful example, broken the boundary between animals and plants species, caused great interest and the great attention of biosphere.From this, Secreted antibody, complete antibody, chimeric antibody, Fab fragment, scFv fragment and double specific activity scFv fragments etc. are the most successfully Expressing in the organs such as blade, root, tuber and the seed at Nicotiana tabacum L., Rhizoma Solani tuber osi, Oryza sativa L., Semen Tritici aestivi or arabidopsis, expression is usual Account for total soluble protein 0.5%～2% (Barta et al., 1986；De Muynck et al.,2010；Desai et al.,2010；Xu et al.,2011；Huang and McDonald,2012).Nineteen ninety, (the During et such as During Al., 1990) heavy chain of antibody and light chain gene are loaded same expression vector box, obtain after proceeding to Nicotiana tabacum L. and have antigen combination to live The antibody of property.1991 Benvenuto (Benvenuto et al., 1991) the VH gene of a kind of neuropeptide monoclonal antibody inserted plant Thing expression vector, obtains in Nicotiana tabacum L. and expresses, and this single domain antibody has antigenic binding property, and expression accounts for soluble protein 1%.1992, Owen etc. (Owen et al., 1992) was built into the scFv gene of anti-phytochrome, after importing Nicotiana tabacum L., and scFv Gene is high level expression in transfer-gen plant.De Wilde etc. (De Wilde et al., 1998) are mouseearcress and Nicotiana tabacum L. In have expressed the Fab fragment of IgG antibody 1, and confirm that the antibody that two kinds of plants give expression to all has antigen binding capacity, but express Amount difference.Kathuria etc. (Kathuria et al., 2002) successfully have expressed the full length antibody of anti-HCG in Nicotiana tabacum L., anti- The scale of construction is up to the 4% of tobacco leaf total protein.Rodriguez etc. (Rodriguez et al., 2005) instantaneous table in tobacco leaf Reach the recombinant antibodies of anti-epidermal growth factor (EGF2R), and confirm the EGF2R of its recognizable A431 tumor cell surface.? Nearly Chen Lei and pass through codon successful expression bevacizumab in Oryza sativa L. in for often (Chen Lei and in for normal, 2015) (Bevacizumab) antibody, for laying the foundation by plant production anticancrin medicine.

Expressing Antibodies in Plants development includes following procedure: (1) Cloning Human Immunoglobulin Genes；(2) build antibody gene plant to express Carrier；(3) antibody gene imports plant cell or obtains genetic transformation plant；(4) expression of plantibody and purification.Antibody weight Chain and variable region of light chain (VH and VL) and constant region (Fc) genetic fragment all can pass through the PCR method hybridization from secretory antibody Oncocyte or obtain by expanding in phage antibody library.Antibody gene is expressed in plant to be needed to add plant expression startup Son, CaMV35S is conventional composition promoter, it is possible to utilize tissue-specific promoter to make antibody table in certain organs Reach.As with ScFv special accumulation in tobacco seed of LeB4 and the USP promoter expression from Semen Viciae fabae (Vicia faba).Anti- Body is secreted into the folding effective for antibody of specific organelle and is stably needs.(can plus signal peptide before antibody gene Deriving from plant, yeast, mice) antagonist synthesizes in specific subcellular components and accumulates is important.Existing research table Bright scFv fragment C end adds ER retention signal peptide KDEL or KDEI, it is possible in the accumulation of intracellular acquisition higher level.One A little whole antibodies or Fab and scFv all differ in cell inner expression amount, and the height of expression is to a great extent by antibody fragment The impact of skeleton own.It is generally acknowledged that action target is optimum selection at apoplast then IgG or Fab, and in scFv is endoplasmic reticulum The immunoregulatory optimum selection of target.

Express complete antibody with plant and can use different strategies.One is heavy chain and light chain gene to be converted respectively plant Thing, then obtain coexpression plant by sexual hybridization；Also can screen obtain altogether with the carrier cotransformation same plant of two genes Express plant, also can be gene constructed on same T-DNA two, render transgenic work is easier to.Antibody gene express with First Function Identification can be carried out with protoplast.Also the fermenting and producing setting up antibody secreting cell system for antibody can be screened.Anti- The approach of body channel genes recipient plant is typically agrobacterium-mediated transformation and particle bombardment, and other multiple transgenic method is also All can use.The recovery of plantibody and purification generally comprise multinomial step, as extracted, precipitate, adsorb, chromatograph and diafiltration etc..

At present, the expression system of genetic engineering antibody mainly have mammalian cell, escherichia coli, yeast, insecticide and Plant.1) mammalian cell expression system: rat bone marrow tumour cell is optimal expressive host, in addition with some B lymphs The cell line of cell derived.These cells have a whole set of complete synthesis, assembling, the cell device of immunoglobulin,exocrine, Complete antibody molecule can be produced.The advantage of mammalian cell expression is can correctly to be assembled by antibody polypeptides chain, fold And glycosyl is melted into activated entire molecule, the antibody of q.s can be expressed, but production cost is high.2) Escherichia coli system: Escherichia coli are widely used for expressing antibody function segment, and such as Fab, Fv and scFv, but it can't be expressed completely at present Antibody molecule.Utilize escherichia coli expression small molecular antibody, have that scale is big, speed fast and the advantage of low cost, before application Scape is gratifying.But a major issue in prokaryotic expression is how to efficiently control transcribe initial, carries out the expression envisioned.Mesh Front existing two kinds of expression vector is for the expression of antibody: IPTG induction type and temperature inducible, the startup of each of which Sub-tac2 and KPL is controlled by IPTG and temperature respectively.3) yeast expression system: yeast can effectively express, assembles and divide Secrete and there is immunocompetent antibody or its functional fragment, but owing to yeast is to the glycosylation of polypeptide and mammalian cell Modification situation is different, thus have impact on ACDC (relying on the cytotoxicity of the complement-mediated of the antibody) effect of antibody.It is now recognized that The expression system that yeast has been not.4) plant expression system: United States Medicine biologist Hiatt reported first in 1989 is anti- Body expression in plant.His result of study is the successful example that human use's foreign cell expresses antibody, has broken dynamic planting Boundary between species genus, causes great interest and the great attention of biosphere.Be currently used for express complete antibody, Fab and The plant of the antibody function segments such as scFv has Nicotiana tabacum L. and mouseearcress.The one of Expressing Antibodies in Plants is big, and advantage is can be by total length heavy chain It is assembled into the whole antibody (and the maximum antibody fragment that escherichia coli can assemble is monovalent Fab fragment) with Fc region with light chain, These antibody can functional identification antigen conjugated antigen.

Monoclonal antibody is the tetramer being assembled into by 2 heavy chains and 2 light chains.Heavy chain and light chain the most all pass through difference Gene expression frame express respectively.Or by IRES (internal ribosome entry site) system, by two bases Because expressing in same transcripton, but the equivalent that these methods all cannot realize heavy chain and light chain is expressed.As by different Gene expression frame, although using identical promoter and terminator sequence, but due to during gene transformation transgenic at base Because the on position in group is different, expressing of they is affected generation position effect by surrounding genes group sequence, and makes weight Chain is different with the expression of light chain protein.Although and use IRES system heavy chain and light chain in same transcripton, but at egg In white matter translation process due to after IRES the translation efficiency of gene lower than previous, therefore the expression of heavy chain and light chain is still Different.Although as in the past by Oryza sativa L. expression system also can be formed complete Bevacizumab antibody (Chen Lei, Yu Weichang, 2015), but due in expression system heavy chain and the light chain ratio of antibody the best, affect yield and antibody mass, such as complete antibody Tetrameric packaging efficiency is low, antibody degradation etc..

The recombiant protein additionally expressed plant, is often subject to the impact of plant-specific glycosylation.And plant specificity Protein glycosylation, such as β 1,2-xylose and α 1,3-fucose occur at Golgi body (Golgi).Plant-specific glycosylation Some characteristics of antibody can be affected, such as the adhesion of antibody, antibody stability in blood circulation and cause human body antagonist Immunoreation etc..These characteristics are the major defects that plant expresses recombiant protein, need to overcome.

Summary of the invention

It is an object of the invention to the deficiency overcoming prior art to exist, propose the plant source restructuring of brand-new a kind of optimization Humanization shellfish cuts down preparation method and the medical applications of monoclonal antibody.

An object of the present invention is: heavy chain and the light chain expressing equivalent by 2A, makes antibody packaging efficiency improve, subtracts Few degraded, thus improve yield and the quality of antibody；

Another object of the present invention is to retain sequence KDEL by design endoplasmic reticulum, thus be reduced or avoided Gorky Internal is glycosylation modified；

A further object of the present invention is, by design Furin protease cutting site, to excise 2A sequence, it is to avoid 2A sequence pair The impact of antibody；

A further object of the invention is that the shellfish that new method is developed is cut down monoclonal antibody is used for medicine, is used for treating mammary gland Cancer, is used for treating pulmonary carcinoma, glioblastoma, renal carcinoma, cervical cancer, ovarian cancer, colon cancer and rectal cancer.

In order to reach these purposes, the present invention proposes following technical scheme:

One, optimize plant source to express as a example by Oryza sativa L. is expressed

Bevacizumab Oryza sativa L. is expressed the technical scheme of optimization system and comprises the following steps:

Step 1. design contains 2A sequence and the bevacizumab heavy chain at Furin point of contact and light chain fusion protein.Such as sequence table Shown in SEQ ID NO:1, sequence comprises successively:

1) antibody heavy chain sequence BHC

MEFGLSWLFLVAILKGVQCEVQLVESGGGLVQPGGSLRLSCAASGYTFTNYGMNWVRQAPGKGLEWVGWINTYTGEP TYAADFKRRFTFSLDTSKSTAYLQMNSLRAEDTAVYYCAKYPHYYGSSHWYFDVWGQGTLVTVSSASTKGPSVFPLA PSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKP SNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVE VHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTK NQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYT QKSLSLSPGK(SEQ ID NO:1)

2) heavy chain endoplasmic reticulum retains sequence (KDEL) (SEQ ID NO:2)

3) Furin proteolytic cleavage site (RRKR) (SEQ ID NO:3)

4) connection peptides (GSG)

5) 2A sequence (QLLNFDLLKLAGDVESNPGP) (SEQ ID NO:4)

6) antibody light chain sequences BLC

MKYLLPTAAAGLLLLAAQPAMADIQMTQSPSSLSASVGDRVTITCSASQDISNYLNWYQQKPGKAPKVLIYFTSSLH SGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYSTVPWTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASV VCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSF NRGEC(SEQ ID NO:5)

7) light chain endoplasmic reticulum retains sequence (KDEL)

Step 2. is codon optimized with Oryza sativa L. and synthesizes above-mentioned antigen-4 fusion protein gene, such as sequence 2) shown in；Wherein comprise 5 ' PacI (TTAATTAA) and 3 ' MluI (ACGCGT) restriction endonuclease sites.

Step 3. builds bevacizumab plant binary expression vector.By antigen-4 fusion protein gene DNA fragmentation by PacI and MluI restriction endonuclease sites is inserted into the gene expression frame on binary vector pUN1390, and this gene is positioned at Semen Maydis After ubiquitin promoter, and before Nos terminator (Fig. 1).

Step 4. uses agrobacterium-mediated transformation by antibody gene Introduced into Rice callus cell and to pass through hygromycin selection Obtain genetic transformation rice plant.

Step 5. uses Southern Blot and Western Blot method detection bevacizumab table in Oryza sativa L. Reach.

Step 6. detects activity and the content of antibody in Oryza sativa L. by enzyme linked immunological (ELISA).

Step 7. is isolated and purified by ProteinA affinity column, obtains shellfish and cuts down monoclonal antibody.

Step 8. detects the antibody adhesion to antigen by SPR.

Step 9. is analyzed shellfish by protease hydrolysis and mass spectrometer and is cut down monoclonal antibody glycosylation.

Further, the shellfish that preparation method of the present invention obtains is cut down monoclonal antibody and prepares biological medicament with common drug carrier Thing is applied to people's body source.

Further, shellfish is cut down bio-pharmaceutical prepared by monoclonal antibody and is used for treating breast carcinoma, pulmonary carcinoma, glioblastoma Tumor, renal carcinoma, cervical cancer, ovarian cancer, colon cancer and rectal cancer.

The key problem in technology point of the present invention is:

Expressed heavy chain and the light chain of equivalent by 2A, make antibody packaging efficiency improve, reduce degraded, thus improve antibody Yield and quality；

By design endoplasmic reticulum retain sequence KDEL, thus be reduced or avoided in Golgi body glycosylation modified, subtract Few plant-specific glycosylation；

By design Furin protease cutting site, excise 2A sequence, do not affect structure and the function of antibody.

Advantages of the present invention and good effect

The present invention by the heavy chain of expressing recombinant antibody Bevacizumab in plant and light chain fusion protein, due to Fusion protein adds 2A sequence so that the heavy chain of antibody and light chain obtain the expression close to 1:1 ratio, and this ratio is to completely Obvious facilitation is played in the assembling of antibody, our result indicate that the antibody production applying this system expression is higher.Simultaneously It is assembled into more stable tetramer structure due to the heavy chain of recombinant antibodies and light chain equal proportion, and it is degradable to decrease comparison Unassembled polypeptide, thus obtain purer consistent monoclonal antibody.Additionally, due to devising Furin albumen before 2A sequence Enzyme action point so that 2A sequence is cut after expression, thus the antibody obtained does not contains 2A sequence, it is to avoid because introduce extra Sequence and the antibody structure that causes and the change of function.By adding KDEL endoplasmic reticulum at heavy chain of antibody and light chain polypeptide C-terminal Retain sequence so that antibody is retained in endoplasmic reticulum, and without Golgi body secretory pathway, decreases and produce through Golgi body Raw plant-specific glycosylation.And plant-specific glycosylation typically can affect the structure of recombiant protein, function, antibody is people The stability of systemic circulatory system and cause the human body immunoreation to this antibody.

Accompanying drawing explanation

Fig. 1 is the structure figure of the bevacizumab plant expression vector of the present invention, wherein antigen-4 fusion protein gene BHC-KDEL- After Furin-GSG-2A-BLC-KDEL is positioned at maize transcription promoter PUbi, before Nos transcription terminator TNos.HptII gene exists Hygromycin gene is expressed, for the sieve of transgenic plant between CaMV35S promoter (P35S) and 35S terminator (T35S) Choosing.LB and RB is respectively the right boundary of Agrobacterium binary vector pUN1390.

Fig. 2 be the bevacizumab of the present invention at transgenic paddy rice--hygromycin Rice Callus

Fig. 3 be the bevacizumab of the present invention at transgenic paddy rice--transgenic test tube Seedling

Fig. 4 be the bevacizumab of the present invention at transgenic paddy rice--Transgenic Rice Plants

Fig. 5 is that the Southern Blot of the present invention identifies bevacizumab transfer-gen plant figure；

Wherein, (a) EcoRI enzyme action produces～3kb BHC+BLC fusion protein fragment, utilizes the hybridization of BLC gene probe aobvious Show that all transgenic paddy rices have this transgenic；

B () EcoRI enzyme action, utilizes hygromycin gene (hptII) as probe, the different transgenic plant transfer of hybridization display The change of gene copy number, wherein 3,5,6,10 is single copy transgenic event；7,9,11,13,15 is two copy transgenic Event；Other are multicopy transgenic event；2 be wild rice DNA be negative control, 1 to be plasmid DNA right as the positive According to.

Fig. 6 is that the present invention uses Western Blot to analyze bevacizumab expression in transgenic paddy rice.

Wherein, A) degeneration glue, B) non denatured glue.PC:Bevacizumab monoclonal antibody (Avastin, Lot No: H0129；Roche Pharma, Switzerland) as positive control, NC be wild rice albumen be negative control, #1-9 For transgenic paddy rice.

Fig. 7 is that the ELISA of the present invention detects the active schematic diagram of antibody in transgenic plant.Wherein, PC is Arastin Positive control, NC is non-transgenic plant negative control, and other are transgenic plant；

Fig. 8 is that the ELISA detection of the present invention utilizes Arastin standard substance to draw canonical plotting；

Fig. 9 is that the ELISA of the present invention detects antibody content analysis schematic diagram in different transgenic plant；

Figure 10 be the present invention different transgenic plants in SDS-PAGE detection purify antibody (～150kD) schematic diagram.

Wherein, 1-2:5 μ g antibody；3-4:2 μ g antibody；5-6:1 μ g antibody；7:0.5 μ g Arastin compares.

Figure 11 is the combination situation map of surface plasma resonance (SPR) detection Arastin antibody and antigen；

Figure 12 is the antibody combination situation map with antigen of surface plasma resonance (SPR) the detection present invention.

Wherein, HL is the Bevacizumab antibody purified from transgenic paddy rice.

Detailed description of the invention

Technical scheme is further described in conjunction with drawings and Examples

The design of embodiment 1Bevacizumab antibody fusion protein, synthesis and the structure of plant expression vector

As shown in Figure 1, design contains 2A sequence and the bevacizumab heavy chain at Furin point of contact and light chain fusion protein.As Shown in sequence table SEQ ID NO:1, sequence comprises successively: antibody heavy chain sequence BHC, and endoplasmic reticulum retains sequence (KDEL) (SEQ ID NO:2), Furin proteolytic cleavage site (RRKR) (SEQ ID NO:3), connection peptides (GSG), 2A sequence (QLLNFDLLKLAGDVESNPGP) (SEQ ID NO:4), antibody light chain sequences BLC (SEQ ID NO:5) endoplasmic reticulum retains sequence Row (KDEL).Codon optimized with Oryza sativa L. and synthesize above-mentioned antigen-4 fusion protein gene, as shown in sequence 2.Wherein comprise 5 ' PacI And 3 ' MluI (ACGCGT) restriction endonuclease sites (TTAATTAA).

Antigen-4 fusion protein gene DNA fragmentation is inserted into binary vector by PacI and MluI restriction endonuclease sites Gene expression frame on pUN1390, this gene is positioned at after Semen Maydis ubiquitin promoter, and before Nos terminator.

Sequence 1: antibody fusion protein sequence

MEFGLSWLFLVAILKGVQCEVQLVESGGGLVQPGGSLRLSCAASGYTFTNYGMNWVRQAPGKGLEWVGWINTYTGEP TYAADFKRRFTFSLDTSKSTAYLQMNSLRAEDTAVYYCAKYPHYYGSSHWYFDVWGQGTLVTVSSASTKGPSVFPLA PSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKP SNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVE VHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTK NQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYT QKSLSLSPGKKDELRRKRGSGQLLNFDLLKLAGDVESNPGPMKYLLPTAAAGLLLLAAQPAMADIQMTQSPSSLSAS VGDRVTITCSASQDISNYLNWYQQKPGKAPKVLIYFTSSLHSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQY STVPWTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDS KDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECKDEL*(SEQ ID NO:9)；

Sequence 2: antibody fusion protein gene nucleic acid sequence (SEQ ID NO:10)

TTAATTAA (PacI restriction enzyme site) ATG (protein translation initiates) GAG TTC GGT CTC TCA TGG CTT TTT TTG GTT GCA ATT CTC AAA GGG GTT CAG TGC GAG GTG CAG TTG GTT GAA TCT GGT GGG GGG CTC GTT CAA CCG GGC GGG TCA CTT AGG CTC TCG TGT GCG GCT TCG GGA TAC ACG TTT ACG AAC TAC GGA ATG AAT TGG GTG AGG CAA GCA CCC GGA AAG GGC CTT GAG TGG GTT GGG TGG ATC AAT ACG TAC ACG GGT GAA CCT ACC TAC GCT GCT GAC TTT AAG AGA AGA TTT ACA TTC AGC CTG GAC ACA AGC AAG AGC ACC GCA TAT CTT CAG ATG AAC AGC CTT AGG GCT GAA GAT ACC GCG GTG TAC TAC TGC GCT AAA TAC CCT CAC TAC TAC GGG TCA AGT CAT TGG TAT TTC GAC GTT TGG GGG CAA GGG ACA TTG GTC ACA GTC TCT TCA GCT AGT ACT AAG GGA CCA TCC GTC TTT CCC CTG GCT CCA AGC AGT AAA AGC ACT AGC GGA GGG ACA GCG GCT TTG GGT TGT TTG GTC AAG GAT TAC TTT CCC GAA CCA GTC ACT GTG TCC TGG AAT TCG GGA GCT CTT ACC TCC GGG GTC CAC ACG TTC CCC GCC GTG CTG CAG AGC TCG GGG CTG TAC TCG CTG TCC TCC GTC GTC ACA GTT CCA TCG TCC TCG CTT GGA ACG CAG ACC TAT ATA TGC AAC GTT AAC CAT AAA CCT TCT AAC ACC AAA GTG GAT AAA AAA GTG GAG CCA AAA TCA TGT GAT AAA ACT CAT ACC TGC CCA CCT TGT CCA GCC CCG GAA TTG TTG GGT GGG CCG TCT GTT TTT CTG TTT CCC CCA AAA CCG AAG GAT ACA CTT ATG ATC TCC CGG ACG CCG GAG GTT ACG TGC GTT GTC GTT GAT GTG AGT CAT GAA GAC CCG GAG GTG AAA TTT AAC TGG TAC GTC GAT GGT GTC GAG GTC CAT AAC GCA AAA ACA AAG CCC CGC GAG GAA CAA TAC AAC TCG ACC TAC CGG GTC GTC AGC GTG CTG ACG GTC CTG CAC CAA GAT TGG CTC AAT GGA AAA GAA TAC AAG TGC AAG GTG AGC AAC AAA GCA CTG CCC GCG CCA ATC GAA AAG ACT ATC TCC AAA GCG AAA GGT CAG CCG AGG GAA CCC CAA GTG TAC ACC CTG CCG CCT AGT CGC GAG GAG ATG ACC AAG AAC CAG GTC AGT CTT ACG TGC CTG GTG AAG GGA TTT TAT CCA TCT GAT ATC GCA GTC GAG TGG GAA TCC AAT GGC CAG CCA GAA AAC AAT TAT AAG ACT ACC CCA CCT GTT CTC GAT AGC GAT GGC TCG TTT TTC CTG TAC AGC AAA CTG ACT GTC GAT AAA AAA GAC GAG CTC AGT AGG TGG CAA CAA GGC AAC GTG TTC TCT TGC TCG GTG ATG CAC GAA GCA CTC CAC AAC CAT TAC ACC CAG AAG TCA TTG TCC CTG TCA CCC GGG AAG CGC CGC AAA AGG GGT AGC GGA CAG CTT CTT AAC TTC GAT CTG TTG AAG CTT GCT GGA GAC GTC GAA AGT AAC CCT GGA CCA ATG AAG TAT TTG CTT CCG ACA GCA GCG GCG GGG TTG TTG CTT CTC GCC GCG CAA CCG GCT ATG GCC GAC ATA CAG ATG ACT CAG AGC CCA TCA TCG CTG AGT GCG TCC GTT GGT GAT CGC GTC ACA ATT ACT TGC TCA GCA AGT CAG GAT ATT TCA AAT TAT TTG AAC TGG TAC CAA CAG AAG CCG GGA AAG GCT CCT AAA GTT CTC ATC TAC TTC ACT AGT AGC TTG CAC AGC GGG GTC CCA TCC AGA TTC TCG GGC TCC GGG TCC GGC ACC GAT TTC ACG CTC ACC ATT TCA TCC CTT CAG CCC GAA GAC TTT GCC ACG TAT TAT TGT CAA CAA TAT TCT ACT GTG CCA TGG ACA TTT GGA CAG GGT ACC AAA GTC GAG ATT AAA AGA ACA GTT GCC GCA CCA TCA GTG TTT ATT TTT CCA CCA TCG GAC GAG CAA CTC AAA TCG GGT ACT GCC AGC GTC GTG TGC TTG CTT AAC AAC TTT TAC CCC AGA GAA GCA AAG GTT CAG TGG AAA GTT GAT AAT GCC CTC CAG TCG GGA AAC TCT CAA GAA TCC GTG ACC GAA CAA GAT AGC AAA GAT TCT ACA TAT TCC CTT AGT TCA ACA CTC ACA CTG TCA AAA GCA GAC TAC GAA AAG CAT AAG GTT TAT GCA TGT GAA GTT ACG CAC CAG GGT TTG TCC TCT CCA GTC ACG AAG AGT TTC AAC CGC GGG GAG TGT AAA GAC GAG CTC TAA (protein translation termination) ACGCGT (MluI restriction enzyme site)

The bevacizumab vector Rice Callus that embodiment 2 is agriculture bacillus mediated

1) induction of Rice Callus: the ripe fine seed of Japan is shelled, the alcohol disinfecting with 75% 30 seconds, then use 5% liquor natrii hypochloritis sterilizes 25 minutes, rinsed with sterile water 3～5 times, puts on aseptic paper, dries up on superclean bench, inoculation On N6D2 callus inducing medium, each culture dish inoculates 12～15, and in 28 DEG C of light culture 4 weeks, period excised embryo Root, subculture is once every two weeks.

2) activation of Agrobacterium tumefaciems: 1) Agrobacterium (EHA105) having converted bevacizumab plasmid is seeded in containing The card of 50mg/L is received in mycin and the flat LB fluid medium of 50mg/L profit fluorine, 28 DEG C, 200 rpms of shaken cultivation 18 of rotating speed Hour；2) take the cultured Agrobacterium of 1ml to be placed in the centrifuge tube of 2ml sterilizing, rotating speed 6000 rpms, within centrifugal 5 minutes, receive Collection thalline, with the resuspended thalline of AAM fluid medium of 200 μ l, takes 10 μ l bacterium solution and is inoculated into 50ml and contains 200 μMs of AS (acetyl fourths Ketone musk) AAM fluid medium in, 28 DEG C, 160 rpms of shaken cultivation of rotating speed 2 hours, in case convert be used.

3) Agrobacterium-mediated Transformation Oryza sativa L. embryo callus: 1) contaminate, choose the wound healing of the compact structure of a diameter of 3～5mm Tissue particles, contaminates 10min in the AAM bacterium solution prepared；2) co-culturing, the callus after contaminating is placed on aseptic filter paper On suck the bacterium solution of tissue surface, be forwarded to co-culture on base N6D2-Co, 26 DEG C of light culture 3 days；3) select to cultivate, will training altogether Support the callus rinsed with sterile water containing 0.01% tween 5 times of 3 days, then aseptic containing 500mg/L cephamycin Water rinses 10 minutes, callus is placed on aseptic filter paper, superclean bench dries the water on callus, then turns It is connected on Selective agar medium N6D-Se, 28 DEG C of light culture surroundings (Fig. 2)；4) differentiation culture, is forwarded to resistant calli point Changing in culture medium MS-Re, illumination in 16 hours, 8 hours dark, cultivates to differentiating green Seedling (Fig. 3) for 28 DEG C.It is forwarded to seedling use On the root media MS-Hf that sterilizing is bottled, after cultivating 2 weeks, clean the culture medium on little shoot root and forward cultivation 3 days in water to, so After be transplanted to land for growing field crops (Fig. 4).

4) culture medium needed for rice tissue is cultivated:

N6D2 culture medium: 3.9g/L Chus N6 (Chu, 1975) (CHP01-50LT, 10402809, Caisson, USA), N6 vitamin, (2mg/L glycine, 0.5mg/L nicotinic acid, 1.0mg/L vitamin B1,0.5mg/L vitamin B6), 0.1g/L flesh Alcohol, 1.0g/L caseinhydrolysate, 0.5g/L proline, 0.5g/L glutamine, 2mg/L 2,4-dichlorphenoxyacetic acid (2,4- D), 30g/L sucrose, 1M KOH regulates pH5.8,3.0g/L plant gel, 121 DEG C, 220KPa, sterilizing 20 minutes.

N6D2-Co culture medium: adding 10g/L glucose in N6D2,1M KOH regulates pH5.5,3.0g/L plant gel, 121 DEG C, 220KPa, sterilizing 10 minutes, treat that culture medium is cooled to 50 DEG C, add the acetosyringone of 200uM (Acetosyringone, AS).

N6D2-Se culture medium: N6D2 culture medium is cooled to 50 DEG C, 121 DEG C, 220KPa, sterilizing 20 minutes, add 50mg/L HYG and 500mg/L cephamycin.

MS-Re culture medium: 4.6g/L MS (M10400-50.0, P06968, rpi, USA), 2.0mg/L6-benzyl amino gland is fast Purine (6-BA), 0.5mg/L naphthalene acetic acid (NAA), 1.0mg/L kinetins (KT), 30g/L sucrose, 30g/L sorbitol, 1M KOH adjusts Joint pH5.8,3.0g/L Gelrite, 121 DEG C, 220KPa, sterilizing 20 minutes, be cooled to 50 DEG C, add 50mg/L HYG and 500mg/L cephamycin.

MS-HF culture medium: 2.3g/L MS (M10400-50.0, P06968, rpi, USA), 30g/L sucrose, 1M KOH adjusts PH5.8,3.0g/L Gelrite, 121 DEG C, 220KPa, sterilizing 20 minutes, it is cooled to 50 DEG C, adds 50mg/L HYG.

AAM culture medium: 0.5g/L caseinhydrolysate, 68.5g/L sucrose, 36g/L glucose, 0.9g/L glutamine, 0.3g/L aspartic acid, 3g/L potassium chloride, 10mg/L manganese sulfate pentahydrate, 3.0mg/L boric acid, 2.0mg/L zinc sulphate heptahydrate, 0.25mg/L molybdate dihydrate acid is received, 0.025mg/L copper sulphate pentahydrate, 0.025mg/L cobalt chloride hexahydrate, 0.75mg/L potassium iodide, 15mg/L calcium chloride dihydrate, 25mg/L Magnesium sulfate heptahydrate, 4mg/L ethylenediaminetetraacetic acid (EDTA), 15mg/L dihydrogen phosphate dihydrate Sodium, 1mg/L nicotinic acid, 1mg/L vitamin B6,10mg/L vitamin B1,100mg/L inositol, 176mg/L arginine, 75mg/L is sweet Propylhomoserin, 1M KOH regulates pH to 5.2, and 0.22uM membrane filtration is degerming.

Embodiment 3Southern blot identifies bevacizumab transgenic paddy rice (Fig. 5)

1) probe of digoxigenin labeled is prepared: with bevacizumab binary expression vector DNA as template, light chain (BLC) and tide The probe of the digoxigenin labeled of light chain and hptII is prepared in forward and reverse primer amplification respectively of mould plain gene (hptII).

PCR reaction solution is: plasmid DNA 10ng as template, each 1 μ L of forward and reverse primer, Premix Ex Taq Hot Start Version 15 μ l, the DIG-dUTP of 1 μ L, distilled water constant volume to 30 μ L.

Pcr amplification reaction program is: 95 DEG C 5 minutes, 95 DEG C 20 seconds, 60 DEG C 20 seconds, 72 DEG C 1.5 minutes, 30 circulations, 72 DEG C 5 minutes.After PCR reaction terminates, take 3 μ L reactant liquor electrophoresis detection.

2) turn the enzymolysis of bevacizumab trans-genetic hybrid rice STb gene: turn bevacizumab trans-genetic hybrid rice blade STb gene 15ug, use EcoRI carries out enzymolysis, and 37 DEG C of reactions are overnight.The enzyme digestion reaction system of each sample is: STb gene 15 μ g, CutSmart buffer 10 μ L, the EcoRI of 100U, distilled water constant volume 100 μ L.Wherein cut down list using wild rice genomic DNA as negative control, shellfish Anti-plasmid is positive control.After enzyme digestion reaction terminates, loading 5 μ L carries out electrophoresis detection, purification, is dissolved in 15 μ L distilled waters, 4 DEG C standby or-20 DEG C of preservations.

3) electrophoresis of enzymatic hydrolysate: enzymatic hydrolysate after purification is splined on 1.0% agarose gel, with 5V/cm voltage Electrophoresis 3 hours；Take pictures, the position of labelling molecular weight；Gel is put in 0.2N hydrochloric acid process 10 minutes, rinses one with distilled water Under；Put in denaturation buffer and process 30 minutes；Put in neutralization buffer and process 30 minutes.

4) DNA is imprinted onto hybond membrane: according to the size of agarose gel, the sizeable hybond membrane of clip, uses distilled water Moistening；Glass plate is placed on pallet, takes 1 3M filter paper, soaks with transfering buffering liquid, puts on a glass, removes filter paper and glass Bubble in glass plate.Appropriate transfering buffering liquid is added in pallet；Gel is placed on filter paper, removes between gel and imprinting surface Bubble；Hybond membrane is placed on above gel, and aerofluxus bubble again；By 2 3M filter paper (more slightly larger than hybond membrane) transfering buffering liquid It is placed on hybond membrane after Shi Tou；Around glue, put a circle rupture disk, prevent the liquid in liquid pool from flowing directly to above gel Tissue layer (i.e. " short-circuit " phenomenon).Putting one on filter paper and fold absorbent paper, then press the object weighing about 300g, transfer is overnight； Hybond membrane is rinsed in the transfering buffering liquid of dilution 10 times, is placed under super-clean bench and dries, dry 2 hours in 80 DEG C of baking ovens.

5) hybridization: the film aquesterilisa moistening after baking is rolled, puts in hybrid pipe, add 30ml hybridization solution, be placed on 42 DEG C of prehybridizations 2 hours；Outwell prehybridization solution, add hybridization solution new for 10ml in 42 DEG C of preheatings；Take out the spy being ready for Pin, thawed on ice, join in the hybridization solution having been warmed up, 42 DEG C of hybridized overnight.

6) hybridization post processing: pour out hybridization solution, pour 100ml elution buffer W1 in hybrid pipe into, eluting 5 points under room temperature Clock, is repeated once.Outwell elution buffer W1, add 100ml elution buffer W2, in 65 DEG C of eluting 15 minutes, be repeated once； Take out film, put in the little square position being covered with preservative film, add elution buffer W3, eluting 5 minutes；Outwell elution buffer W3, Add 50ml confining liquid S1, incubated at room 15 minutes；Outwell confining liquid S1, add 10ml DigiTAb reactant liquor S2, hatch 15 minutes；Take the film out and put into another box, add 100ml elution buffer W3, eluting 15 minutes under room temperature, repeat eluting Once；Add the detection buffer S3 of 20mL, hatch 5 minutes；Outwell detection buffer S3, add 2ml CSPD and hatch 5 minutes； Take the film out and be laid on preservative film, wrapped up, be placed on 37 DEG C and hatch 5 minutes.

7) development is with fixing: is placed in camera obscura by the film wrapped, is placed on by X-film on the film wrapped in dark place, closes Close dark folder, place 30 minutes for 37 DEG C；Return and darkroom is taken out film, put into and developer solution soaks 2 minutes, at Shui Zhongchong once, then Putting into fixative solution to soak 2 minutes, develop photographic film in flowing water, taking-up is dried.

8) reagent needed for Southern blot:

Denaturation buffer: 20g/L NaOH, 87.75g/L NaCl；

Neutralization buffer: 60.05g/L Tris (trishydroxymethylaminomethane), 87.75g/L NaCl, the concentrated hydrochloric acid of 37% Regulation pH7.2；

Transfering buffering liquid (20 × SSC): 175.3g/L NaCl, 88.2g/L trisodium citrate 4M hydrochloric acid regulation pH7.0；

Elution buffer W1:2 × SSC；0.1%SDS (dodecyl sodium sulfate) (M/V)；

Elution buffer W2:0.5 × SSC；0.1%SDS；

Elution buffer W3:11.607g/L maleic acid, 8.775g/L NaCl, with solid NaoH regulation pH value to 7.5, uses Front addition 0.3% polysorbas20；

Detection buffer S3:12.1g/L Tris, 5.85g/L NaCl, regulate pH 9.5；

Hybridization solution: 70g/L SDS, 50% Methanamide, 25%20XSSC, 2% defatted milk powder (Roche), 50ml 1M phosphoric acid Sodium buffer (pH7.2), 1.0g/L dodecyl musculamine acid sodium；

Confining liquid S1:1% defatted milk powder (M/V) is dissolved in elution buffer W3；

DigiTAb reactant liquor S2: be centrifuged 5 minutes with 10000 rpms by Anti-Digoxigenin-AP, draws Upper solution is dissolved in confining liquid S1 with 1:10000.

Embodiment 4Western Blot analyzes bevacizumab expression in transgenic paddy rice

1) prepared by sample:

A) with transgenic paddy rice blade as material, grind in liquid nitrogen.Weigh the ground leaf tissue of 30mg and add 30 μ l Extraction buffer (200mM Tris-HCl, pH 8.0,100mM NaCl, 400mMsucrose, 10mM EDTA, 1mM Phenylmethylsulfonyl fluoride, 0.05%Tween20), vortex vibrates, and places on ice 10 minutes, then exists 4 DEG C, 13000 rpms centrifugal 10 minutes, takes supernatant in new centrifuge tube.

2) electrophoretic separation: 25 μ g are extracted total protein and the mixing of SDS sample-loading buffer, then under Denaturing, sample Add 5% mercaptoethanol 2-mercaptoethanol before loading, boil degeneration in 5 minutes) or non denatured under the conditions of run 10%SDS-PAGE Electrophoresis.100V electrophoresis 2 hours.

3) transferring film: unload offset plate, peels off glue, and glue is dipped in transfering buffering liquid balance 10min.Size clip according to glue Film and filter paper 6, put into pvdf membrane methanol immersion and within saturated 3-5 second, put into balance 10min in transfering buffering liquid.Assembling transfer Sandwich: be followed successively by 3 metafiltration paper, glue, pvdf membrane, 3 metafiltration paper from bottom to top, after every layer is put well, rushes bubble with test tube.Plug Electrode, 20V, 45 minutes.After transferring film terminates, cut off the electricity supply, take out hybond membrane.

4) immuning hybridization and colour developing: put into by film in distilled water and soak 10 minutes, takes out and puts into 20ml 5% defatted milk powder Confining liquid in incubated at room 1 hour.Add horseradish peroxidase (HRP) the labelling goat anti-human igg (LC+HC) of dilution 5000 times (ProteinTech Group, USA), incubated at room 2 hours.15mlTBS-T washes 3 times, each 5min.Protein Detection (according to Thermo Pierce ECL Western BlottingSubstrate test kit operates).

5) degeneration SDS-PAGE demonstrates 2 bands, be respectively～50kDa heavy chain and～25kDa light chain (Fig. 6 A).

6) non denatured SDS-PAGE demonstrates 1～150kDa band, for the antibody tetramer (Fig. 6 B), does not observe fall Hydrolysis products.

7) by the gray value of band in ImageJ software analysis Fig. 6 A, the ratio such as table 1 of light chain/heavy chain is calculated.9 turn In gene strain, the average of relatives number of light chain/heavy chain is 0.99, identical with the ratio of comparison commodity Arastin.

Light chain/heavy chain expression amount ratio in the different transgenic plant of table 1.

Transgenic plant line	LC:HC ratio
		Positive control PC	0.99
1	0.68
		2	1.18
3	1.00
		4	0.99
5	1.00
		6	0.11
7	0.60
		8	1.49
9	1.90
		Meansigma methods+variance	0.99±0.52

The activity analysis and quantitatively of Bevacizumab antibody in embodiment 5 transgenic paddy rice

1) extraction of transgenic plant total protein is with example 4, after then pressing 1:100 dilution, goes 100 μ L to add ELISA enzyme In mark version hole, hatch 1 hour in 25 DEG C, allow the hVEGF antigen protein in antibody and enzyme mark version hole fully combine.In enzyme mark version hole Be pre-coated with hVEGF growth factor protein (SHIKARIQ-BEVA, Matriks Biotechnology Co.Ltd., Turkey)。

2), after washing 3 times with 1xPBST, the streptavidine of horseradish peroxidase (HRP) labelling is added, and at 25 DEG C Hatch 0.5 hour.

3), after washing 3 times with 1xPBST, add tmb substrate, and hatch 20 minutes at 25 DEG C.

4) add stop buffer and terminate reaction, by microplate reader in the readings of 450nm wavelength detecting color reaction.

5) react as negative control, transgenic plant using commodity Arastin as positive control, non-transgenic plant It is all the positive (Fig. 7), it was demonstrated that the activated antibody of Expressed in Transgenic Plant, can combine with antigen.

6) utilize Arastin for standard substance, draw standard curve (Fig. 8) by above-mentioned ELISA method.

7) by comparing with standard curve, the content (Fig. 9) of antibody in transgenic plant, the minimum every public affairs of content are calculated Jin fresh weight, containing 160.7mg, reaches as high as 242.8mg.

Embodiment 6 purifies Bevacizumab monoclonal antibody by affinity chromatograph from transgenic paddy rice

1) taking 50 grams of transgenic plant materials, the extraction of total protein is with embodiment 4.

2) protein crude extract is through 0.45 μm membrane filtration.

3) with protein binding buffer (0.02M sodium phosphate buffer, pH 7.0) balance HiTrap Protein A HP post.

4) the protein extract upper prop after filtering, and wash post with the protein binding buffer of 5 times of column volumes.

5) with the antibody combined on eluent (0.1M Glycine-HCl, pH 2.7) eluting post, and it is collected in and washes containing 1/5 In the 1M Tris-HCl (pH 9.0) that lift-off is long-pending, neutralize pH value 7.0.

6) filter membrane (molecular weight, 30kDa, Millipore, the USA) ultrafiltration of the antibody molecular weight 30kDa collected is pure Change.

7) utilizing ELISA method in examples detailed above 5 to measure antibody concentration, the concentration being recovered to antibody in two samples is suitable In every gram of flesh tissue, antibody content is 135.1 and 101.1 μ g, and the response rate is respectively 55.6% and 59.8%.

8) utilize in examples detailed above 4 and separate on non denatured SDS-PAGE glue, coomassie brilliant blue staining method detection antibody Quality and purity.Found that overwhelming majority antibody is about the tetramer (Figure 10) of 150kDa and a small amount of catabolite.Purity is big In 95%.

9) antibody purified can be used for following SPR Binding experiment and glycosylation analysis.

Embodiment 7 surface plasma resonance SPR detection antibody and the adhesion of antigen

1) hVEGF antigen protein is incorporated on GE CM5 induction chip.

2) with variable concentrations, Arastin antibody and above-mentioned purification antibody are combined with the antigen on chip respectively, detect table Face plasma resonance situation (Figure 11,12) calculating antibody and the combination of antigen and the coefficient that dissociates (table 2).

3) antibody as can be seen from Table 2, purified from transgenic paddy rice is better than A Wasi to the adhesion of antigen hVEGF Spit of fland antibody, it is possible to the substitute products as Arastin are used for treatment of cancer.

The combination (ka) of table 2. antibody and antigen and dissociate (kd) coefficient and adhesion (KD)

	ka(1/Ms)	kd(1/s)	KD(M)
				HL	1.90E+04	5.35E-04	2.81E-08
PC	9.21E+03	1.82E-03	1.98E-07

Embodiment 8 transgenic paddy rice purifies the glycosylation analysis of antibody

1) the antibody Trypsin protease hydrolysis purified in above-described embodiment 4 becomes polypeptide.

2) polypeptide uses quadrupole time-of-flight (Q-TOF) after the reverse chromatography of capillary tube Ultima Global (Waters) mass spectrograph is analyzed.

3) Peptide Mass program (http://www.expasy.org/tools/peptide-is utilized Mass.html) the Bevacizumab aminoacid sequence of mass spectrometric data and Trypsin protease hydrolysis is compared draw glycosylation Data (as shown in table 3).

4) as can be seen from Table 3, plant-specific glycosylation, including MMXF (M3Gn2X1F1), GnMXF (M3Gn3X1F1) The 46.8% of total amount is accounted for GnGnXF (M3Gn4X1F1).

Table 3. transgenic paddy rice expresses the glycosylation of monoclonal antibody Bevacizumab

Claims

1. the plant source recombinant humanized shellfish of an optimization cuts down the preparation method of monoclonal antibody, it is characterised in that: include following Step:

Step 1. design contains 2A sequence and the bevacizumab heavy chain at Furin point of contact and light chain fusion protein, and sequence comprises successively:

1) antibody heavy chain sequence BHC

MEFGLSWLFLVAILKGVQCEVQLVESGGGLVQPGGSLRLSCAASGYTFTNYGMNWVRQAPGKGLEWVGWINTY TGEPTYAADFKRRFTFSLDTSKSTAYLQMNSLRAEDTAVYYCAKYPHYYGSSHWYFDVWGQGTLVTVSSASTKGPSV FPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNV NHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYV DGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRE EMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALH NHYTQKSLSLSPGK(SEQ ID NO:1)

2) heavy chain endoplasmic reticulum retains sequence (KDEL) (SEQ ID NO:2)

3) Furin proteolytic cleavage site (RRKR) (SEQ ID NO:3)

4) connection peptides (GSG)

5) 2A sequence (QLLNFDLLKLAGDVESNPGP) (SEQ ID NO:4)

6) antibody light chain sequences BLC

MKYLLPTAAAGLLLLAAQPAMADIQMTQSPSSLSASVGDRVTITCSASQDISNYLNWYQQKPGKAPKVLIYFT SSLHSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYSTVPWTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSG TASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPV TKSFNRGEC(SEQ ID NO:5)

7) light chain endoplasmic reticulum retains sequence (KDEL)

Step 2. is codon optimized with Oryza sativa L. and synthesizes above-mentioned antigen-4 fusion protein gene；

Step 3. builds bevacizumab plant binary expression vector；

Step 4. uses agrobacterium-mediated transformation by antibody gene Introduced into Rice callus cell and to be obtained by hygromycin selection Genetic transformation rice plant；

Step 5. uses Southern Blot and the detection bevacizumab expression in Oryza sativa L. of Western Blot method；

Step 6. detects activity and the content of antibody in Oryza sativa L. by enzyme linked immunological (ELISA)；

Step 7. passes through the isolated and purified antibody of ProteinA affinity column；

Step 8. detects the antibody adhesion to antigen by SPR；

Step 9. is analyzed shellfish by protease hydrolysis and mass spectrometer and is cut down monoclonal antibody glycosylation；Obtain standardized shellfish Cut down monoclonal antibody.

The plant source recombinant humanized shellfish of a kind of optimization the most according to claim 1 cuts down the preparation method of monoclonal antibody, It is characterized in that: described step 1 determining, Furin proteolytic cleavage site is RXXR (SEQ IDNO:6), RX (R/K) R (SEQ ID NO:7) or RRKR (SEQ ID NO:8), wherein R is arginine, and X is any aminoacid, and K is lysine, its Antibody Fusion Protein sequence table is:

MEFGLSWLFLVAILKGVQCEVQLVESGGGLVQPGGSLRLSCAASGYTFTNYGMNWVRQAPGKGLEWVGWINTY TGEPTYAADFKRRFTFSLDTSKSTAYLQMNSLRAEDTAVYYCAKYPHYYGSSHWYFDVWGQGTLVTVSSASTKGPSV FPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNV NHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYV DGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRE EMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALH NHYTQKSLSLSPGKKDELRRKRGSGQLLNFDLLKLAGDVESNPGPMKYLLPTAAAGLLLLAAQPAMADIQMTQSPSS LSASVGDRVTITCSASQDISNYLNWYQQKPGKAPKVLIYFTSSLHSGVPSRFSGSGSGTDFTLTISSLQPEDFATYY CQQYSTVPWTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVT EQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECKDEL*(SEQ ID NO:9)；

Described antibody fusion protein gene nucleic acid sequence (SEQ ID NO:10) is:

TTAATTAA (PacI restriction enzyme site) ATG (protein translation initiates) GAG TTC GGT CTC TCA TGG CTT TTT TTG GTT GCA ATT CTC AAA GGG GTT CAG TGC GAG GTG CAG TTG GTT GAA TCT GGT GGG GGG CTC GTT CAA CCG GGC GGG TCA CTT AGG CTC TCG TGT GCG GCT TCG GGA TAC ACG TTT ACG AAC TAC GGA ATG AAT TGG GTG AGG CAA GCA CCC GGA AAG GGC CTT GAG TGG GTT GGG TGG ATC AAT ACG TAC ACG GGT GAA CCT ACC TAC GCT GCT GAC TTT AAG AGA AGA TTT ACA TTC AGC CTG GAC ACA AGC AAG AGC ACC GCA TAT CTT CAG ATG AAC AGC CTT AGG GCT GAA GAT ACC GCG GTG TAC TAC TGC GCT AAA TAC CCT CAC TAC TAC GGG TCA AGT CAT TGG TAT TTC GAC GTT TGG GGG CAA GGG ACA TTG GTC ACA GTC TCT TCA GCT AGT ACT AAG GGA CCA TCC GTC TTT CCC CTG GCT CCA AGC AGT AAA AGC ACT AGC GGA GGG ACA GCG GCT TTG GGT TGT TTG GTC AAG GAT TAC TTT CCC GAA CCA GTC ACT GTG TCC TGG AAT TCG GGA GCT CTT ACC TCC GGG GTC CAC ACG TTC CCC GCC GTG CTG CAG AGC TCG GGG CTG TAC TCG CTG TCC TCC GTC GTC ACA GTT CCA TCG TCC TCG CTT GGA ACG CAG ACC TAT ATA TGC AAC GTT AAC CAT AAA CCT TCT AAC ACC AAA GTG GAT AAA AAA GTG GAG CCA AAA TCA TGT GAT AAA ACT CAT ACC TGC CCA CCT TGT CCA GCC CCG GAA TTG TTG GGT GGG CCG TCT GTT TTT CTG TTT CCC CCA AAA CCG AAG GAT ACA CTT ATG ATC TCC CGG ACG CCG GAG GTT ACG TGC GTT GTC GTT GAT GTG AGT CAT GAA GAC CCG GAG GTG AAA TTT AAC TGG TAC GTC GAT GGT GTC GAG GTC CAT AAC GCA AAA ACA AAG CCC CGC GAG GAA CAA TAC AAC TCG ACC TAC CGG GTC GTC AGC GTG CTG ACG GTC CTG CAC CAA GAT TGG CTC AAT GGA AAA GAA TAC AAG TGC AAG GTG AGC AAC AAA GCA CTG CCC GCG CCA ATC GAA AAG ACT ATC TCC AAA GCG AAA GGT CAG CCG AGG GAA CCC CAA GTG TAC ACC CTG CCG CCT AGT CGC GAG GAG ATG ACC AAG AAC CAG GTC AGT CTT ACG TGC CTG GTG AAG GGA TTT TAT CCA TCT GAT ATC GCA GTC GAG TGG GAA TCC AAT GGC CAG CCA GAA AAC AAT TAT AAG ACT ACC CCA CCT GTT CTC GAT AGC GAT GGC TCG TTT TTC CTG TAC AGC AAA CTG ACT GTC GAT AAAAAA GAC GAG CTC AGT AGG TGG CAA CAA GGC AAC GTG TTC TCT TGC TCG GTG ATG CAC GAA GCA CTC CAC AAC CAT TAC ACC CAG AAG TCA TTG TCC CTG TCA CCC GGG AAG CGC CGC AAA AGG GGT AGC GGA CAG CTT CTT AAC TTC GAT CTG TTG AAG CTT GCT GGA GAC GTC GAA AGT AAC CCT GGA CCA ATG AAG TAT TTG CTT CCG ACA GCA GCG GCG GGG TTG TTG CTT CTC GCC GCG CAA CCG GCT ATG GCC GAC ATA CAG ATG ACT CAG AGC CCA TCA TCG CTG AGT GCG TCC GTT GGT GAT CGC GTC ACA ATT ACT TGC TCA GCA AGT CAG GAT ATT TCA AAT TAT TTG AAC TGG TAC CAA CAG AAG CCG GGA AAG GCT CCT AAA GTT CTC ATC TAC TTC ACT AGT AGC TTG CAC AGC GGG GTC CCA TCC AGA TTC TCG GGC TCC GGG TCC GGC ACC GAT TTC ACG CTC ACC ATT TCA TCC CTT CAG CCC GAA GAC TTT GCC ACG TAT TAT TGT CAA CAA TAT TCT ACT GTG CCA TGG ACA TTT GGA CAG GGT ACC AAA GTC GAG ATT AAA AGA ACA GTT GCC GCA CCA TCA GTG TTT ATT TTT CCA CCA TCG GAC GAG CAA CTC AAA TCG GGT ACT GCC AGC GTC GTG TGC TTG CTT AAC AAC TTT TAC CCC AGA GAA GCA AAG GTT CAG TGG AAA GTT GAT AAT GCC CTC CAG TCG GGA AAC TCT CAA GAA TCC GTG ACC GAA CAA GAT AGC AAA GAT TCT ACA TAT TCC CTT AGT TCA ACA CTC ACA CTG TCA AAA GCA GAC TAC GAA AAG CAT AAG GTT TAT GCA TGT GAA GTT ACG CAC CAG GGT TTG TCC TCT CCA GTC ACG AAG AGT TTC AAC CGC GGG GAG TGT AAA GAC GAG CTC TAA (protein translation termination) ACGCGT (MluI restriction enzyme site), wherein comprises 5 ' PacI (TTAATTAA) and 3 ' MluI (ACGCGT) restriction endonuclease sites.

The plant source recombinant humanized shellfish of a kind of optimization the most according to claim 1 cuts down the preparation method of monoclonal antibody, It is characterized in that: described step 3 builds bevacizumab plant binary expression vector, be that antigen-4 fusion protein gene DNA fragmentation is passed through PacI and MluI restriction endonuclease sites is inserted into the gene expression frame on binary vector pUN1390, and this gene is positioned at Semen Maydis After ubiquitin promoter, and before Nos terminator；Above-described restriction enzyme site, it is also possible to be any restricted Restriction enzyme site, is inserted into the two ends of recombination fragment by gene chemical synthesis, PCR primer, or catenation sequence, in order to will restructuring Gene is inserted into corresponding site；Above-mentioned promoter can be any promoter being conducive to gene expression, as general in above-mentioned Semen Maydis Element (ubiquitin) promoter, Oryza sativa L. Actin promoter, cauliflower mosaic virus (CaMV) 35S promoter, Agrobacterium Nos opens The composition promoteres such as mover；Tissue-specific promoter, such as Gt1 specific expressed in paddy endosperm, GluA, GluB open Mover；Inducible promoter, such as rice drought evoked promoter Oshox24P, estradiol abduction delivering promoter XVE；Above-mentioned end Only son refers to any DNA sequence enabling to genetic transcription termination, such as above-mentioned Nos terminator and T35 terminator.

The plant source recombinant humanized shellfish of a kind of optimization the most according to claim 1 cuts down the preparation method of monoclonal antibody, It is characterized in that: described step 4 uses particle gun, Agrobacterium or other gene transformation by antibody gene Introduced into Rice wound healing group Knitting cell and obtain genetic transformation rice plant by hygromycin selection, it specifically comprises the following steps that

1) induction of Rice Callus: the ripe fine seed of Japan is shelled, the alcohol disinfecting with 75% 30 seconds, then with 5% Liquor natrii hypochloritis sterilizes 25 minutes, rinsed with sterile water 3～5 times, puts on aseptic paper, dries up on superclean bench, is inoculated in On N6D2 callus inducing medium, each culture dish inoculates 12～15, and in 28 DEG C of light culture 4 weeks, period excised radicle, Subculture is once every two weeks；

2) activation of Agrobacterium tumefaciems: 1) Agrobacterium (EHA105) having converted bevacizumab plasmid is seeded in containing 50mg/L Card receive in mycin and the flat LB fluid medium of 50mg/L profit fluorine, 28 DEG C, 200 rpms of shaken cultivation of rotating speed 18 hours； 2) take the cultured Agrobacterium of 1ml to be placed in the centrifuge tube of 2ml sterilizing, rotating speed 6000 rpms, within centrifugal 5 minutes, collect bacterium Body, with the resuspended thalline of AAM fluid medium of 200 μ l, takes 10 μ l bacterium solution and is inoculated into 50ml and contains 200 μMs of AS (acetyl Flos Caryophyllis Ketone) AAM fluid medium in, 28 DEG C, 160 rpms of shaken cultivation of rotating speed 2 hours, in case convert be used；

3) Agrobacterium-mediated Transformation Oryza sativa L. embryo callus: 1) contaminate, choose the callus of the compact structure of a diameter of 3～5mm Granule, contaminates 10min in the AAM bacterium solution prepared；4) co-culturing, the callus after contaminating is placed on aseptic filter paper suction Go the bacterium solution of tissue surface, be forwarded to co-culture on base N6D2-Co, 26 DEG C of light culture 3 days；3) select to cultivate, 3 days will be co-cultured The callus rinsed with sterile water containing 0.01% tween 5 times, then float in the sterilized water containing 500mg/L cephamycin Wash 10 minutes, callus is placed on aseptic filter paper, superclean bench dries the water on callus, then is forwarded to choosing Select in culture medium N6D-Se, 28 DEG C of light culture surroundings；4) differentiation culture, is forwarded to division culture medium MS-by resistant calli On Re, illumination in 16 hours, 8 hours dark, cultivates to differentiating green Seedling for 28 DEG C；Seedling is forwarded to the take root training bottled with sterilizing Support on base MS-Hf, after cultivating 2 weeks, clean the culture medium on little shoot root and forward to water is cultivated 3 days, be then transplanted to land for growing field crops.

The plant source recombinant humanized shellfish of a kind of optimization the most according to claim 4 cuts down the preparation method of monoclonal antibody, It is characterized in that: described rice tissue is cultivated required culture medium and is:

N6D2 culture medium: 3.9g/L Chus N6 (Chu, 1975) (CHP01-50LT, 10402809, Caisson, USA), N6 tie up Raw element, (2mg/L glycine, 0.5mg/L nicotinic acid, 1.0mg/L vitamin B1,0.5mg/L vitamin B6), 0.1g/L inositol, 1.0g/L caseinhydrolysate, 0.5g/L proline, 0.5g/L glutamine, 2mg/L 2,4-dichlorphenoxyacetic acid (2,4-D), 30g/L sucrose, 1M KOH regulates pH5.8,3.0g/L plant gel, 121 DEG C, 220KPa, sterilizing 20 minutes；

N6D2-Co culture medium: adding 10g/L glucose in N6D2,1M KOH regulates pH5.5,3.0g/L plant gel, 121 DEG C, 220KPa, sterilizing 10 minutes, treat that culture medium is cooled to 50 DEG C, add 200uM acetosyringone (Acetosyringone, AS)；

N6D2-Se culture medium: N6D2 culture medium is cooled to 50 DEG C, 121 DEG C, 220KPa, sterilizing 20 minutes, add 50mg/L tide mould Element B and 500mg/L cephamycin；

MS-Re culture medium: 4.6g/L MS (M10400-50.0, P06968, rpi, USA), 2.0mg/L 6-benzyl aminoadenine (6-BA), 0.5mg/L naphthalene acetic acid (NAA), 1.0mg/L kinetins (KT), 30g/L sucrose, 30g/L sorbitol, 1M KOH regulates PH5.8,3.0g/L Gelrite, 121 DEG C, 220KPa, sterilizing 20 minutes, be cooled to 50 DEG C, add 50mg/L HYG and 500mg/L cephamycin；

MS-HF culture medium: 2.3g/L MS (M10400-50.0, P06968, rpi, USA), 30g/L sucrose, 1M KOH adjusts PH5.8,3.0g/L Gelrite, 121 DEG C, 220KPa, sterilizing 20 minutes, it is cooled to 50 DEG C, adds 50mg/L HYG；

AAM culture medium: 0.5g/L caseinhydrolysate, 68.5g/L sucrose, 36g/L glucose, 0.9g/L glutamine, 0.3g/L Aspartic acid, 3g/L potassium chloride, 10mg/L manganese sulfate pentahydrate, 3.0mg/L boric acid, 2.0mg/L zinc sulphate heptahydrate, 0.25mg/L bis- Water molybdic acid is received, 0.025mg/L copper sulphate pentahydrate, 0.025mg/L cobalt chloride hexahydrate, 0.75mg/L potassium iodide, 15mg/L bis-water chlorine Change calcium, 25mg/L Magnesium sulfate heptahydrate, 4mg/L ethylenediaminetetraacetic acid (EDTA), 15mg/L sodium dihydrogen phosphate dihydrate, 1mg/L nicotinic acid,

1mg/L vitamin B6,10mg/L vitamin B1,100mg/L inositol, 176mg/L arginine, 75mg/L glycine, 1M KOH regulates pH to 5.2, and 0.22uM membrane filtration is degerming.

The plant source recombinant humanized shellfish of a kind of optimization the most according to claim 1 cuts down the preparation method of monoclonal antibody, It is characterized in that: light chain of antibody and the expression ratio 0.99 of heavy chain in the transgenic line of described step 1, or ratio range exists 0.11-1.9。

The plant source recombinant humanized shellfish of a kind of optimization the most according to claim 1 cuts down the preparation method of monoclonal antibody, It is characterized in that: in described plant source transgenic paddy rice, the content of Bevacizumab antibody is in per kilogram fresh weight 160.7mg---242.8mg。

The plant source recombinant humanized shellfish of a kind of optimization the most according to claim 1 cuts down the preparation method of monoclonal antibody, It is characterized in that: use this preparation method to prepare and the shellfish that purifies to cut down monoclonal antibody be the tetramer, its molecular weight is 140- 160kDa, purity is more than 95%.

The plant source recombinant humanized shellfish of a kind of optimization the most according to claim 1 cuts down the preparation method of monoclonal antibody, It is characterized in that: described shellfish cuts down the plant-specific glycosylation of monoclonal antibody, including MMXF (M3Gn2X1F1), GnMXF (M3Gn3X1F1) and GnGnXF (M3Gn4X1F1), the 46.8% of glycosylated antibodies total amount is accounted for.

10. a shellfish cuts down monoclonal antibody drug, it is characterised in that: by the plant source of a kind of optimization described in claim 1--9 Recombinant humanized shellfish is cut down the shellfish that the preparation method of monoclonal antibody prepares and cuts down monoclonal antibody and prepare with common drug carrier Bio-pharmaceutical；This medicine may replace commodity medicine Arastin, is applied to people's body source；This medicine is used for treating breast carcinoma, lung Cancer, glioblastoma, renal carcinoma, cervical cancer, ovarian cancer, colon cancer and rectal cancer.