CN109371049A - Application of the molecular chaperones in the formation for promoting monoclonal antibody and/or the expression quantity for improving monoclonal antibody - Google Patents
Application of the molecular chaperones in the formation for promoting monoclonal antibody and/or the expression quantity for improving monoclonal antibody Download PDFInfo
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- CN109371049A CN109371049A CN201811354834.7A CN201811354834A CN109371049A CN 109371049 A CN109371049 A CN 109371049A CN 201811354834 A CN201811354834 A CN 201811354834A CN 109371049 A CN109371049 A CN 109371049A
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/70—Vectors or expression systems specially adapted for E. coli
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- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
Abstract
The present invention relates to field of biotechnology, in particular to application of the molecular chaperones in the formation for promoting monoclonal antibody and/or the expression quantity for improving monoclonal antibody.The present invention is using E.coli MG1655 as starting strain, a kind of method in expression in escherichia coli overall length IgG antibody is provided, while having co-expressed molecular chaperones, promotes being properly formed for overall length IgG, the yield for improving IgG antibody, provides theoretical foundation for the industrialized production in future.
Description
Technical field
The present invention relates to field of biotechnology, in particular to molecular chaperones are promoting the formation of monoclonal antibody and/or are mentioning
Application in the expression quantity of high monoclonal antibody.
Background technique
Monoclonal antibody is one of most important component part of bio-pharmaceutical industry, especially in autoimmune disease and
Tumor area occupies main status.Cut-off 2013 has 35 kinds of monoclonal antibodies to obtain the approval of U.S. FDA, wherein immune ball
Protein G (IgG) accounts for the overwhelming majority.Therefore, people pay close attention to the production of IgG the most.
Monoclonal antibody is the different tetramer protein being assembled by light chain LC and heavy chain HC.According to different weights
Monoclonal antibody can be divided into 5 immunoglobulin like protein: IgG, IgM, IgD, IgA and IgE by chain.IgG antibody is identical by two
The symmetrical antibody of heavy chain (HC) light chain (LC) composition identical with two, is each connected by disulfide bond.
EGFR is one of Epidermal Growth Factor Receptor Family member, is the expression product of proto-oncogene C-ERBB1.EGFR can
To accumulate aggregation in nucleus, and cell proliferation, DNA reparation, tumour generation, inflammation etc. have an impact.Cut-off 2017 1
Month, it shares 10 kinds and obtains FDA approval listing using EGFR as the drug of target spot, including 4 kinds of macromolecular drugs (cetuximab,
Nimotuzumab, panitumumab and necitumumab) and 6 kinds of small-molecule drugs (erlotinib, gefitinib,
Lcotinib, afatinib, osimertinib and olmutinib).
Cetuximab (Cetuximab) is produced by Merck KGaA Lyons drugmaker, is mainly used for treating colorectum
Cancer, trade name Erbitux are the IgG monoclonal antibodies for EGF receptor.Its predecessor's antibody 225 is by hybridoma skill
What art generated.For Cetuximab in conjunction with the extracellular domain of Human epidermal growth factor receptor, competitiveness blocks the combination of receptor and native ligand,
Such as EGF and TGF- α.The Cetuximab of higher affinity prevents EGFR from forming the activity conformation and dimerization of its opening, causes
The cascade protein of the phosphorylation and follow-up signal of EGFR is reduced.After Cetuximab and EGFR specific binding, it can block thin
Intracellular signalling pathway, and then inhibit the proliferation of cancer cell, induce the apoptosis of cancer cell.
Currently, the example of successful expression overall length IgG antibody is limited in Escherichia coli.2002, Simmons et al. was delivered
Document Expression of full-length on Journal of Immunological Methods
immunoglobulins in Escherichia coli:rapid and efficient production of
Aglycosylated antibodies. is realized for the first time in expression in escherichia coli overall length IgG monoclonal antibody, and is optimized
The light chain of IgG and the ratio of heavy chain, to obtain the colibacillus engineering strain of high yield IgG.2007, Yarv M et al. was delivered
Document Isolation of engineered, full-length antibodies on Nature Biotechnology
From libraries expressed in Escherichia coli. discloses the combinatorial libraries system of building IgG monoclonal antibody,
Filter out the E. coli mutant bacterial strain of high yield IgG.
Molecular chaperones are class proteins, it, which can identify and combine, folds incomplete protein, are helping polypeptide just
Really transhipment, folding.Folding enzymes and periplasmic space chaperone are common molecular chaperones, their coexpression can prevent albumen
The aggregation of matter, and improve the expression quantity of recombinant protein in periplasmic space.Express the recombination large intestine bar of anti-TNF antibodies Fab segment
This Chinese patent of bacterium engineering bacteria was in Shen Qing Publication in 2016, and which disclose multiple molecular chaperones such as DsbC, PPIB, Skp
The influence that equal molecular chaperones express Fab.
However, being co-expressed in the different kinds of molecules companion of anti-egfr antibodies IgG in Escherichia coli, which kind of molecular chaperones is most suitable
It closes, the expression quantity for improving IgG and the formation for promoting complete IgG can be maximized, a large amount of experimental study is needed to determine.
Summary of the invention
Resist in view of this, the present invention provides molecular chaperones in the formation and/or raising monoclonal for promoting monoclonal antibody
Application in the expression quantity of body.This experiment provides one kind in expression in escherichia coli using E.coliMG1655 as starting strain
The method of overall length IgG antibody, while molecular chaperones have been co-expressed, promote being properly formed for overall length IgG, improves the production of IgG antibody
Amount, provides theoretical foundation for the industrialized production in future.
In order to achieve the above-mentioned object of the invention, the present invention the following technical schemes are provided:
The present invention provides molecular chaperones in the formation for promoting monoclonal antibody and/or the expression quantity for improving monoclonal antibody
In application；
The molecular chaperones have any one in nucleotide sequence as follows:
The present invention provides the nucleotide sequences with DsbA, DsbC and/or Skp；
II, it modified, replaced, missed or added one or more bases obtain with the nucleotide sequence as shown in I
Nucleotide sequence；
III, with the nucleotide sequence such as I shown in have at least 80% homology sequence or translation after gained albumen and
The same or similar nucleotide sequence of the protein function of DsbA, DsbC and/or Skp expression；
The complementary series of IV, the sequence as shown in I, II or III.
In some specific embodiments of the invention, the monoclonal antibody is IgG.
On this basis, sea of the present invention provides a kind of expression module, including molecular chaperones and monoclonal antibody；
The molecular chaperones have any one in nucleotide sequence as follows:
I, with the nucleotide sequence of DsbA, DsbC and/or Skp；
II, it modified, replaced, missed or added one or more bases obtain with the nucleotide sequence as shown in I
Nucleotide sequence；
III, with the nucleotide sequence such as I shown in have at least 80% homology sequence or translation after gained albumen and
The same or similar nucleotide sequence of the protein function of DsbA, DsbC and/or Skp expression；
The complementary series of IV, the sequence as shown in I, II or III.
In some specific embodiments of the invention, the monoclonal antibody is IgG.
The present invention also provides the expression modules in the formation for promoting monoclonal antibody and/or to improve monoclonal antibody
Expression quantity in application.
The present invention also provides expression vectors, including expression module of the present invention.
On this basis, the present invention also provides the expression vectors to promote the formation of monoclonal antibody and/or mention
Application in the expression quantity of high monoclonal antibody.
The present invention also provides bacterial strains, including expression vector of the present invention.
In some specific embodiments of the invention, starting strain is E.coli MG1655, promoter Bad.
The present invention also provides the bacterial strains in the formation for promoting monoclonal antibody and/or the table for improving monoclonal antibody
Up to the application in amount.
Monoclonal antibody and its derivative are widely used in research and clinic, have been developed as and prevent, treat and diagnose examination
Agent.This experiment provides a kind of side in expression in escherichia coli overall length IgG antibody using E.coli MG1655 as starting strain
Method, while molecular chaperones have been co-expressed, promote being properly formed for overall length IgG, improve the yield of IgG antibody, is the industry in future
Metaplasia, which produces, provides theoretical foundation.
Detailed description of the invention
In order to more clearly explain the embodiment of the invention or the technical proposal in the existing technology, to embodiment or will show below
There is attached drawing needed in technical description to be briefly described.
Fig. 1 shows the plasmid schematic diagram of expression in escherichia coli IgG；
Fig. 2 (A) shows DsbA and DsbC mechanism of action schematic diagram；
Fig. 2 (B) shows the plasmid schematic diagram of coexpression molecular chaperones；
Fig. 2 (C) shows the influence that molecular chaperones express monoclonal antibody；
The biostatistics result of the corresponding dsbC of Fig. 3 diagram 2 (C)；Wherein, 1.00- is wild type data, and 2.00- is
Data after transformation；P=0.004 < 0.01；
The biostatistics result of the corresponding dsbA of Fig. 4 diagram 2 (C)；Wherein, 1.00- is wild type data, and 2.00- is
Data after transformation；P=0.000 < 0.01；
The biostatistics result of the corresponding skp of Fig. 5 diagram 2 (C)；Wherein, 1.00- is wild type data, and 2.00- is to change
Make rear data；P=0.019 < 0.05.
Specific embodiment
The invention discloses molecular chaperones in the formation for promoting monoclonal antibody and/or the expression quantity for improving monoclonal antibody
In application, those skilled in the art can use for reference present disclosure, be suitably modified realization of process parameters.In particular
It is that all similar substitutions and modifications are apparent to those skilled in the art, they are considered as being included in this
Invention.Method and application of the invention is described by preferred embodiment, and related personnel can obviously not depart from this
Method described herein and application are modified or appropriate changes and combinations in summary of the invention, spirit and scope, realizing and
Using the technology of the present invention.
The external synthesis and amplification of gene
The light chain and heavy chain amino acid sequence (deriving from source of people) of monoclonal antibody are obtained from ncbi database, right in the website JCAT
Sequence is optimized, and XbaI, NdeI, SpeI and HindIII restriction enzyme site are avoided in the sequence of optimization.
DsbC gene order (deriving from Escherichia coli MG1655) is also obtained from ncbi database, to sequence in the website JCAT
Column are optimized, and XbaI, NdeI, SpeI and HindIII restriction enzyme site are avoided in the sequence of optimization.dsbA
It is identical as the acquisition pattern of dsbC gene with skp gene.
Light chain (as shown in SEQ ID No.1), heavy chain (as shown in SEQ ID No.2), dsbC are (such as SEQ ID No.5 institute
Show), dsbA (as shown in SEQ ID No.4) and skp (as shown in SEQ ID No.6) gene synthesize in vitro.
Coli strain and plasmid used
In all experiments, Escherichia coli TransT1 is used for gene cloning, and Escherichia coli MG1655 is expressed for monoclonal antibody.
Monoclonal antibody gene is connected in the p2A4 carrier comprising pBR322 replicon.DsbC, dsbA and skp gene are connected to multiple comprising p15A
The p3C5bad carrier of system and bad promoter.
Construction method:
Monoclonal antibody gene: the protein expression region of p2A4 empty plasmid vector is guided by lacUV5 promoter, behind connect
Lactose operon, there is one section of ribosome bind site later, in sequence later comprising EcoRI, Xba I, Nde I, Spe I and
Pst I restriction enzyme site.By with connecting the above-mentioned monoclonal antibody gene synthesized in vitro after Nde I and Spe I digestion empty plasmid vector；
Molecular chaperones: the protein expression region of p3C5bad empty plasmid vector is guided by bad promoter, behind connect
Operon has one section of ribosome bind site later, includes EcoR I, Xba I, Nde I, Spe I and Pst in sequence later
I restriction enzyme site.By with connecting above-mentioned dsbC, dsbA or skp synthesized in vitro after Nde I and Spe I digestion empty plasmid vector
Gene.
Noun and term are explained
E.coli: Escherichia coli
Monoclonal antibody: abbreviation monoclonal antibody
IgG: immunoglobulin G
Light chain (LC): the light chain region of monoclonal antibody is made of VL and CL
Heavy chain (HC): the heavy chain region of monoclonal antibody, by VH, CH1, the region CH2 and CH3 is constituted
PCR: polymerase chain reaction
PBS: phosphate buffered saline solution
U.S. FDA: U.S. Food and Drug Administration
LB culture medium: 5g/L yeast extract；10g/L peptone；10g/L sodium chloride
IPTG: isopropylthiogalactoside
L-ara:L- arabinose.
Molecular chaperones provided by the invention are in the formation for promoting monoclonal antibody and/or the expression quantity for improving monoclonal antibody
In application in starting strain and reagent used be available on the market.
Below with reference to embodiment, the present invention is further explained:
1 construction method of embodiment
Monoclonal antibody gene: the protein expression region of p2A4 empty plasmid vector is guided by lacUV5 promoter, behind connect
Lactose operon has one section of ribosome bind site later, includes EcoR I, Xba I, Nde I, Spe I and Pst I in sequence
Etc. a series of restriction enzyme sites；Wherein, promoter and operon are located between EcoR I and Xba I restriction enzyme site, and ribosomes combines
Site is located between Xba I and Nde I restriction enzyme site.First by with Nde I and Spe I digestion by light chain (LC) and heavy chain
(HC) gene is separately connected on p2A4 plasmid empty carrier；Then the p2A4 that will contain heavy chain (HC), passes through Xba I and Pst
I digestion is connected on the p2A4 plasmid vector containing light chain (LC), so that light chain (LC) and heavy chain (HC) gene are connected to together
On one p2A4 plasmid.There is one section of ribosome bind site on this plasmid, before light chain and heavy chain, light chain is in preceding, weight
Chain is rear, and a general lacUV5 promoter carries out inducing expression.As shown in Figure 1.
Molecular chaperones: the protein expression region of p3C5bad empty plasmid vector is guided by bad promoter, behind connect
Operon has one section of ribosome bind site later, includes EcoR I, Xba I, Nde I, Spe I and Pst I etc. one in sequence
Serial restriction enzyme site；Wherein, promoter and operon are located between EcoR I and Xba I restriction enzyme site, ribosome bind site
Between Xba I and Nde I restriction enzyme site.By the way that dsbC, dsbA or skp gene are connected respectively with Nde I and Spe I digestion
It accesses on p3C5bad plasmid empty carrier.As shown in Fig. 2 (B).
2 shake flask fermentation of embodiment
By single Bacillus coli cells LB liquid medium and antibiotic appropriate (100 μ g/ml ampicillins and/
Or 34 μ g/ml chloramphenicol) in culture, be incubated overnight under 37 DEG C and 220rpm；Culture is transferred with 1:100 ratio new into 50mL
In fresh LB culture medium (250mL shaking flask), and grown at 37 DEG C.Optical density (OD at 600nm600) when reaching about 0.6, training
It supports box temperature degree and is down to 25 DEG C, equilibrium temperature 20 minutes.It is added isopropylthiogalactoside (IPTG), makes its final concentration of 1mM.
When co-expressing molecular chaperones, the L-arabinose that final concentration of 1mM is added while IPTG induction is being added, cell is at 25 DEG C
Lower growth 16 hours.
The purifying of 3 monoclonal antibody of embodiment
By the culture after fermentation, 6500rpm and 4 DEG C is collected and is centrifuged.Cell is with 0.01M PBS (pH=7.2-7.4)
Washing.Cell is resuspended with PBS and carries out ultrasonication processing, 5s pulse, 10s interval, net cycle time is 20 minutes.Then,
Cell lysate is centrifuged 30 minutes at 10,000rpm and 4 DEG C, collects supernatant.Then it was carried out using 0.45 μm of filter membrane
Filter.
In this experiment, antibody purification magnetic bead kit is used for monoclonal antibody purification.
1. sample treatment: taking the sample of antibody content about 0.1-0.15mg to be placed in new 1.5mL EP pipe, antibody is added
Combination buffer to total volume is 500 μ L (such as sample volume is greater than 500 μ L, then without being added), is uniformly mixed.2. magnetic bead is located in advance
Reason: antibody purification magnetic bead is vortexed and shakes 30s, magnetic bead is resuspended sufficiently；100 μ L suspension containing magnetic beads are taken to be placed in another new 1.5mL
, which carries out Magnetic Isolation to suspension containing magnetic beads, in EP pipe (makes magnetic bead be attracted to tube wall to solution to clarify；Operation description is following to be saved
Slightly, supernatant is abandoned.EP pipe is removed from magnetic separator, magnetic bead can be directly used for antibody separation in pipe).3. antibody adsorbs:
The sample solution that step 1 is handled is added in the pretreated magnetic bead pipe of step 2, is vortexed concussion uniformly, (about 25 DEG C) are set at room temperature
In on overturning mixed instrument, promote sample and magnetic bead to come into full contact with and adsorb, carries out Magnetic Isolation after overturning about 15min, move on abandoning
Clear liquid.4. magnetic bead washs: 1mL antibody combination buffer being added into EP pipe, oscillation carries out Magnetic Isolation after magnetic bead is resuspended, moves and abandon
Supernatant；The operation is repeated twice.5. antibody elution: 1mL antibody is added and combines in the EP pipe of above-mentioned completion magnetic bead washing and delays
Fliud flushing, the concussion that is vortexed are resuspended rapidly, are then placed on overturning mixed instrument at room temperature, carry out Magnetic Isolation after overturning 10min, received
Collect supernatant to new EP pipe.6. antibody is dialysed: because antibody elution buffer contains the salinity of higher concentration, the antibody of collection is molten
Liquid cannot be directly used to SDS-PAGE detection, but can be returned to zero with antibody elution buffer and carry out antibody concentration measurement.Antibody is washed
De- buffer is in slant acidity, carries out dialysis treatment to the antibody-solutions of collection immediately with the neutral low salt solutions voluntarily prepared, with
Antibody inactivation rate is reduced, the antibody-solutions of greater activity and stability are obtained.
The medium-sized low salt solutions voluntarily prepared are 0.01M PBS solution: sodium chloride 137mmol/L；Potassium chloride
2.7mmol/L；Disodium hydrogen phosphate 10mmol/L；Potassium dihydrogen phosphate 2mmol/L.
4 SDS-PAGE of embodiment
After fermentation, culture solution 1ml to be tested is sucked out, with 10,000rpm centrifugation 2 minutes, discards supernatant liquid.With
0.01M PBS solution is washed cell 3 times, and liquid is discarded supernatant.Cell is resuspended with 200ul PBS again.Then suitable loading is delayed
Fliud flushing, which is added, to be resuspended in solution and is sufficiently mixed.Mixed liquor is boiled 10 minutes and is placed on -80 DEG C of storages.Before point sample, with 12,
000rpm is centrifuged 2min, then draws suitable supernatant loading dispensing.
5 ELSIA of embodiment (enzyme-linked immunosorbent assay)
1. dilute sample (choosing of this step is done).
2. 100 μ l Assay Buffer are added in every hole, corresponding aperture is added in 10 μ l samples or various concentration standard items respectively
In, gently rocker is simply mixed, and seals reacting hole with sealing plate film, and (18-25 degree) hatches 30min at room temperature.
3. taking sealing plate film off, inhales and abandon liquid in hole.It is washed 3 times with the diluted Wash Buffer of 300 μ l, in clean paper
On pat dry.
4. the ready HRP Conjugate of 100 μ l is added in every hole.
5.. it is sealed with new sealing plate film.Incubation at room temperature 30min.
6.. step 3 is repeated.
7. 100 μ l TMB Substrate Solution are added in every hole.
8. incubation at room temperature 10 minutes (not sealer) under dark condition.
9. 100 μ l Stop Solution, which are added, stops reaction.Gently rocker is simply mixed, and color is become by indigo plant in hole
It is yellow.
10. returning to zero with blank well, the absorbance in every hole is measured in 30min in 450nm using microplate reader.
Effect example
The expression of anti-egfr antibodies IgG in Escherichia coli
Fig. 1 is the plasmid schematic diagram of expression in escherichia coli anti-egfr antibodies IgG.LacUV5 promoter is selected to be used for herein
Express IgG.In the front end of light chain (LC) and heavy chain (HC), one section of STII signal peptide sequence is introduced respectively (such as SEQ ID No.3 institute
Show), guarantee that light chain and heavy chain can be secreted into periplasmic space.Periplasmic space refers to the narrow sky between cell wall and cell membrane
Between.Protease is fewer than in cytoplasm and active low in periplasmic space, and therefore, destination protein, which is in periplasmic space, can reduce its drop
Solution, is stabilized conducive to it.
Co-express folding factor and molecular chaperones
Folding, secretion and the assembling of protein are vital for the formation of soluble recombinant protein.Usually with folding
Folded enzyme and molecular chaperones coexpression are to increase the solubility of protein and improve the yield of recombinant protein.In this experiment, it grinds
These three molecular chaperones of DsbA, DsbC and Skp are studied carefully, to assess their influences in Escherichia coli to IgG expression.DsbA
(periplasmic protein disulfide isomerase I) and DsbC (protein disulfide isomerase
II) it is protein disulfide isomerase (belonging to folding enzymes), is located at periplasmic space.Wherein, the molecular weight of DsbA is 23.1kD,
And the heterodimeric protein matter that DsbC is made of two molecular weight for 23.3kD subunit.They can be catalyzed light chain and heavy chain
Between between disulfide bond and two heavy chains disulfide bond formation, to obtain complete IgG antibody, mechanism of action is as shown in Figure 2 A.
Skp (periplasmic chaperone) is called and is OmpH, is a kind of molecular chaperones, is located at periplasmic space.It can promote weight
The folding of histone, and increase the solubility and secreting, expressing of light chain and heavy chain.
Fig. 2 B is the plasmid schematic diagram for co-expressing molecular chaperones, selects Bad promoter for co-expressing molecular chaperones.
The coexpression of experiment discovery these three molecular chaperones of DsbA, DsbC and Skp produces influence to the expression of IgG monoclonal antibody, such as schemes
Shown in 2C, table 1, ELISA result confirms that the coexpression of DsbC, DsbA and Skp improve the yield of complete IgG.In particular, working as
When co-expressing DsbA, IgG yield increases at most, and being 2 times of blank control or so, (blank control is not co-express molecular chaperones
Wild type E. coli strain).The result shows that the generation of IgG depends on the coexpression of above-mentioned three kinds of chaperones, especially height
Coexpression dependent on DsbA.From the results, it was seen that DsbA, which expresses IgG, influences maximum in three kinds of chaperones, it can be most
Bigization improves the expression quantity of IgG and promotes the formation of complete IgG.
Table 1
| OD450 mean value | Standard deviation | |
| WT | 0.243 | 0.012 |
| dsbC | 0.316 | 0.018 |
| dsbA | 0.483 | 0.016 |
| skp | 0.285 | 0.015 |
Biometric result is shown in Fig. 3~5.Wherein, Fig. 3 show dsbC compared with wild type data, P=0.004 < 0.01；
Fig. 4 show dsbA compared with wild type data, P=0.000 < 0.01；Fig. 5 show skp compared with wild type data, P=0.019 <
0.05.Show that the coexpression of DsbC, DsbA and Skp improve the yield of complete IgG.In particular, when co-expressing DsbA,
IgG yield increases at most, and being 2 times of blank control or so, (blank control is the wild type large intestine bar for not co-expressing molecular chaperones
Bacteria strain).
The above is only a preferred embodiment of the present invention, it is noted that for the ordinary skill people of the art
For member, various improvements and modifications may be made without departing from the principle of the present invention, these improvements and modifications are also answered
It is considered as protection scope of the present invention.
Sequence table
<110>University Of Tianjin；Yangzhou Ao Ruite pharmaceutcal corporation, Ltd；Yangzhou Lian Ao biological medicine Co., Ltd
<120>application of the molecular chaperones in the formation for promoting monoclonal antibody and/or the expression quantity for improving monoclonal antibody
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atttctgata atgtgaagaa aaaactgccg gaaggcgtga agatgactaa ataccacgtc 240
aacttcatgg gtggtgacct gggcaaagat ctgactcagg catgggctgt ggcgatggcg 300
ctgggcgtgg aagacaaagt gactgttccg ctgtttgaag gcgtacagaa aacccagacc 360
attcgttctg cttctgatat ccgcgatgta tttatcaacg caggtattaa aggtgaagag 420
tacgacgcgg cgtggaacag cttcgtggtg aaatctctgg tcgctcagca ggaaaaagct 480
gcagctgacg tgcaattgcg tggcgttccg gcgatgtttg ttaacggtaa atatcagctg 540
aatccgcagg gtatggatac cagcaatatg gatgtttttg ttcagcagta tgctgataca 600
gtgaaatatc tgtccgagaa aaaataa 627
<210> 5
<211> 711
<212> DNA
<213> dsbC
<400> 5
atgaagaaag gttttatgtt gtttactttg ttagcggcgt tttcaggctt tgctcaggct 60
gatgacgcgg caattcaaca aacgttagcc aaaatgggca tcaaaagcag cgatattcag 120
cccgcgcctg tagctggcat gaagacagtt ctgactaaca gcggcgtgtt gtacatcacc 180
gatgatggta aacatatcat tcaggggcca atgtatgacg ttagtggcac ggctccggtc 240
aatgtcacca ataagatgct gttaaagcag ttgaatgcgc ttgaaaaaga gatgatcgtt 300
tataaagcgc cgcaggaaaa acacgtcatc accgtgttta ctgatattac ctgtggttac 360
tgccacaaac tgcatgagca aatggcagac tacaacgcgc tggggatcac cgtgcgttat 420
cttgctttcc cgcgccaggg gctggacagc gatgcagaga aagaaatgaa agctatctgg 480
tgtgcgaaag ataaaaacaa agcgtttgat gatgtgatgg caggtaaaag cgtcgcacca 540
gccagttgcg acgtggatat tgccgaccat tacgcacttg gcgtccagct tggcgttagc 600
ggtactccgg cagttgtgct gagcaatggc acacttgttc cgggttacca gccgccgaaa 660
gagatgaaag aattcctcga cgaacaccaa aaaatgacca gcggtaaata a 711
<210> 6
<211> 486
<212> DNA
<213> skp
<400> 6
atgaaaaaat ggctgctggc tgctggtctg ggtctggctc tggctacctc tgctcaggct 60
gctgacaaaa tcgctatcgt taacatgggt tctctgttcc agcaggttgc tcagaaaacc 120
ggtgtttcta acaccctgga aaacgaattc aaaggtcgtg cttctgaact gcagcgtatg 180
gaaaccgacc tgcaggctaa aatgaaaaaa ctgcagtcta tgaaagctgg ttctgaccgt 240
accaaactgg aaaaagacgt tatggctcag cgtcagacct tcgctcagaa agctcaggct 300
ttcgaacagg accgtgctcg tcgttctaac gaagaacgtg gtaaactggt tacccgtatc 360
cagaccgctg ttaaatctgt tgctaactct caggacatcg acctggttgt tgacgctaac 420
gctgttgctt acaactcttc tgacgttaaa gacatcaccg ctgacgttct gaaacaggtt 480
aaataa 486
Claims (10)
1. application of the molecular chaperones in the formation for promoting monoclonal antibody and/or the expression quantity for improving monoclonal antibody；
The molecular chaperones have any one in nucleotide sequence as follows:
I, with the nucleotide sequence of DsbA, DsbC and/or Skp；
II, it modified with the nucleotide sequence as shown in I, replace, miss or add the nucleosides that one or more bases obtain
Acid sequence；
III, have after the sequence or translation of at least 80% homology with the nucleotide sequence such as I shown in obtained by albumen and DsbA,
The same or similar nucleotide sequence of the protein function of DsbC and/or Skp expression；
The complementary series of IV, the sequence as shown in I, II or III.
2. application as described in claim 1, which is characterized in that the monoclonal antibody is IgG.
3. expressing module, which is characterized in that including molecular chaperones and monoclonal antibody；
The molecular chaperones have any one in nucleotide sequence as follows:
I, with the nucleotide sequence of DsbA, DsbC and/or Skp；
II, it modified with the nucleotide sequence as shown in I, replace, miss or add the nucleosides that one or more bases obtain
Acid sequence；
III, have after the sequence or translation of at least 80% homology with the nucleotide sequence such as I shown in obtained by albumen and DsbA,
The same or similar nucleotide sequence of the protein function of DsbC and/or Skp expression；
The complementary series of IV, the sequence as shown in I, II or III.
4. expression module as claimed in claim 3, which is characterized in that the monoclonal antibody is IgG.
5. expression module as described in claim 3 or 4 is in the formation for promoting monoclonal antibody and/or improves monoclonal antibody
Application in expression quantity.
6. expression vector, which is characterized in that including expression module as described in claim 3 or 4.
7. expression vector as claimed in claim 6 is in the formation for promoting monoclonal antibody and/or the table for improving monoclonal antibody
Up to the application in amount.
8. bacterial strain, which is characterized in that including expression vector as claimed in claim 6.
9. bacterial strain as claimed in claim 8, which is characterized in that starting strain is E.coli MG1655, promoter Bad.
10. bacterial strain as claimed in claim 8 or 9 is in the formation for promoting monoclonal antibody and/or the table for improving monoclonal antibody
Up to the application in amount.
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| CN109355303A (en) * | 2018-11-14 | 2019-02-19 | 天津大学 | Inhibit and/or knock out application of the gene in the expression quantity for improving monoclonal antibody |
| CN109439683A (en) * | 2018-11-14 | 2019-03-08 | 天津大学 | Inhibit, knockout and/or expressing gene are improving metabolism of pyruvate path product and improving the application in monoclonal antibody expression quantity |
| CN111733178A (en) * | 2020-07-13 | 2020-10-02 | 山西中医药大学 | Recombinant expression vector for improving soluble expression quantity of disease course related protein of astragalus mongholicus |
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