CN106244612B - A kind of method that genetic engineering IgG antibody is prepared in Escherichia coli - Google Patents
A kind of method that genetic engineering IgG antibody is prepared in Escherichia coli Download PDFInfo
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- CN106244612B CN106244612B CN201610755474.6A CN201610755474A CN106244612B CN 106244612 B CN106244612 B CN 106244612B CN 201610755474 A CN201610755474 A CN 201610755474A CN 106244612 B CN106244612 B CN 106244612B
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- 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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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
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- C—CHEMISTRY; METALLURGY
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- C12N2800/00—Nucleic acids vectors
- C12N2800/10—Plasmid DNA
- C12N2800/101—Plasmid DNA for bacteria
Abstract
The invention belongs to antibody engineering field, more particularly to a kind of method that genetic engineering IgG antibody is prepared in Escherichia coli, pass through phage display elutriation and technique for gene engineering, heavy chain of antibody and light chain prokaryotic expression carrier are built respectively, then the expression vector of structure is transformed into Escherichia coli, passes through IPTG expression profile engineering IgG antibodies.Genetic engineering IgG antibody can apply in immunity detection reagent caused by this method, so as to realize the immune detection related to the residues such as food, agricultural product additive and agricultural chemicals/antibiotic, pathogenic microorganism and medical domain, the immunity detection reagent that high quality is produced for commercialized development is laid a good foundation.
Description
Technical field
The invention belongs to antibody engineering field, and in particular to a kind of that genetic engineering IgG antibody is prepared in Escherichia coli
Method.
Background technology
Since monoclonal antibody technique in 1975 is founded, due to can be secreted with unrestricted by cell hydridization and screening
The monoclonal antibody of high specificity and produced in enormous quantities, the characteristics of due to the antibody molecule high specificity and high affinity and depth
Liked by people.However, monoclonal antibody also has many problems, current monoclonal antibody is from mouse source, thus
Very strong HAMA reactions can be produced in human body therapy and use.The serious application for limiting monoclonal antibody of this defect.
It is often unstable that the hybridoma cell strain of acquisition is screened by cell fusion, it is easy to influenceed by various factors and
Antibody-secreting ability is lost, or so-called atavism occurs.In addition, monoclonal antibody technology of preparing is very time-consuming takes
Power.The mammal as mouse is not allowed to be also easy to produce the antibody of high-affinity particularly when preparing some specific antigens, from
And high-sensitivity detection can not be realized to special antigen molecule.Therefore, one kind is established in vitro prepare gene based on Escherichia coli
The method of engineering IgG antibody is necessary！
Genetic engineering IgG antibody is a kind of novel IgG antibodies type, the antibody molecule can in Escherichia coli efficient table
Reach, it has similar structure to traditional antibody molecule, still maintains the binding activity of antigen.However, its practical significance
But considerably beyond this point.Elutriation obtains antibody gene from the natural phage antibody library of structure so that we can cross over
The step for animal immune, substantially reduce the time of antibody acquisition.In addition, it is unstable existing for hybridoma technology, yield poorly
Problem, can be by this technology by from the antibody cloning that natural Phage Antibody Library obtains to Escherichia coli table
It is overcome up to carrier and in expression in escherichia coli.Because antibody gene is placed under human control by this technology, antibody
Whether bulk of molecule, the height of affinity, the power of cytotoxicity, antibody connect other useful molecules etc. all can be according to controlling
Treat and the needs of diagnosis are designed and operated.This is that hybridoma technology is irreplaceable, and its efficiency is also chemical method institute
It can not compare.
By contrast, escherichia expression system factor affected by environment is less, can with more stable express antibody point
Son.Under the conditions of simple, it is possible to achieve the large-scale expression and purification of antibody, so as to improve antibody life to a certain extent
The controllability of production, to save substantial amounts of manpower, financial resources and material resources, realize more considerable economic benefits.Escherichia coli table simultaneously
Up to the remote supracellular antibody expression amount of amount, therefore, the technology platform can substantially reduce the production cost of antibody, with suitable for not
The detection of same level, different purposes is needed, while can also be used as antibody starting material production platform, and customization is produced for different users
Antibody starting material.By the platform uniqueness the characteristics of, the platform can be used for the sieve for any antigen molecule in theory
Choosing, some antigen molecules for not having immunogenicity can not be screened by animal immune, there is pole so as to embody the system
Big application prospect.In addition, it can further establish the immune inspection based on novel gene engineering IgG antibody using the technology platform
Test agent method.Establish a set of quick and precisely complete for the residual such as various food, agricultural product additive and agricultural chemicals/antibiotic
The related immune detection of thing, pathogenic microorganism and medical domain, early warning, the system for preventing science.
The content of the invention
It is an object of the invention to provide a kind of method that genetic engineering IgG antibody is prepared in Escherichia coli.In large intestine bar
High efficient expression purifying is resisted with high-affinity, strong specific genetic engineering IgG antibody and foundation based on genetic engineering IgG in bacterium
The immunological detection method of body, established for final commercialization immunity detection reagent of the exploitation based on genetic engineering IgG antibody
Basis.
The present invention protects a kind of method that genetic engineering IgG antibody is prepared in Escherichia coli first, passes through phage display technology
Show elutriation and technique for gene engineering, heavy chain of antibody and light chain prokaryotic expression carrier are built respectively, then by the expression vector of structure
It is transformed into Escherichia coli, passes through IPTG expression profile engineering IgG antibodies.
One plant of protection simultaneously builds heavy chain of antibody and light chain is former respectively by phage display elutriation and technique for gene engineering
Nuclear expression carrier, then the expression vector of structure is transformed into the expressing gene engineering IgG obtained in e. coli bl21 resisted
The coli strain of body is ETEC（Escherichia coli）BAC-hFLIG-Ab-001, in 2016 06
The moon 20 is in China Committee for Culture Collection of Microorganisms's common micro-organisms center's preservation, preserving number CGMCC
No.12641, address are the institute of Chaoyang District Beijing North Star West Road 1.
Secondly genetic engineering IgG caused by a kind of method that genetic engineering IgG antibody is prepared in Escherichia coli of protection
Antibody.
A kind of described method that genetic engineering IgG antibody is prepared in Escherichia coli, is to obtain phage display elutriation
To heavy chain of antibody and chain variable region gene expanded, be cloned into respectively containing heavy chain constant region (CH) and constant region of light chain
In the prokaryotic expression carrier of genetic fragment (CL), and convert e. coli bl21（DE3）Then IPTG induced expression purifying is carried out,
Obtain the genetic engineering IgG antibody with antigen-binding activity.
The present invention is at least 10 by constructing storage capacity using genetic engineering means in vitro9Natural bacteriophage above
Antibody library, the antibody gene for specific antigen is obtained using phage display and the elutriation of solid phase panning technique.By the weight of antibody
Chain variable region gene VHWith chain variable region gene VLCH containing heavy chain constant region or constant region of light chain CL two differences are inserted respectively
Have plasmid compatibility prokaryotic expression carrier in, and using Calcium Chloride Method conversion Escherichia coli.Induced, co-cultured using IPTG
Recombination fusion protein is expressed, the heavy chain of antibody and light chain protein molecule of expression are respectively in the presence of signal peptide by cytoplasm
To periplasmic space, complete IgG antibody molecule is assembled into an oxidizing environment.Assemble the restructuring IgG antibody molecule obtained
With the structure composition similar to native IgG antibodies, and remain stronger antigen binding ability and antibody specificity.Using upper
The method of stating is truly realized high efficient expression and purifying of the IgG antibody molecule in bacterium, and this method has stronger novelty.Together
When, genetic engineering IgG antibody can apply in immunity detection reagent caused by this method, so as to realize to food, agriculture
The related immune detection of the residue such as product additive and agricultural chemicals/antibiotic, pathogenic microorganism and medical domain, is commercialization
The immunity detection reagent of Development and Production high quality is laid a good foundation.
The advantage of the invention is that：
（1）This method has extremely strong novelty.
Display technique of bacteriophage, technique for gene engineering and escherichia expression system are combined by this method, being capable of human nature
The design and screening of change are directed to the specific IgG antibodies of the high-affinity of specific antigen, so as to efficiently solve antibody screening
With expressing the problem of difficult.Therefore the antibody is the novel gene engineered antibody more superior than conventional monoclonal antibody property.
（2）It this method solve the technical bottleneck present in hybridoma technology.
Elutriation obtains antibody gene from the natural antibody storehouse of structure so that we can be across this step of animal immune
Suddenly, the time of antibody acquisition is substantially reduced.In addition, it is unstable existing for hybridoma technology, yield poorly problem, this can be passed through
One technology is by the antibody cloning for screening to obtain from natural antibody storehouse to coli expression carrier and in Escherichia coli
Express and be overcome.Because antibody gene is placed under human control by this technology, the size of antibody molecule, the height of affinity
Whether low, cytotoxicity power, antibody connect other useful molecules etc. can all be set according to the needs for the treatment of and diagnosis
Meter and operation.This is that hybridoma technology is irreplaceable, and its efficiency is also that chemical method can not compare.
（3）Antibody be this method solve in expression in escherichia coli issues of purification.
It is that cost is low the characteristics of Escherichia coli compared to other host expression systems, it is simple to operate and can train on a large scale
Support.Therefore, it is expressive host to improve solubility expression of protein be optimal using Escherichia coli.This method passes through spy for many years
Rope and effort, antibody molecule solubility expression, transhipment and antibody point in Escherichia coli are realized using technique for gene engineering
The assembling of son so that we are easy to obtain the functioning gene engineering IgG antibody of high-purity in escherichia expression system.
（4）The Platform Screening antibody molecule has high efficiency and plasticity.
Compared to traditional antibody screening techniques, this method need not be by animal immune, as long as obtaining the anti-of respective concentration
Original can carry out screening and the expression identification of antibody.Because storage capacity is big, screening obtains the general of the specific antibody of high-affinity
Rate can obtain the more plants of high-affinity antibodies for being directed to specific antigen, to meet difference considerably beyond conventional antibodies triage techniques
Demand and the antibody conjugates detection of detection type.In addition, after the antibody strain of certain affinity is obtained, can be by construct outside
Molecular evolution and gene recombination technology are artificial reconstructed to antibody progress, to realize the antibody demand of different purposes and research field,
So as to provide guarantees for the acquisition of antibody, this point be often conventional antibodies technology it is incomparable with realization.
（5）The platform has wide market application value.
Escherichia expression system factor affected by environment is less, can with more stable express antibody molecule.Simple
Under conditions of, it is possible to achieve the large-scale expression and purification of antibody, so as to improve the controllable of antibody producing to a certain extent
Property, to save substantial amounts of manpower, financial resources and material resources, realize more considerable economic benefits.Bacillus coli expression amount is far super simultaneously
The antibody expression amount of cell, therefore, the technology platform can substantially reduce the production cost of antibody, with suitable for different levels,
The detection of different purposes is needed, while can also be used as antibody starting material production platform, and the antibody of customization is produced for different users
Raw material.By the platform uniqueness the characteristics of, the platform can be used for the screening for any antigen molecule in theory, can be with
Some antigen molecules for not having immunogenicity are not screened by animal immune, there is greatly application so as to embody the system
Prospect.
Brief description of the drawings
Fig. 1 is genetic engineering IgG antibody molecule construction schematic diagram in the present invention.
Fig. 2 is that the present invention builds natural phage antibody library PCR qualification results.（M：DNA marker DL-2000；Swimming lane
1-6：The sub- PCR amplifications of random picking monoclonal）.
Fig. 3 is that the present invention builds natural phage antibody library DNA sequence analysis result.
Fig. 4 is genetic engineering IgG antibody Prokaryotic expression, purification result of the present invention.（M：Middle-molecular-weihydroxyethyl albumen marker；Swimming
Road 1,3：Heavy chain and light chain empty carrier expression product；Swimming lane 2,4：Heavy chain and light chain recombinant expression carrier expression product；Swimming lane 5-
6：Heavy chain and light chain recombinant expression carrier expression product purification result）.
Fig. 5-1 is genetic engineering IgG antibody ELISA qualification results of the present invention（Real figure）.
Fig. 5-2 is genetic engineering IgG antibody ELISA qualification results of the present invention（Block diagram）.
Fig. 6-1 is genetic engineering IgG antibody antigen-binding activity testing result of the present invention（Real figure）.（1：The IgG of purifying resists
Body HRP marks sheep anti-mouse igg antibody testing result；2：The IgG antibody HRP of purifying marks anti-His6 tag antibodies testing result；
3：Negative control）.
Fig. 6-2 is genetic engineering IgG antibody antigen-binding activity testing result of the present invention（Block diagram）.
Fig. 7 is genetic engineering IgG antibody specific detection result of the present invention.
Embodiment
The preparation and identification of the natural phage antibody library of the present invention of embodiment 1.
1）, spleen total serum IgE extraction
Spleen Total RNAs extraction is carried out using Trizol methods.Draw neck to put to death animal, mouse abdomen is cut off with DEPC processing scissors
Portion, spleen is taken out rapidly, be placed in addition liquid nitrogen in the mortar of Liquid nitrogen precooler and smash grind into powder to pieces.Powder is moved at DEPC
In the EP pipes of reason, 1 mL Trizol and 200 μ L chloroform are added, acutely vibration mixes, 10000 r/min centrifugations 5-10
Min, supernatant is extracted, the isopropanol for adding 2 times of volume absolute ethyl alcohols or 0.6 times of volume carries out RNA precipitate, precipitation is blown after centrifugation
It is dry, add 50-100 μ L DEPC water dissolving RNA.
2）, RT-PCR and antibody gene expand
Using the spleen total serum IgE of extraction as template, according to the synthesis that the chains of CDNA first are carried out the step of Promega kits.
Utilize heavy chain of antibody（Primer sequence is VH1BACK：5’-AGGTSMARCTGCAGSAGTCWGG-3’；VH1FOR：5’-
tgaggagacggtgacggt ggtcccttggcccc-3’）With the primer of light chain（Primer sequence is vk2back：5’-
GACATTGAGCTC ACCCAGTCTCCA-3’；MJk1FONX: 5’-CCGTTTGATTTCCAGCCTGGTGCC-3’;
MJk2FONX: 5’-CCGTTTTATTTCCAGCCTGGTGCC-3’; MJk4FONX: 5’-CCG
TTTTATTTCCAACCTTGTGCC-3’; MJk5FONX：5’-CCGTTTCAGCTCCAGCCT GGTGCC-3’）VH is expanded respectively
With VL genes, with VH the and VL genes of recovery, enter performing PCR restructuring with a certain amount of Linker DNA genes, pass through overlap-extension PCR
PCR expands scFv genes.
3）, the natural phage antibody library of genetic engineering preparation
For the product that PCR is obtained after agarose gel electrophoresis detects, the purpose band cut on gel is public through Fermentas
Department's glue reclaim kit carries out recovery target DNA fragment.Obtained PCR primer warp will be reclaimedSfiI andNotAfter I double digestions
It is attached with the carrier pCANTAB-5E DNA of same digestion, the coupled reaction molar ratio of the two is 3:1.16 DEG C are connected
Take over night.By the DNA of overnight connection by electroporated 100 μ L e. coli tg1 competent cells, adding 10 mL immediately
2 × YTG (containing 2% glucose) culture medium, 37 DEG C of cultures, when thalli growth to OD600 is 0.8, add M13KO7 auxiliary
Bacteriophage, to final concentration 1010pfu/mL.Amp antibiotic is added after 30 min to the μ g/mL of final concentration 100, continues culture 1
H, 1000 g, 4 DEG C are collected by centrifugation bacterial sediment；Fresh YT culture mediums are added into precipitation（Contain 100 μ g/mL Amp and 50
μg/mL Kana）, 37 DEG C of overnight incubations；Under low temperature, supernatant is collected by centrifugation in 1000 g, utilizes PEG/NaCl precipitating phages（Body
Product ratio is supernatant：PEG/NaCl=5 :1）, bacteriophage is dissolved with fresh YT culture mediums, the solution of gained is at the beginning of phage antibody
Level storehouse.Using the bacteriophage of different gradient dilutions, respectively take 10 μ L to be coated on the LB flat boards of top layer agarose, calculate storage capacity.
4）, the natural phage antibody library of genetic engineering identification
The random bacterium single bacterium colony transformant selected on 20 SOB-AG flat boards, inoculation (contain 2% with 42 × YTG of mL
Glucose) culture medium, 37 DEG C of overnight incubations, enters performing PCR amplification identification using the PCR transformants different to 20.Meanwhile utilize
Phage vector universal primer (pCANTAB5-R1:5 '-CCATGATTACGCCAAGCTTTGGAGCC-3 ') to institute's picking
Transformant carries out DNA sequencing analysis.
The elutriation of antibody gene
1）, single-chain antibody elutriation
Antigen diluent is 5 μ g/mL into concentration by the albumen coating buffer for being 9.6 with pH, carries out 96 orifice plate coatings, 100 μ
L/ holes, it is coated with overnight in 4 DEG C of environment.Albumen coating buffer is siphoned away with pipettor, PBS is washed 3 times, to remove free antigen point
Son, the PBSM confining liquids in 200 μ L/ holes are added into hole, be placed in 37 DEG C of 2 h of closing.Confining liquid is discarded, is added into hole
The phage antibody library prepared, 100 μ L/ holes, it is placed in 37 DEG C of 2 h of reaction.Respectively with PBST and PBS hole flushings 10 times, 200 μ
L/ holes.Gly-HCL is added into reacting hole, using acid condition elution and the bacteriophage of antigen binding of the solution, then with 1
mol/L Tris-HCL（pH 8.0）Neutralize Gly-HCL.The bacteriophage progress TG1 cells that above-mentioned elution neutralizes are taken out immediately to invade
Dye, to expand the elutriation that bacteriophage number is easy to next round.Method carries out the enrichment elutriation of continuous 6 wheel like this, is opened from third round
Begin, often take turns the bacteriophage that elutriation obtains and take 10 μ L to be coated.
2）, antigentic specificity single-chain antibody ELISA detection
Third and fourth, five, six wheel coatings SOB-AG flat boards on each 25 single-chain antibodies of picking single bacterium colony, according to upper
The method stated carries out the preparation of phage antibody.The albumen coating buffer for being 9.6 with pH by antigen diluent into concentration be 5 μ g/mL,
96 orifice plate coatings are carried out, 100 μ L/ holes, are coated with overnight in 4 DEG C of environment.Albumen coating buffer is siphoned away with pipettor, PBS is washed 3 times,
To remove free antigen molecule, the PBSM confining liquids in 200 μ L/ holes are added into hole, are placed in 37 DEG C of 2 h of closing.Discard closing
Liquid, the phage antibody of above-mentioned single bacterium colony is separately added into hole, 100 μ L/ holes, is made a record, be placed in 37 DEG C of 2 h of reaction.Point
Not Yong PBST and PBS hole flushings 10 times, 200 μ L/ holes.Add the monoclonal antibody of the albumen of anti-p III of PBSM dilutions（1：4000 is dilute
Release）, it is placed in 37 DEG C of 2 h of reaction.Repeat step 6 once, adds the sheep anti-mouse igg antibody of the HRP marks of PBSM dilutions（1：
8000 dilutions）, it is placed in 37 DEG C of 2 h of reaction.Repeat step 6 once.100 μ L of the addition TMB colorbuffers per hole, 37 DEG C
15 min are reacted, add 2M H2SO4 terminating reactions.OD450 nm determine absorption value.All experiment progress 3 are parallel, with
M13KO7 is set to positive, and BSA is set to negative control.
3）, antigentic specificity single-chain antibody identification
The DNA sequencing of all positive single-chain antibody strain recombinant plasmids is carried out in Huada gene company, the sequence after sequencing
Row are directly compared with ncbi database, the primer sequence used during sequencing for：pCANTAB5-R1: 5’-CCA TGA
TTA CGC CAA GCT TTG GAG CC-3’.Heavy chain of antibody and chain variable region gene sequence are obtained by DNA sequencing.
The construction and expression purifying of genetic engineering IgG antibody prokaryotic expression carrier
UtilizeEcoRI andHind Antibody VH genes are inserted pET by III digestion site-his6-sp-ch-his6In carrier,
Form recombinant vector pET-his6-sp-vh-ch-his6, correct recombinant plasmid will be sequenced in e. coli bl21（DE3）In
Carry out expressing fusion protein (as shown in Fig. 1).The albumen of expression contains His6 tag labels.Utilize Sph I and Cla I digestions
Vl gene is inserted pACYC- by sitesp-cl-c-mycIn carrier, recombinant vector pACYC- is formedsp-vl-cl-c-myc,
Correct recombinant plasmid will be sequenced expressing fusion protein is carried out in Escherichia coli BAC-hFLIG-Ab-001.The albumen of expression contains
There are c-myc labels (as shown in Fig. 1).Protein SDS-PAGE analysis result is as shown in Figure 4：With control strain and empty vector control
Compare, there are two obvious expressing fusion protein bands at 55 kDa and 25 kDa respectively, the protein band of purifying is also distinguished
At 55kDa and 25 kDa.Because the heavy chain of the IgG antibody molecule of expression contains His-6 tag labels, and light chain segments are then
His-6 tag labels are not contained.This research and utilization nickel ion affinity chromatograph and antigen antibody interaction building block principle carry out IgG
Antibody purification, finally obtain complete IgG antibody molecule.SDS-PAGE analysis results have two protein expression bars of heavy chain and light chain
Band, show, the structure of prokaryotic expression carrier is successful, and the heavy chain and light chain segments of antibody can interact and assemble
Into IgG antibody, follow-up related experiment can be carried out.
The ELISA identifications of expression in escherichia coli genetic engineering IgG antibody
By the genetic engineering IgG antibody of purifying（1：50； 1：100；1：200 dilutions）It is dilute with coating buffer solution with BSA antigens
Release to 5.0 μ g/mL concentration, be coated with 96 hole elisa Plates respectively, 100 μ L/ holes, closed after rinsing and with 4% PBSM, 200 μ
L/ holes.With 5% PBSM 1:The goat anti-human igg antibody that the 8000 anti-6 × His antibody and HRP for diluting HRP marks respectively mark, 37
DEG C 1.5 h are incubated, repeated washing step, add the μ L/ holes of TMB nitrite ions 100,37 DEG C of 10 ~ 15 min of incubation, and add 2
mol/L H2SO4Terminate liquid.The OD values surveyed with ELIASA at 450 nm.As shown in Fig. 5, ELISA results are shown, the antibody of expression
Heavy chain and light chain occur to assemble and be utilized affinity chromatography method successful purification and come out, the genetic engineering IgG antibody energy of purifying
The goat anti-human igg antibody of enough anti-6 × His antibody and HRP marks by HRP marks identifies respectively, illustrates to express by this method
It is successful with purifying gene engineering IgG antibody.
The determination of activity of expression in escherichia coli genetic engineering IgG antibody
With coating buffer solution（pH 9.6）By target antigen（TLH）2.5 μ g/mL concentration is diluted to, washing closing is such as the same
Face ELISA modes are carried out, and are separately added into the genetic engineering IgG antibody of the purifying of different diluted concentrations, and goat-anti people is marked with HRP
The binding activity of IgG antibody indirect detection IgG antibody and antigen, the specificity of antibody is analyzed with OD450 nm values.Such as Fig. 6 institutes
Show, the IgG antibody can combine with target antigen, and with add antibody being proportionate property of concentration, meanwhile, the antibody without
Combined with Negative antigens.ELISA results show that there is this method high efficient expression the IgG antibody of antigen-binding activity to be can
Capable.
The specific assay of expression in escherichia coli genetic engineering IgG antibody
With coating buffer solution（pH 9.6）By different antigen（1-2:TLH, feminine gender:BSA、Control 1: OVA、
Control 2: KLH）It is diluted to 2.5 μ g/mL concentration, washing closing is carried out such as above ELISA modes, and anti-TLH's is anti-
Serum is used to do positive control, and the specificity of antibody is analyzed with OD450 nm values.As shown in fig. 7, the IgG antibody only know by specificity
Other TLH antigens, and the antigen molecule related to other does not react.Show genetic engineering IgG antibody specificity preferably, with
Other antigens almost do not have cross reaction.
The foregoing is only presently preferred embodiments of the present invention, all equivalent changes done according to scope of the present invention patent with
Modification, it should all belong to the covering scope of the present invention.
SEQUENCE LISTING
<110>Foochow Kang Heer bio tech ltd
<120>A kind of method that genetic engineering IgG antibody is prepared in Escherichia coli
<130> 9
<160> 9
<170> PatentIn version 3.3
<210> 1
<211> 22
<212> DNA
<213>Artificial sequence
<400> 1
aggtsmarct gcagsagtcw gg 22
<210> 2
<211> 32
<212> DNA
<213>Artificial sequence
<400> 2
tgaggagacg gtgacggtgg tcccttggcc cc 32
<210> 3
<211> 24
<212> DNA
<213>Artificial sequence
<400> 3
gacattgagc tcacccagtc tcca 24
<210> 4
<211> 24
<212> DNA
<213>Artificial sequence
<400> 4
ccgtttgatt tccagcctgg tgcc 24
<210> 5
<211> 24
<212> DNA
<213>Artificial sequence
<400> 5
ccgttttatt tccagcctgg tgcc 24
<210> 6
<211> 24
<212> DNA
<213>Artificial sequence
<400> 6
ccgttttatt tccaaccttg tgcc 24
<210> 7
<211> 24
<212> DNA
<213>Artificial sequence
<400> 7
ccgtttcagc tccagcctgg tgcc 24
<210> 8
<211> 26
<212> DNA
<213>Artificial sequence
<400> 8
ccatgattac gccaagcttt ggagcc 26
<210> 9
<211> 26
<212> DNA
<213>Artificial sequence
<400> 9
ccatgattac gccaagcttt ggagcc 26
Claims (3)
1. Escherichia coli（Escherichia coli）BAC-hFLIG-Ab-001, on 06 20th, 2016 in the micro- life of China
The common micro-organisms center preservation of thing culture presevation administration committee, preserving number are CGMCC No.12641.
A kind of 2. genetic engineering IgG antibody caused by bacterial strain as described in claim 1.
3. antibody as claimed in claim 2 is in the pathogenic microorganism examination kit in preparing food, agricultural product or medical science
Using.
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