CN106146658B - A kind of preparation method recombinating Porcine epidemic diarrhea virus antibody - Google Patents
A kind of preparation method recombinating Porcine epidemic diarrhea virus antibody Download PDFInfo
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- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
- C07K16/10—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from RNA viruses
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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
- C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
- C12N15/1034—Isolating an individual clone by screening libraries
- C12N15/1037—Screening libraries presented on the surface of microorganisms, e.g. phage display, E. coli display
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- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
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- C07K2317/00—Immunoglobulins specific features
- C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
- C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
- C07K2317/622—Single chain antibody (scFv)
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- C12N2800/101—Plasmid DNA for bacteria
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Abstract
The present invention relates to a kind of preparation methods for recombinating Porcine epidemic diarrhea virus antibody, the preparation method of this recombination Porcine epidemic diarrhea virus antibody: the mode of antigen-antibody coexpression is chosen, PEDV antigen gene S1 and anti-PEDV ScFv gene co-expression is intracavitary in colibacillus periplasm, utilize flow cytometry to carry out three-wheel elutriation；With PEDV antibody is purified after Bacillus coli expression ScFv gene, PEDV ScFv antibody specificity is detected with ELISA method, is named as PEDV-ScFv；It is carried out especially by two steps of preparation of the building in the library anti-PEDV ScFv, anti-PEDV antibody.Pig source PEDV antibody provided by the invention will not generate immune response, it is with strong points, be easier to screen the high antibody of neutralization activity, short preparation period, speed it is fast.
Description
One, technical field
The present invention relates to the preparations of the genetic engineering of Porcine epidemic diarrhea virus (PEDV) antibody, and in particular to Yi Zhongchong
The preparation method of group Porcine epidemic diarrhea virus antibody.
Two, background technique
Antibody is the natural endogenous protein of body, and toxicity is low, and specificity is high, can be done directly on target spot, in disease
It is widely applied in treatment, becomes the important biotech drug of one kind of clinical application.By 2010, U.S. FDA approved
25 kinds of antibody are used for disease treatment, and more than 240 kinds antibody are applied to clinic, and the disease for the treatment of includes autoimmune disease and inflammation
Disease, cancer, organ transplant, cardiovascular disease, infectious disease and ophthalmology disease etc..The clinical success use of antibody drug produces huge
Big commercial profit, sales volume in 2007 are more than 27,000,000,000 dollars, account for 8 in 20 most situation of selling well biotech drugs, biology
The share of medicine 20%.
Pig epidemic diarrhea (porcine epidemic diarrhea, PED) is one kind by Porcine epidemic diarrhea virus
Intestines problem highly infectious caused by (porcine epidemic diarrhea virus, PEDV).The pig at any age
It can infect, especially serious on piglet influence, case fatality rate is very high；For the farrowing sow of adult, reproductive performance after infection
It is affected, will appear miscarriage after infecting such as the sow of early pregnancy, pregnancy rate reduces；Weight loss after growing and fattening pigs infection.Mesh
Before, although commercialization PEDV vaccine, the Combined vaccine of PEDV and transmissible gastroenteritis of swine (TGEV) have generally used, the disease is still
It is popular.2007, Thailand broke out PED, and large quantities of piglets occurs death, causes serious financial consequences.Meanwhile PEDV conduct
A possibility that a member of coronavirus, viral genome is big, and mutation rate is high, attenuated vaccine back mutation is big.Subunit vaccine
It develops also without the infection problems for fundamentally solving PEDV, piglet is usually since maternal antibody is low, disappears or without breast milk
And lose passive immunity；Or the antibody that piglet self injections vaccine generates has little time the PEDV to play a protective role, and traditional
Treatment method is unable to satisfy people to green food demand again.Therefore, a kind of antibody of effective prevention and control pig epidemic diarrhea is developed
Drug is very necessary.
Three, summary of the invention
It is an object of the present invention to provide a kind of preparation method for recombinating Porcine epidemic diarrhea virus antibody, this Recombinant Swine streams
The preparation method of row diarrhea virus antibody can generate immune response, manufacturing cycle to solve traditional source of mouse PEDV antibody
Problem long, antibody neutralization is low.
To achieve the goals above, the specific technical solution that the present invention uses is as follows: this recombination Porcine Epidemic Diarrhea
The preparation method of malicious antibody: the mode of antigen-antibody coexpression is chosen, by PEDV antigen gene S1 and anti-PEDV ScFv base
It is intracavitary in colibacillus periplasm because co-expressing, three-wheel elutriation is carried out using flow cytometry；With Bacillus coli expression ScFv base
PEDV antibody is purified because after, is detected PEDV ScFv antibody specificity with ELISA method, is named as PEDV-ScFv；Especially by
The building in the library anti-PEDV ScFv, two steps progress of preparation of anti-PEDV antibody.
The method of the building in the library anti-PEDV ScFv is as follows in above scheme:
1. the detection of animal immune and serum antibody titer:
3-6 age in days piglet attacks poison, front and back totally 3 times later first with PEDV attenuated vaccine immunity with medium virulence strain；Every time
It is immune or attack before poison 1 day and last time it is 1 week immune after take a blood sample, indirect ELISA detects serum antibody titer；96 hole elisa Plates packets
By the S1 albumen of recombination PEDV, primary antibody is test serum, and every plate sets blank control, negative control and each 2 hole of positive control, horseradish
The rabbit-anti pig IgG of peroxidase labelling is secondary antibody, and TMB colour developing reads each hole OD value with microplate reader；Last time is 1 week immune
Afterwards, Sodium azide is added to final concentration of 0.2%, 4 in the positive serum for selecting antibody titer highoC places stand-by；
Higher three piglets of antibody titer are put to death, spleen is taken, places it on 300 mesh nylon wires and grind, take cell
Suspension；Splenocyte suspension adds ACK lysing buffer, is placed at room temperature for 5 min, and 2000 g are centrifuged 10 min, collects leucocyte
Precipitating；By 106Trizol is added in the amount of a cell/ml, and lytic cell extracts total serum IgE；With the lymph of three pig spleens of extraction
Total serum IgE after mixing with cells is template, and Oligo (dT) -18 is primer, referring to M-MLV reverse transcriptase specification, carries out cDNA the
One chain synthesis, and carry out 1% agarose gel electrophoresis with 1 μ l reverse transcription product and check its quality；
According to the both ends constant-region sequences of the pig heavy chain of antibody VH, light chain VL variable region gene that have been delivered on GenBank, divide
Not Li Yong primer-design software Primer 5.0 analyze design primer, and be separately added into heavy chain upstream and downstreamNhe I、NdeI enzyme
Enzyme site, light chain downstream are separately added intoBam HI、NotI restriction enzyme site；Using immune rear pig spleen cDNA as template, respectively on VH
Swim primer, VH downstream primer and VL upstream primer, VL downstream primer carries out grads PCR amplification；VH and VL amplified production is carried out
Glue recycling, and connect with pMD18-T carrier, DH5 α is converted, and random 1 single colonie of picking is sequenced, and determines the base obtained
Because sequence is pig source antibody.
2. the building of ScFv bacteria display antibody library:
Using immune rear pig spleen cDNA as template, heavy chain variable region gene is expanded with VH upstream primer, VH downstream primer, wherein
5 ' the ends of VH introduceNheI restriction enzyme site, 3 ' introduceNdeI restriction enzyme site, PCR product utilize Ago-Gel DNA reclaim reagent
Box is purified；It takes VH PCR purified product to connect with pMD18-T carrier, converts DH5 α, and random 1 single colonie of picking carries out
Sequencing；It carries outNhe I、NdeI double digestion, 37oC water-bath 3h, the identification of 1% agarose gel electrophoresis, and it is anti-to carry out conversion building VH
Body library；Next day collects all bacterium colonies and extracts plasmid, carries outBam HI、NotThe identification of I double digestion；Then it usesBamH I、Not
I carries out double digestion to VL PCR recovery product；Glue recycling is carried out after digestion, connects and convert DH5 α, spread plate, 37oC is stayed overnight
Culture collects bacterium colony and extracts plasmid, as PEDV-ScFv antibody library；
It takes the PEDV-ScFv antibody library of building to carry out digestion identification and PCR identification, uses respectivelyNhe I、NdeI andBam
HI、NotI carries out double digestion to antibody library；Taking PEDV-ScFv antibody library plasmid is template, uses the upstream and downstream of VH and VL respectively
Its progress PCR identification of primer pair.
In above scheme anti-PEDV antibody the preparation method is as follows:
1. flow cytometry screens Escherichia coli pBSD-PEDV ScFv display libraries:
All bacterium colonies in the library of above-mentioned collection are taken to be diluted to OD with LB liquid medium600≈ 0.2,37oC cultivates 2h, makes
OD600IPTG to final concentration of 2.5 mmol/L is added in ≈ 0.4, induces 4 h.It is added in 1.5 ml Eppendorf pipes
1.5 ml cultures, 6000 rpm are centrifuged 3 min, remove supernatant；
Bacterial sediment is resuspended with 350 μ l Sucrose/Tris solution, and the concentration of Sucrose is 0.75mmol/
The concentration of L, Tris are 0.1mol/L；The lysozyme mother liquor that 35 μ l concentration are 10 mg/ml Fresh is added；It is added dropwise
700 μ l, 1 mmol/L EDTA solution, while the mixing that is vortexed；It is incubated for 15 min on ice；50 μ l 0.5mol/L MgCl are added2
Solution, and it is incubated for 10 min on ice；4oC, 10000 rpm are centrifuged 10 min；Spheroplast precipitates 1 ml PBS solution weight
Outstanding washing, 6000 rpm are centrifuged 3 min, remove supernatant；Spheroplast precipitating is resuspended with 100 μ l PBS；
It is that 1% BSA stores liquid, 1.5 that 10 μ l mass volume ratios are added in the bacterium solution of induction that 100 μ l PBS are resuspended
S1 after μ g FITC label, is protected from light is incubated for 1 h on ice；8000 rpm, are centrifuged 3 min, and 1 ml PBS resuspension washed once, sink
It forms sediment and is resuspended with 100 μ l PBS.Negative control is set, blank bacterium DH5 α is treated as spheroplast, in blank bacterium DH5 α not
The recombinant plasmid of ScFv containing pBSD-IBDV is incubated for the VP2 antigen after label；
With flow cytometer under 488 nm wavelength lasers, the fluorescence intensity of test sample and negative control collects sample
Middle fluorescence intensity is higher than the part of negative control；The bacterium sorted out is subjected to plasmid preparation, it is electroporated to Escherichia coli
DH5 α constructs secondary screens library, carries out the second wheel by above-mentioned method and screens, the cell sorting in each peak ranges is gone out
Come, converts bacillus coli DH 5 alpha after preparing plasmid, construct secondary screens library, after carrying out third round screening by above-mentioned method, choose
Single colonie 20 are taken, is detected one by one with flow cytometer after spreading cultivation, the fluorescence signal clone stronger than negative control is selected, into
Row is sequenced and analyzes；
2. expression and purification and activity of the anti-PEDV ScFv antibody in Escherichia coli:
Sequence alignment will be carried out with online pig source antibody after plasmid order-checking, utilizes primer-design software Primer
Analysis design PCR primer P1, P2 of Premier 5.0, chooses two progress PCR amplification, purifying, connects into pMD18-T carrier, turn
Change to bacillus coli DH 5 alpha competent cell, picking positive colony is inoculated in LB liquid medium and is incubated overnight, and extracts matter
Sequencing is sent to after grain, PCR and digestion identification；After sequencing result is correct, double digestion is carried out to plasmid, is connected into pET-27b carrier, is turned
It dissolves into Rosetta；
Picking contains the Rosetta single colonie of expression vector pET-27b-ScFv, is inoculated in respectively containing 100 μ g/ml
Amp+LB liquid medium in, 37oC is incubated overnight；Next day takes above-mentioned overnight culture to be inoculated in respectively with 1% ratio to contain
100 μg/ml Amp+LB liquid medium in, 37oC shaken cultivation 2h, adds IPTG 37oC continues to cultivate, and induces 4h.It takes
1ml culture thallus；Pre-cooling PBS is added after washing to be resuspended；5 × SDS sample buffer is added, boils 5 min；Take 20 μ l samples
Carry out SDS-PAGE；It after electrophoresis, dyes through coomassie brilliant blue R_250, is decolourized with methanol-glacial acetic acid destainer, observe result；
Picking conversion recombinant plasmid pET-27b-ScFv single bacterium colony is inoculated in the LB liquid containing 100 μ g/ml Amp+
In culture medium, overnight incubation；It takes above two overnight culture to be inoculated in 1L with 1% ratio and contains 100 μ g/ml Amp+'s
In fresh LB liquid medium, 37oC shaken cultivation is to OD600When value reaches 0.3, add IPTG to 0.25 mmol/L of final concentration,
37 oC continues 4 h of Fiber differentiation；Culture is taken out in 4oC is centrifuged 5 min with 12000 r/min, collects thallus；Supernatant is abandoned, often
20 ml bacterium solutions precipitating is added 1ml Binding buffer and thallus is resuspended, and addition lysozyme is high on ice to final concentration 1mg/ml
Intensity ultrasonic is crushed about 40 min, each 10s, every minor tick 10s；Thallus 12000 rpm 4 after ultrasonic disruptionoC centrifugation
30 min, collect inclusion body respectively；Respectively with denaturing liquid dissolution 4 after washingoC is stayed overnight；Albumen after dissolution is added to 10 times of bodies
In long-pending renaturation solution, 4oC renaturation 24 h, pH are adjusted to 7.4 or so；In the PBS through pH 7.4 in 4oC dialyses 3 times, every time 8 h；
Albumen after purification carries out SDS-PAGE, and with its concentration of UV spectrophotometer measuring；By antigen protein with concentration gradient packet
By 96 orifice plates, in 4oC coating is overnight.PBST washes 96 orifice plate 3 times, every time 2 min, with 5% skimmed milk power 37oC closes 2h, respectively
The diluted concentration gradient of confining liquid is added to be 100 μ g/mL, antibody is not added in 10 μ g/mL, 5 μ g/mL, 1 μ g/mL negative control
S1 albumen, 37oC is incubated for 2 h, and PBST washes 96 orifice plate 3 times, adds secondary antibody HRP-goat anti-porcine IgG, and 37oC incubates 1 h, after PBS washes 96 orifice plate 4 times, tmb substrate liquid is added, in 37oC is protected from light 5 min of colour developing, and it is whole that 50 μ L are added in every hole
Only liquid detects its OD value with microplate reader under 450 nm of wavelength.
The primer that S1 is expanded in above scheme:
Upstream primer 5 '-GGTCTCTAGGTGTTACTTTGCCATCATTTAA-3 ' (SEQ ID NO:1)
Downstream primer 5 '-ATGGATCCTTAAACGTCCGTGACACCTTCAA-3 ' (SEQ ID NO:2).
The composition of Binding buffer in above scheme are as follows: 8 mol/L Urea, 0.1 mol/LNaH2PO4, 0.01
Mol/L Tris, the pH 8.0 of Binding buffer.
The composition of denaturing liquid in above scheme are as follows: 8 mol/L urea, 0.1 mol/L Na2HPO4, 0.01 mol/L
Tris-HCL, pH 8.0。
Renaturation formula of liquid is reduced glutathione and oxidized form of glutathione to final concentration of 2 mmol/ in above scheme
L and 0.2 mmol/L, arginine monohydrochloride to 0.5 mol/L of final concentration, glycerol to final concentration 10%, Tris to final concentration 0.4
Mol/L, renaturation solution pH are adjusted to 7.4 or so.
Terminate liquid is the H of 2 mol/L in above scheme2SO4。
The utility model has the advantages that
1, pig source PEDV antibody provided by the invention will not generate immune response, it is with strong points, be easier to screen neutralization
The high antibody of activity, short preparation period, speed are fast.
2, the present invention, by the screening of flow cytometry three-wheel, has obtained anti-PEDV in a manner of E. coli display
Pig source antibody, have neutralize PEDV ability, can be used as the clinical treatment antibody of PEDV.
3, PEDV antibody provided by the invention is pig source antibody, is resisted to avoidable in grice diarrhoea therapeutic process by source of mouse
Pig anti-mouse antibody caused by body generates.
4, preparation method is simple by the present invention, is suitble to expand scale up test, wide market.
Four, Detailed description of the invention
Fig. 1 is the blank control that flow cytometry sorts anti-PEDV single-chain antibody in the present invention；
Fig. 2 is the single-chain antibody that the flow cytometry first round sorts anti-PEDV in the present invention；
Fig. 3 is the single-chain antibody that the wheel of flow cytometry second sorts anti-PEDV in the present invention；
Fig. 4 is the single-chain antibody that flow cytometry third round sorts anti-PEDV in the present invention；
Fig. 5 is the expression and purification of PEDV antibody in the present invention；
Fig. 6 is the compatibility analysis of PEDV antibody in the present invention.
Five, specific embodiment
The present invention is described further below:
The preparation method of this recombination Porcine epidemic diarrhea virus antibody: choosing the mode of antigen-antibody coexpression, will
PEDV antigen gene S1 and anti-PEDV ScFv gene co-express it is intracavitary in colibacillus periplasm, using flow cytometry into
Row three-wheel elutriation.With PEDV antibody is purified after Bacillus coli expression ScFv gene, PEDV ScFv antibody is detected with ELISA method
Specificity is named as PEDV-ScFv.
The specific preparation method of above-mentioned PEDV antibody:
One, the building in the library anti-PEDV ScFv
1. the detection of animal immune and serum antibody titer
3-6 age in days piglet attacks poison, front and back totally 3 times later first with PEDV attenuated vaccine immunity with medium virulence strain.Every time
It is immune or attack before poison 1 day and last time it is 1 week immune after take a blood sample, indirect ELISA detects serum antibody titer.96 hole elisa Plates packets
By the S1 albumen of recombination PEDV, primary antibody is test serum, and every plate sets blank control, negative control and each 2 hole of positive control, horseradish
The rabbit-anti pig IgG of peroxidase labelling is secondary antibody, and TMB colour developing reads each hole OD value with microplate reader.Last time is 1 week immune
Afterwards, Sodium azide is added to final concentration of 0.2%, 4 in the positive serum for selecting antibody titer highoC places stand-by.By antibody titer
Higher three piglets are put to death, and are taken spleen, are placed it on 300 mesh nylon wires and grind, take cell suspension.Splenocyte suspension adds
ACK lysing buffer is placed at room temperature for 5 min, and 2000 g are centrifuged 10 min, collects leukocyte cell pellet.By 106A cell/
Trizol is added in the amount of ml, and lytic cell extracts total serum IgE.With the mixed total serum IgE of lymphocyte of three pig spleens of extraction
For template, Oligo (dT) -18 is primer, referring to M-MLV reverse transcriptase specification, carries out first chain synthesis of cDNA, and with 1
μ l reverse transcription product carries out 1% agarose gel electrophoresis and checks its quality.According to the pig heavy chain of antibody delivered on GenBank
(VH), the both ends constant-region sequences of light chain (VL) variable region gene are utilized respectively the analysis of primer-design software Primer 5.0 and set
Primer is counted, and is separately added into heavy chain upstream and downstreamNhe I、NdeI restriction enzyme site, light chain downstream are separately added intoBam HI、Not I
Restriction enzyme site.Using immune rear pig spleen cDNA as template, respectively under VH upstream primer, VH downstream primer and VL upstream primer, VL
It swims primer and carries out grads PCR amplification.Glue recycling is carried out to VH and VL amplified production, and is connect with pMD18-T carrier, DH5 is converted
α, and random 1 single colonie of picking is sequenced, and determines that the gene order obtained is pig source antibody.
The primer of S1 amplification:
Upstream primer 5 '-GGTCTCTAGGTGTTACTTTGCCATCATTTAA-3 ' (SEQ ID NO:1)
Downstream primer 5 '-ATGGATCCTTAAACGTCCGTGACACCTTCAA-3 ' (SEQ ID NO:2)
2. the building of ScFv bacteria display antibody library
Using pig spleen cDNA as template, heavy chain variable region gene is expanded with VH upstream primer, VH downstream primer, wherein the 5 ' of VH
End introducesNheI restriction enzyme site, 3 ' introduceNdeI restriction enzyme site, PCR product are carried out using Ago-Gel DNA QIAquick Gel Extraction Kit
Purifying.It takes VH PCR purified product to connect with pMD18-T carrier, converts DH5 α, and random 1 single colonie of picking is sequenced.
It carries outNhe I、NdeI double digestion, 37o3 h of C water-bath, the identification of 1% agarose gel electrophoresis, and carry out conversion building VH antibody
Library.Next day collects all bacterium colonies and extracts plasmid, carries outBam HI、NotThe identification of I double digestion.Then it usesBamH I、Not I
Double digestion is carried out to VL PCR recovery product, digestion system is same as above.Glue recycling is carried out after digestion, connects and convert DH5 α, is coated with
Plate, 37oC is incubated overnight, and is collected bacterium colony and is extracted plasmid, as PEDV-ScFv antibody library.Take the PEDV-ScFv of building anti-
Body library carries out digestion identification and PCR identification, uses respectivelyNhe I、NdeI andBam HI、NotI carries out double digestion to antibody library.
Taking PEDV-ScFv antibody library plasmid is template, carries out PCR identification to it using the upstream and downstream primer of VH and VL respectively.
Sequence 3 in the ScFV gene order of PEDV antibody such as sequence table indicates.
Two, the preparation of anti-PEDV antibody
1. flow cytometry screens Escherichia coli (pBSD-PEDV ScFv) display libraries
All bacterium colonies in the library of above-mentioned collection are taken to be diluted to OD with LB liquid medium600≈ 0.2,37oC cultivates 2h, makes
OD600IPTG to final concentration of 2.5 mmol/L is added in ≈ 0.4, induces 4 h.It is added in 1.5 ml Eppendorf pipes
1.5 ml cultures, 6000 rpm are centrifuged 3 min, remove supernatant.350 μ l Sucrose (0.75mmol/ of bacterial sediment
L)/Tris (0.1mol/L) solution is resuspended；The lysozyme mother liquor that 35 μ l concentration are 10 mg/ml Fresh is added；
700 μ l, 1 mmol/L EDTA solution, while the mixing that is vortexed is added dropwise；It is incubated for 15 min on ice；50 μ l are added
0.5mol/L MgCl2Solution, and it is incubated for 10 min on ice；4oC, 10000 rpm are centrifuged 10 min；Spheroplast is precipitated with 1
Washing is resuspended in ml PBS solution, and 6000 rpm are centrifuged 3 min, remove supernatant；Spheroplast precipitating is resuspended with 100 μ l PBS.?
10 μ l, 1% (mass volume ratio) BSA is added in the bacterium solution (induction) that 100 μ l PBS are resuspended and stores liquid, 1.5 μ g FITC
S1 after label is protected from light is incubated for 1 h on ice；8000 rpm, are centrifuged 3 min, and 1 ml PBS resuspension washed once, precipitate with 100
μ l PBS is resuspended.Negative control is set, blank bacterium DH5 α (being free of pBSD-IBDV ScFv recombinant plasmid) is treated as primary
Matter ball is incubated for the VP2 antigen after label.With flow cytometer under 488 nm wavelength lasers, test sample and negative control
Fluorescence intensity, collect sample in fluorescence intensity be higher than negative control part.The bacterium sorted out is subjected to plasmid preparation,
It is electroporated to construct secondary screens library to bacillus coli DH 5 alpha, the second wheel is carried out by above-mentioned method and is screened, by each peak value model
Cell sorting in enclosing comes out, and converts bacillus coli DH 5 alpha after preparing plasmid, constructs secondary screens library, carries out by above-mentioned method
It after third round screening, picking single colonie 20, is detected one by one with flow cytometer after spreading cultivation, selects fluorescence signal than negative
Strong clone is compareed, be sequenced and is analyzed.
2. expression and purification and activity of the anti-PEDV ScFv antibody in Escherichia coli
Sequence alignment will be carried out with online pig source antibody after plasmid order-checking, utilizes primer-design software Primer
Analysis design PCR primer P1, P2 of Premier 5.0, chooses two progress PCR amplification, purifying, connects into pMD18-T carrier, turn
Change to bacillus coli DH 5 alpha competent cell, picking positive colony is inoculated in LB liquid medium and is incubated overnight, and extracts matter
Sequencing is sent to after grain, PCR and digestion identification.After sequencing result is correct, double digestion is carried out to plasmid, is connected into pET-27b carrier, is turned
It dissolves into Rosetta.Picking contains the Rosetta single colonie of expression vector pET-27b-ScFv, is inoculated in respectively containing 100 μ
g/ml Amp+LB liquid medium in, 37oC is incubated overnight.Next day takes above-mentioned overnight culture to be inoculated with respectively with 1% ratio
In containing 100 μ g/ml Amp+LB liquid medium in, 37o2 h of C shaken cultivation, adds IPTG 37oC continues to cultivate, induction 4
h.Take 1 ml culture thallus.Pre-cooling PBS is added after washing to be resuspended.5 × SDS sample buffer (containing DTT) is added, boils 5
min.20 μ l samples are taken to carry out SDS-PAGE.It after electrophoresis, is dyed through coomassie brilliant blue R_250, with methanol-glacial acetic acid destainer
Result is observed in decoloration.Picking conversion recombinant plasmid pET-27b-ScFv single bacterium colony is inoculated in containing 100 μ g/ml Amp+'s
In LB liquid medium, overnight incubation.It takes above two overnight culture to be inoculated in 1 L with 1% ratio and contains 100 μ g/ml
Amp+Fresh LB liquid medium in, 37oC shaken cultivation is to OD600When value reaches 0.3, add IPTG to final concentration 0.25
Mmol/L, 37oC continues 4 h of Fiber differentiation.Culture is taken out in 4oC is centrifuged 5 min with 12000 r/min, collects thallus.It abandons
1 ml Binding buffer (8 mol/L Urea, 0.1 mol/LNaH are added in supernatant, every 20 ml bacterium solution precipitating2PO4,
0.01 mol/L Tris, pH 8.0) thallus is resuspended, lysozyme is added to 1 mg/ml of final concentration, high strength supersonic is broken on ice
Broken about 40 min, every time 10 s, every 10 s of minor tick.Thallus 12000 rpm 4 after ultrasonic disruptionoC is centrifuged 30 min,
Inclusion body is collected respectively.Denaturing liquid (8 mol/L urea, 0.1 mol/L Na are used after washing respectively2HPO4, 0.01 mol/L
Tris-HCL, pH 8.0) dissolution 4oC is stayed overnight.Albumen after dissolution is added in the renaturation solution of 10 times of volumes, 4oC renaturation 24
H, renaturation formula of liquid are reduced glutathione and oxidized form of glutathione to final concentration of 2 mmol/L and 0.2 mmol/L, essence
Propylhomoserin hydrochloride to 0.5 mol/L of final concentration, glycerol to final concentration 10%, Tris to 0.4 mol/L of final concentration, pH be adjusted to 7.4 a left side
It is right.Through PBS (pH 7.4) 4oC dialyses 3 times, every time 8 h.Albumen after purification carries out SDS-PAGE, and uses ultraviolet spectrometry
Photometer detects its concentration.Antigen protein is coated with 96 orifice plates with concentration gradient, in 4oC coating is overnight.PBST washes 96 orifice plates 3
It is secondary, 2 min every time, with 5% skimmed milk power 37oC closes 2 h, and respectively plus the diluted concentration gradient of confining liquid is 100 μ g/mL,
10 μ g/mL, the S1 albumen (antibody is not added in negative control) of 5 μ g/mL, 1 μ g/mL, 37oC is incubated for 2 h, and PBST washes 96 holes
Plate 3 times, adding secondary antibody HRP-goat anti-porcine IgG (H+L), (Positive control wells add HRP-goat anti-
Mouse antibody is purchased from R&D company), 37oC incubates 1 h, after PBS washes 96 orifice plate 4 times, tmb substrate liquid is added, in 37oC
5 min of colour developing are protected from light, the 50 μ L terminate liquid (H of 2 mol/L are added in every hole2SO4), it is detected under 450 nm of wavelength with microplate reader
OD value.
Embodiment 1:
It selects the strain of medium virulence to carry out attacking poison to piglet first, the antibody titer of piglet is detected, later to extraction
Simultaneously reverse transcription constructs antibody library at cDNA to the RNA of piglet spleen.Before preparing antigen, biology has been carried out to PEDV S gene
Bioinformatics analysis finds that its glycosylation site is more, and expression difficulty is big, low output, thus later experiments have chosen antigen-antibody
The mode of coexpression has carried out ScFv antibody library three-wheel using flow cytometry and has screened.Then extract plasmid, after PCR amplification, gram
It is grand in carrier T, serving the raw work sequencing in sea.Sequence analysis is carried out using Primer5.0 and DNAMAN software, after plasmid order-checking
Sequence alignment is carried out with online pig source antibody, design PCR is analyzed using primer-design software Primer Premier 5.0 and draws
Object P1, P2 choose two progress PCR amplification, purifying, connect into pMD18-T carrier, it is thin to be transformed into bacillus coli DH 5 alpha competence
Born of the same parents, picking positive colony, are inoculated in LB liquid medium and are incubated overnight, and extract plasmid, send to survey after PCR and digestion identification
Sequence.After sequencing result is correct, double digestion is carried out to plasmid, expression vector is connected into later, is transformed into IPTG induction table in Rosetta
It reaches.1 ml culture thallus is taken, pre-cooling PBS is added after washing and is resuspended, SDS sample buffer (containing DTT) is added, boils.Take 20
μ l sample carries out SDS-PAGE.It after electrophoresis, dyes through coomassie brilliant blue R_250, is decolourized with methanol-glacial acetic acid destainer, it can be with
See ScFv in expression in escherichia coli.Picking conversion positive recombinant plasmid pET-27b-ScFv single bacterium colony be inoculated in containing
Amp+LB liquid medium in, by continuous overnight incubation, IPTG is added to induce, thalline were collected by centrifugation.Lysozyme is added, in ice
Upper high strength supersonic is broken.Thallus is centrifuged after ultrasonic disruption, collects inclusion body respectively.Denaturation and renaturation are carried out after washing,
SDS-PAGE is carried out after obtaining the albumen of purifying, and uses its concentration of UV spectrophotometer measuring, with the anti-PEDV of ELISA detection
ScFv, it was demonstrated that PEDV ScFv has the ability for specially neutralizing virus.
Sequence table 1
<110>Heilongjiang Bayi Agricultural Reclamation University
<120>a kind of preparation method for recombinating Porcine epidemic diarrhea virus antibody
<160> 3
<210> 1
<211> 31
<212> DNA
<213>artificial sequence
<400> 1
GGTCTCTAGGTGTTACTTTGCCATCATTTAA 31
Sequence table 2
<110>Heilongjiang Bayi Agricultural Reclamation University
<120>a kind of preparation method for recombinating Porcine epidemic diarrhea virus antibody
<160> 3
<210> 2
<211> 31
<212> DNA
<213>artificial sequence
<400> 2
ATGGATCCTTAAACGTCCGTGACACCTTCAA 31
Sequence table 3
<110>Heilongjiang Bayi Agricultural Reclamation University
<120>a kind of preparation method for recombinating Porcine epidemic diarrhea virus antibody
<160> 3
<210> 3
<211> 753
<212> DNA
<213>artificial sequence
<400> 3
1 GAGGTGAAGCTGGTGGAGTCTGGAGGAGGCCTGGTGCAGCCTGGGGGGTCTCTG
55 AGACTCTCCTGTGTCGGCTCTGGATTCACCTTCAGTAGTTATGCAATGAGCTGG
109 GTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGCTCGCATCTATTTATAGTAGT
163 GGTTGTAGCACCTACTACACAGACTCTGTGAAGGGCCGATTCACGTTCTCCATC
217 CACGACTCCCAGAACAAGGTGTATCTGCAAATCAACAGCCTCAGAACCGAAGAT
271 ACGGCCCGGTATTACTGTGCGATAGGCCCAGCGGTTGCTATAGCGGTTACTGTG
325 CAAGAGGCCGAATTCGGTGGCGGTGGCTCCGGCGGTGGTGGGTCGGGTGGCGGC
379 GGATCTGGTGGCGGTGGCTCGGGCGGTGGTGGGTCGGGTGGCGGCGGATCACTC
433 GAGGCCATCGTGCTGACCCAGACTCCAGCCTCCCTGGCTGCATCTCTAGGAGAC
487 ACGGTCTCCATCACTTGCCGGGCCAGTCAGAGTGTTAGGAGTTATTTAGCCTGG
541 TATCAACAACAACCAGGGAAGGCTCCGAAACTCGTGATCTATGCTACATCCAAT
595 TTAGAAAGTGGGGTCCCATCCCGCTTCAAGGGCAGTGGATCTGGCACCGATTTC
649 ACCCTCACCATCAGTGGCCTGCAGGCTGAAGATGTTGCAACTTATTACTGTTTG
703 CAGTGGAGCGGTGTATATGGTTTCGGCGCGGGGACCAAACTGGAGCTCAAA
Sequence table 1
<110>Heilongjiang Bayi Agricultural Reclamation University
<120>a kind of preparation method for recombinating Porcine epidemic diarrhea virus antibody
<160> 3
<210> 1
<211> 31
<212> DNA
<213>artificial sequence
<400> 1
GGTCTCTAGGTGTTACTTTGCCATCATTTAA 31
Sequence table 2
<110>Heilongjiang Bayi Agricultural Reclamation University
<120>a kind of preparation method for recombinating Porcine epidemic diarrhea virus antibody
<160> 3
<210> 2
<211> 31
<212> DNA
<213>artificial sequence
<400> 2
ATGGATCCTTAAACGTCCGTGACACCTTCAA 31
Sequence table 3
<110>Heilongjiang Bayi Agricultural Reclamation University
<120>a kind of preparation method for recombinating Porcine epidemic diarrhea virus antibody
<160> 3
<210> 3
<211> 753
<212> DNA
<213>artificial sequence
<400> 3
1 GAGGTGAAGCTGGTGGAGTCTGGAGGAGGCCTGGTGCAGCCTGGGGGGTCTCTG
55 AGACTCTCCTGTGTCGGCTCTGGATTCACCTTCAGTAGTTATGCAATGAGCTGG
109 GTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGCTCGCATCTATTTATAGTAGT
163 GGTTGTAGCACCTACTACACAGACTCTGTGAAGGGCCGATTCACGTTCTCCATC
217 CACGACTCCCAGAACAAGGTGTATCTGCAAATCAACAGCCTCAGAACCGAAGAT
271 ACGGCCCGGTATTACTGTGCGATAGGCCCAGCGGTTGCTATAGCGGTTACTGTG
325 CAAGAGGCCGAATTCGGTGGCGGTGGCTCCGGCGGTGGTGGGTCGGGTGGCGGC
379 GGATCTGGTGGCGGTGGCTCGGGCGGTGGTGGGTCGGGTGGCGGCGGATCACTC
433 GAGGCCATCGTGCTGACCCAGACTCCAGCCTCCCTGGCTGCATCTCTAGGAGAC
487 ACGGTCTCCATCACTTGCCGGGCCAGTCAGAGTGTTAGGAGTTATTTAGCCTGG
541 TATCAACAACAACCAGGGAAGGCTCCGAAACTCGTGATCTATGCTACATCCAAT
595 TTAGAAAGTGGGGTCCCATCCCGCTTCAAGGGCAGTGGATCTGGCACCGATTTC
649 ACCCTCACCATCAGTGGCCTGCAGGCTGAAGATGTTGCAACTTATTACTGTTTG
703 CAGTGGAGCGGTGTATATGGTTTCGGCGCGGGGACCAAACTGGAGCTCAAA
Claims (6)
1. a kind of preparation method for recombinating Porcine epidemic diarrhea virus antibody, it is characterised in that: this recombination pig epidemic diarrhea
The preparation method of antiviral antibody: the mode of antigen-antibody coexpression is chosen, by PEDV antigen gene S1 and anti-PEDV ScFv
Gene co-expression is intracavitary in colibacillus periplasm, and flow cytometry is utilized to carry out three-wheel elutriation；With Bacillus coli expression ScFv
PEDV antibody is purified after gene, is detected PEDV ScFv antibody specificity with ELISA method, is named as PEDV-ScFv；It is specific logical
Two steps of preparation of the building, anti-PEDV antibody of crossing the library anti-PEDV ScFv carry out；
The method of the building in the library anti-PEDV ScFv is as follows:
One, the detection of animal immune and serum antibody titer:
3-6 age in days piglet attacks poison, front and back totally 3 times later first with PEDV attenuated vaccine immunity with medium virulence strain；It is immune every time
Or attack before poison 1 day and last time it is 1 week immune after take a blood sample, indirect ELISA detects serum antibody titer；96 hole elisa Plates coating weight
The S1 albumen of group PEDV, primary antibody is test serum, and every plate sets blank control, negative control and each 2 hole of positive control, horseradish peroxide
The rabbit-anti pig IgG of compound enzyme label is secondary antibody, and TMB colour developing reads each hole OD value with microplate reader；After last time is 1 week immune, choosing
With the high positive serum of antibody titer, Sodium azide is added to final concentration of 0.2%, 4oC places stand-by；
Higher three piglets of antibody titer are put to death, spleen is taken, places it on 300 mesh nylon wires and grind, take cell suspension；
Splenocyte suspension adds ACK lysing buffer, is placed at room temperature for 5 min, and 2000 g are centrifuged 10 min, collects leukocyte cell pellet；
By 106Trizol is added in the amount of a cell/ml, and lytic cell extracts total serum IgE；With the lymphocyte of three pig spleens of extraction
Mixed total serum IgE is template, and Oligo (dT) -18 is primer, referring to M-MLV reverse transcriptase specification, carries out cDNA first
Chain synthesis, and carry out 1% agarose gel electrophoresis with 1 μ l reverse transcription product and check its quality；
It is sharp respectively according to the both ends constant-region sequences of the pig heavy chain of antibody VH, light chain VL variable region gene that have been delivered on GenBank
Design primer is analyzed with primer-design software Primer 5.0, and is separately added into heavy chain upstream and downstreamNhe I、NdeI digestion position
Point, light chain downstream are separately added intoBam HI、NotI restriction enzyme site；Using immune rear pig spleen cDNA as template, drawn respectively with the upstream VH
Object, VH downstream primer and VL upstream primer, VL downstream primer carry out grads PCR amplification；Glue is carried out to VH and VL amplified production to return
It receives, and is connect with pMD18-T carrier, convert DH5 α, and random 1 single colonie of picking is sequenced, and determines the gene sequence obtained
It is classified as pig source antibody；
Two, the building of ScFv bacteria display antibody library:
Using pig spleen cDNA after immune as template, heavy chain variable region gene is expanded with VH upstream primer, VH downstream primer, wherein VH
5 ' ends introduceNheI restriction enzyme site, 3 ' introduceNdeI restriction enzyme site, PCR product using Ago-Gel DNA QIAquick Gel Extraction Kit into
Row purifying；It takes VH PCR purified product to connect with pMD18-T carrier, converts DH5 α, and random 1 single colonie of picking is surveyed
Sequence；It carries outNhe I、NdeI double digestion, 37oC water-bath 3h, the identification of 1% agarose gel electrophoresis, and carry out conversion building VH antibody
Library；Next day collects all bacterium colonies and extracts plasmid, carries outBam HI、NotThe identification of I double digestion；Then it usesBamH I、Not I
Double digestion is carried out to VL PCR recovery product；Glue recycling is carried out after digestion, connects and convert DH5 α, spread plate, 37oC is stayed overnight
Culture collects bacterium colony and extracts plasmid, as PEDV-ScFv antibody library；
It takes the PEDV-ScFv antibody library of building to carry out digestion identification and PCR identification, uses respectivelyNhe I、NdeI andBam HI、NotI carries out double digestion to antibody library；Taking PEDV-ScFv antibody library plasmid is template, is drawn respectively using the upstream and downstream of VH and VL
Object carries out PCR identification to it；
Anti- PEDV antibody the preparation method is as follows:
One, flow cytometry screens Escherichia coli pBSD-PEDV ScFv display libraries:
All bacterium colonies in the library of above-mentioned collection are taken to be diluted to OD with LB liquid medium600≈ 0.2,37oC cultivates 2h, is allowed to OD600
IPTG to final concentration of 2.5 mmol/L is added in ≈ 0.4, induces 4 h；1.5 ml training is added in 1.5 ml Eppendorf pipes
Object is supported, 6000 rpm are centrifuged 3 min, remove supernatant；
Bacterial sediment is resuspended with 350 μ l Sucrose/Tris solution, and the concentration of Sucrose is 0.75mmol/L,
The concentration of Tris is 0.1mol/L；The lysozyme mother liquor that 35 μ l concentration are 10 mg/ml Fresh is added；It is added dropwise 700
1 mmol/L EDTA solution of μ l, while the mixing that is vortexed；It is incubated for 15 min on ice；50 μ l 0.5mol/L MgCl are added2It is molten
Liquid, and it is incubated for 10 min on ice；4oC, 10000 rpm are centrifuged 10 min；Spheroplast precipitating is resuspended with 1 ml PBS solution
Washing, 6000 rpm are centrifuged 3 min, remove supernatant；Spheroplast precipitating is resuspended with 100 μ l PBS；
It is that 1% BSA stores liquid, 1.5 μ g that 10 μ l mass volume ratios are added in the bacterium solution of induction that 100 μ l PBS are resuspended
S1 after FITC label, is protected from light is incubated for 1 h on ice；8000 rpm are centrifuged 3 min, and 1 ml PBS resuspension washed once, and precipitating is used
100 μ l PBS are resuspended；Negative control is set, blank bacterium DH5 α is treated as spheroplast, is free of in blank bacterium DH5 α
PBSD-IBDV ScFv recombinant plasmid is incubated for the VP2 antigen after label；
With flow cytometer under 488 nm wavelength lasers, the fluorescence intensity of test sample and negative control is collected glimmering in sample
Luminous intensity is higher than the part of negative control；The bacterium sorted out is subjected to plasmid preparation, it is electroporated to bacillus coli DH 5 alpha,
Secondary screens library is constructed, the second wheel is carried out by above-mentioned method and screens, the cell sorting in each peak ranges is come out, is prepared
Bacillus coli DH 5 alpha is converted after plasmid, constructs secondary screens library, after carrying out third round screening by above-mentioned method, picking single colonie
It 20, is detected one by one with flow cytometer after spreading cultivation, selects the fluorescence signal clone stronger than negative control, be sequenced simultaneously
Analysis；
Two, expression and purification and activity of the anti-PEDV ScFv antibody in Escherichia coli:
Sequence alignment will be carried out with online pig source antibody after plasmid order-checking, utilizes primer-design software Primer Premier
5.0 analysis design PCR primers P1, P2, choose two progress PCR amplification, purifying, connect into pMD18-T carrier, be transformed into large intestine
Bacillus DH5 α competent cell, picking positive colony are inoculated in LB liquid medium and are incubated overnight, and extract plasmid, PCR and enzyme
Sequencing is sent to after cutting identification；After sequencing result is correct, double digestion is carried out to plasmid, pET-27b carrier is connected into, is transformed into
In Rosetta；
Picking contains the Rosetta single colonie of expression vector pET-27b-ScFv, is inoculated in respectively containing 100 μ g/ml Amp+'s
In LB liquid medium, 37oC is incubated overnight；Next day takes above-mentioned overnight culture to be inoculated in respectively with 1% ratio containing 100 μ
g/ml Amp+LB liquid medium in, 37oC shaken cultivation 2h, adds IPTG 37oC continues to cultivate, and induces 4h；1ml is taken to train
Support object thallus；Pre-cooling PBS is added after washing to be resuspended；5 × SDS sample buffer is added, boils 5 min；20 μ l samples are taken to carry out
SDS-PAGE；It after electrophoresis, dyes through coomassie brilliant blue R_250, is decolourized with methanol-glacial acetic acid destainer, observe result；
Picking conversion recombinant plasmid pET-27b-ScFv single bacterium colony is inoculated in the LB Liquid Culture containing 100 μ g/ml Amp+
In base, overnight incubation；It takes above two overnight culture to be inoculated in 1L with 1% ratio and contains 100 μ g/ml Amp+It is fresh
In LB liquid medium, 37oC shaken cultivation is to OD600When value reaches 0.3, add IPTG to 0.25 mmol/L of final concentration, 37oC
Continue 4 h of Fiber differentiation；Culture is taken out in 4oC is centrifuged 5 min with 12000 r/min, collects thallus；Abandoning supernatant, every 20
Ml bacterium solution precipitating is added 1ml Binding buffer and thallus is resuspended, and addition lysozyme is high-strength on ice to final concentration 1mg/ml
Spend ultrasonication about 40 min, each 10s, every minor tick 10s；Thallus 12000 rpm 4 after ultrasonic disruptionoC centrifugation 30
Min collects inclusion body respectively；Respectively with denaturing liquid dissolution 4 after washingoC is stayed overnight；Albumen after dissolution is added to 10 times of volumes
Renaturation solution in, 4oC renaturation 24 h, pH are adjusted to 7.4 or so；In the PBS through pH 7.4 in 4oC dialyses 3 times, every time 8 h；It is pure
Albumen after change carries out SDS-PAGE, and with its concentration of UV spectrophotometer measuring；Antigen protein is coated with concentration gradient
96 orifice plates, in 4oC coating is overnight；PBST washes 96 orifice plate 3 times, every time 2 min, with 5% skimmed milk power 37oC close 2h, respectively plus
The diluted concentration gradient of confining liquid is 100 μ g/mL, and antibody is not added in 10 μ g/mL, 5 μ g/mL, 1 μ g/mL negative control
S1 albumen, 37oC is incubated for 2 h, and PBST washes 96 orifice plate 3 times, adds secondary antibody HRP-goat anti-porcine IgG (H+
L), 37oC incubates 1 h, after PBS washes 96 orifice plate 4 times, tmb substrate liquid is added, in 37oC is protected from light 5 min of colour developing, and every hole is added 50
μ L terminate liquid detects its OD value with microplate reader under 450 nm of wavelength.
2. the preparation method of recombination Porcine epidemic diarrhea virus antibody according to claim 1, it is characterised in that: described
The primer of S1 amplification:
Upstream primer 5 '-GGTCTCTAGGTGTTACTTTGCCATCATTTAA-3 ' (SEQ ID NO:1)
Downstream primer 5 '-ATGGATCCTTAAACGTCCGTGACACCTTCAA-3 ' (SEQ ID NO:2).
3. the preparation method of recombination Porcine epidemic diarrhea virus antibody according to claim 2, it is characterised in that: described
The composition of Binding buffer are as follows: 8 mol/L Urea, 0.1 mol/LNaH2PO4, 0.01 mol/L Tris, Binding
The pH 8.0 of buffer.
4. the preparation method of recombination Porcine epidemic diarrhea virus antibody according to claim 3, it is characterised in that: described
Denaturing liquid composition are as follows: 8 mol/L urea, 0.1 mol/L Na2HPO4, 0.01 mol/L Tris-HCL, pH 8.0。
5. the preparation method of recombination Porcine epidemic diarrhea virus antibody according to claim 4, it is characterised in that: described
Renaturation formula of liquid is reduced glutathione and oxidized form of glutathione to final concentration of 2 mmol/L and 0.2 mmol/L, smart ammonia
Acid hydrochloride is adjusted to 0.5 mol/L of final concentration, glycerol to final concentration 10%, Tris to 0.4 mol/L of final concentration, renaturation solution pH
7.4 left and right.
6. the preparation method of recombination Porcine epidemic diarrhea virus antibody according to claim 5, it is characterised in that: described
Terminate liquid is the H of 2 mol/L2SO4。
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