CN109517072A - A kind of preparation of typhoid bacillus recombinant protein and its monoclonal antibody - Google Patents
A kind of preparation of typhoid bacillus recombinant protein and its monoclonal antibody Download PDFInfo
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Abstract
The present invention relates to field of biotechnology, the preparation of a kind of typhoid bacillus recombinant protein and its monoclonal antibody is disclosed.Typhoid bacillus recombinant protein of the present invention, amino acid sequence is as shown in SEQ ID No:1.To enhance expression effect of the recombinant protein in prokaryotic cell, the present invention is with one section of soft segment (four glycine) as connection small peptide, it will be repeated three times after the A Dominant Epitopes of typhoid bacillus flagellum (H) antigen and the series connection of B Dominant Epitopes sequence, form a recombinant protein.The invention further relates to the preparations of the recombinant protein monoclonal antibody, obtain monoclonal cell strain by immune, cell fusion and multi-turns screen, monoclonal antibody purification simultaneously distinguishes mark fluorescent microballoon, and orthogonal experiment determines optimum monoclonal antibody combinations of pairs.There is high specific with the immune monoclonal antibody obtained of the recombinant antigen, can be used for the early diagnosis of typhoid bacillus infection.
Description
Technical field
The present invention relates to field of biotechnology, and in particular to a kind of typhoid bacillus recombinant protein encodes the recombinant protein
Nucleotide sequence, the plasmid vector containing above-mentioned nucleotide sequence, conversion contain the bacterial strain of above-mentioned plasmid vector, and with above-mentioned
Recombinant protein prepares monoclonal antibody, and is applied to the quick diagnosis of typhoid bacillus infection.
Background technique
Typhoid fever is a kind of Class B acute infectious intestinal disease as caused by typhoid bacillus through transmission.Cardinal symptom table
It is now high fever, rose color spot etc. occur in abdominal pain, severe diarrhea, headache, body.Intestinal bleeding or perforation are the concurrent of its most serious
Disease.The full course of disease is infectious, maximum with the 2nd~4 week infectiousness of the course of disease.Whole year can fall ill, but be more common in summer, autumn,
Raw and cold food is fond of with people and fly frequent activity is related.Typhoid fever be still at present it is Chinese and in the world many developing countries compared with
For common one of infectious disease, the annual typhoid number in the whole world is more than 30,000,000 people, and death toll is more than due to typhoid fever every year
500000 people.The diagnosis basis of typhoid bacillus is mainly clinical symptoms and laboratory diagnosis, in recent years since typhoidette increases and resists
Rhzomorph is widely used, and clinical symptoms are not often true to type.Therefore, Rapid&Early diagnosis is the key that prevent and treat the disease and emphasis.
Currently, common typhoid fever diagnostic method includes Bacteria Culture, Widal's test, polymerase chain reaction technology and is immunized
Learn detection method etc..Wherein, Bacteria Culture and Widal's test are the conventional methods for diagnosing typhoid fever, but the Bacteria Culture time is longer,
And vulnerable to multifactor impacts such as antibiotic, condition of culture, the quality of collection of specimens and times, carrier's recall rate is lower, as a result
Report also often needs the time of 3d~5d, it is difficult to achieve the purpose that early diagnosis.Widal's test is anti-with the thallus (O) of typhoid bacillus
Former and flagellum (H) antigen and paratyphoid A, second, the third bacterium solution are reacted with diluted test serum, judge blood according to agglutination titer
Whether there is or not the antibody of typhoid bacillus in clear, often there is false positive in this method, and poor specificity, sensibility is low, and antigen-antibody combines
There is coagulation sedimentation and often need 20h or more, also without early diagnosis meaning.Polymerase chain reaction technology needs to utilize specific instrument
Device, specific occasion are detected, not convenient enough and time-consuming.
As a result immunological detection method is easy to determine because its is easy to operate, suitable for great amount of samples on-site quick screening etc.
Feature and obtain very extensive application.This method is mainly to pass through the monoclonal antibody of preparation typhoid bacillus flagellum (H) albumen
To realize the specific detection to typhoid bacillus.Conventionally used for typhoid bacillus H protein monoclonal antibody preparation immunogene be
Whole albumen, due to epitope amino acid sequence homology reason, obtained monoclonal antibody specificity is poor, may identify it
Its albumen, causes testing result to be distorted.
Summary of the invention
In view of this, the purpose of the present invention is pass through a kind of typhoid bacillus recombinant protein of optimization design and prepare its monoclonal
Antibody, the specific recognition for typhoid bacillus detect.
For achieving the above object, the invention provides the following technical scheme:
(1) it using typhoid bacillus flagellum (H) antigen as target antigen, analyzes and selects the antigen two specific Dominant Epitopes A
And B, sequence compare selected two epitopes of display and other protein sequences without obvious homology.(2) for selected by enhancing
Preferentially stimulation of the gesture epitope to Balb/c mouse immune system, with one section of soft segment (four glycine) as connection
Small peptide will repeat three times after the A Dominant Epitopes of typhoid bacillus flagellum (H) antigen and the series connection of B Dominant Epitopes sequence, form a weight
Histone.(3) Escherichia coli preference codon is used, recombinant protein amino acid sequence is converted into corresponding nucleotide sequence,
In favor of expression of the recombinant protein in Escherichia coli, to improve expression quantity.(4) nucleosides that chemical synthesis previous step obtains
Acid sequence, and connected by digestion, nucleotide fragments insertion expression vector pET-28a (+) that synthesis is obtained, building recombination egg
White expression vector.(5) recombinant protein expression vector converts Escherichia coli ER2566 competent cell, and IPTG induction screening obtains weight
Histone expresses bacterial strain.(6) after recombinant protein expression bacterial strain large-scale culture, carrying out ultrasonic bacteria breaking and low-temperature centrifugation are collected supernatant and are used
Nickel agarose affinity chromatography column purification, typhoid bacillus recombinant protein of dialysing to obtain.(7) recombinant protein after purification is repeatedly immunized
After Balb/c mouse, its spleen cell is taken to merge with sp2/0 myeloma cell, finally obtains hybridoma through multi-turns screen
Strain.(8) hybridoma cell strain is prepared to Balb/c mouse ascites respectively, uses octanoic acid-ammonium sulfate precipitation method and ProteinA parent
With chromatography monoclonal antibody purification, and mark fluorescent microballoon respectively in two steps.(9) orthogonal experiment screening display 4F7 monoclonal antibody coating
Marking pairing detection typhoid bacillus with 2A6 fluorescent microsphere is optimal combination.
Compared with the background technology, the present invention, first is that realizing typhoid bacillus flagellum (H) albumen by Protocols in Molecular Biology
The repetition of two dominant antigen epitopes and expressing in series, enhance stimulation of the purpose antigen epitope to mouse immune system, exclude
Unrelated sequences possible interference；Second is that using the corresponding nucleotides sequence of Escherichia coli preference codon optimum combination albumen
Column, greatly improve expression of the recombinant protein in Escherichia coli；Third is that the recombinant protein as immunogene only contains typhoid fever
The distinctive dominant antigen epitope of bacillus H protein, ensure that the high specific of anti-typhoid bacillus H protein monoclonal antibody, and screen
Optimum monoclonal antibody combinations of pairs is obtained, detection sensitivity is improved.
Specific embodiment:
Although following embodiment contrasts detailed verbal description to mentality of designing of the invention, these texts are retouched
State, only the simple text of mentality of designing of the present invention described, rather than the limitation to mentality of designing of the present invention, it is any without departing from
The combination, increase or modification of mentality of designing of the present invention, falls within the protection scope of the present invention.
Embodiment 1: typhoid bacillus flagellum (H) albumen dominant antigen epitope selection
Using typhoid bacillus flagellum (H) albumen as target antigen, its epitope sequence is analyzed using biosoftware DNAssist2.0
The hydrophily and antigenicity of column take into account its specificity, select A dominant antigen epitope (SEQ ID No:2) and B dominant antigen epitope
(SEQ ID No:3).Sequence compares the selected two dominant antigen epitopes of A, B of display with other protein sequences without obvious homologous
Property.
Embodiment 2: the series connection of typhoid bacillus flagellum (H) albumen dominant antigen epitope and sequence optimisation
To reinforce A, B dominant antigen epitope to the effect of Balb/c mouse immune, with one section of soft segment (four sweet ammonia
Acid) as connection small peptide, it is repeated three times after A the and B dominant antigen epitope sequences of typhoid bacillus flagellum (H) albumen are connected, group
At recombinant protein amino acid sequence, amino acid sequence is as shown in SEQ ID No:1.To improve recombinant protein in Escherichia coli
Expression quantity recombinant protein amino acid sequence is converted to by corresponding nucleotide sequence using Escherichia coli preference codon,
Nucleotide sequence adds BamHI and EcoRI restriction enzyme site as shown in SEQ ID No:4, in the nucleotide sequence upstream and downstream respectively
Sequence transfers to Nanjing Genscript Biotechnology Co., Ltd. to synthesize, and the target gene after synthesis is cloned in the (purchase of pMD19-T carrier
From in precious bioengineering Dalian Co., Ltd) in.
Embodiment 3: recombinant protein expression vector establishment
The target gene of synthesis is building up to pET- with restriction enzyme BamHI and EcoRI (being purchased from TaKaRa)
In 28a (+) (being purchased from Novagen) prokaryotic expression carrier.With pET-28a (+) for carrier, BamHI and EcoRI double digestion is anti-
Answer system (20 μ L, 37 DEG C of digestion 2h) as follows:
BamHI the and EcoRI double enzyme digestion reaction system (20 μ L, 37 DEG C of digestion 2h) of the target gene of synthesis is as follows:
Digestion products are carried out using plastic recovery kit (being purchased from Ningbo Zhong Ding Bioisystech Co., Ltd) after digestion to return
It receives, linked system (5 μ L) is as follows:
4 DEG C of connection 12h, connection product convert bacillus coli DH 5 alpha, and are coated on that penicillin resistance (50 μ g/mL) containing card
LB plate, after 37 DEG C of constant temperature are inverted culture 12h, in picking individual colonies on plate to containing that penicillin resistance (50 μ g/mL) of card
LB liquid medium, 37 DEG C of constant-temperature table culture 8h (are had using plasmid purification kit purchased from ancient cooking vessel biotechnology in Ningbo
Limit company) plasmid is extracted, correct recombinant expression carrier is obtained after the identification of BamHI and EcoRI double digestion.
Embodiment 4: recombinant protein expresses strain construction
The recombinant protein expression vector Transformed E .coli ER2566 competent cell that will be built, and be coated on to contain and block that
The LB plate of penicillin resistance (50 μ g/mL), 37 DEG C are inverted culture overnight, and monoclonal bacterial strain is to containing that mould of card on picking plate
The LB liquid medium of plain resistance (50 μ g/mL) after 37 DEG C of constant-temperature table culture 5h, adds isopropylthio-β-D-galactoside
(IPTG) protein electrophoresis sample is prepared after (final concentration of 1.0mmol/L) inducing expression 4h.14% polyacrylamide gel electrophoresis
The result shows that recombinant protein successful expression, obtains recombinant protein expression bacterial strain.
Embodiment 5: recombinant protein inducing expression, purifying and dialysis
Recombinant protein is expressed into strain inoculated to the LB liquid medium for containing that penicillin (final concentration of 50 μ g/mL) of card
In, 37 DEG C of constant-temperature table cultures to OD600=0.6 should with the LB liquid medium of 50 μ g/mL card that penicillin containing final concentration
After bacterium is by 1:100 dilution, dispenses into bacteria culture bottle, set 37 DEG C of shaking table constant temperature and be incubated overnight, next day adds inducer isopropylthio
Thio-β-D- galactoside (IPTG) continues to cultivate 5h to final concentration of 1.0mmol/L.4 DEG C thalline were collected by centrifugation, with 50mM,
The Tris-HCl suspension thalline of pH8.0, ultrasonication, low-temperature centrifugation collect supernatant.Ni is used after 0.45 μm of membrane filtration of supernatant
Column purification is first washed pillar with 20mM imidazole solution (it is 1L that 1.36g imidazoles, which is dissolved in 10mM PBS solution to final volume), then is used
300mM imidazole solution (it is 1L that 20.4g imidazoles, which is dissolved in 10mM PBS solution to final volume) elution typhoid bacillus recombinant protein, is used
Polyacrylamide gel electrophoresis detection is determined as destination protein.Typhoid bacillus recombinant protein after purification is packed into molecular cut off
For in the bag filter of 10kDa, the PBS buffer solution dialysed overnight of pH 7.4, next day takes out packing, saved in -20 DEG C.
Embodiment 6: the acquisition of hybridoma cell strain
4-6 week old female Balb/c healthy mice is taken, after being emulsified completely with 100 μ g recombinant proteins and Freund's complete adjuvant,
Take subcutaneous multi-point injection.Second of same dose and incomplete Freund's adjuvant mixed immunity are carried out after 15 days；15 μ are taken after 30 days
G recombinant protein tail vein booster shots take blood in injection 72h posterior orbit, and put to death mouse, its spleen is taken to prepare cell suspension,
Cell count adds by 1/5 after the quantity of splenocyte takes the good sp2/0 murine myeloma cell of growth conditions, mixing to be centrifuged
Entering polyethylene glycol (PEG-4000) merges the two.In addition, adding isometric feeder cells, 96 holes that are placed in after mixing are thin
Born of the same parents' plate (200 hole μ L/), in 5%CO2Incubator culture.Liquid is changed in later half reservation in 5 days, is examined after 10 days using enzyme linked immunosorbent assay
Survey the Hybridoma Cell Culture supernatant in 96 porocyte plates.Concrete operations are as follows:
(1) it is coated with: diluting recombinant protein to final concentration of 1 μ g/mL with coating buffer, ELISA Plate (Shenzhen is added in 100 holes μ L/
Jin Canhua Industrial Co., Ltd.), 4 DEG C overnight after discard liquid in hole, wash 5 times with cleaning solution and pat dry；
(2) it closes: confining liquid is added with 150 holes μ L/, 37 DEG C of closing 2h abandon liquid in hole, and cleaning solution is washed 5 times and clapped
It is dry；
(3) it is loaded: adding cells and supernatant to be checked and control serum, 100 holes μ L/, 37 DEG C of incubation 1h, cleaning solution washing 5
It is secondary and pat dry；
(4) sheep anti mouse of HRP (horseradish peroxidase) label of diluted fresh enzyme labeling antibody: is added with 100 holes μ L/
IgG, after 37 DEG C are incubated for 30 minutes, cleaning solution is washed 5 times and is patted dry；
(5) add developing solution: every hole adds each 50 μ L of developing solution A and developing solution B, and 37 DEG C are protected from light colour developing 10 minutes；
(6) it terminates reaction: 2M H is added with 50 holes μ L/2SO4；
(7) in microplate reader, at 450nm, OD value result judgement: is read behind blank well school zero.With immune serum
As positive control.Related solution formula is as follows:
Coating buffer: Na2CO31.5g, NaHCO32.9g adds ddH2O is settled to 1000mL (pH9.6).
Confining liquid: Na2HPO4.12H2O 2.68g, NaH2PO4.2H2O 0.39g, NaCl 8.5g, 20g bovine serum albumin
It is white, add ddH2O is settled to 1000mL (pH7.4).
Cleaning solution: Na2HPO4.12H2O 2.68g, NaH2PO4.2H2O 0.39g, NaCl 8.5g, Tween-200.5mL,
Add ddH2O is settled to 1000mL (pH7.4).
Developing solution A:200mg TMB is dissolved in 100mL dehydrated alcohol, adds ddH2O is settled to 1000mL.
Developing solution B: citric acid 2.1g, Na2HPO4.12H2O 71g, adds ddH2O is settled to 1000mL.When use: 1mL is aobvious
Color liquid A+1mL developing solution B+0.4 μ L 30%H2O2
Terminate liquid: 2M H2SO4, the dense H of 21.7mL2SO4Add ddH2O is settled to 1000mL.
For the positive hybridoma cell clone of detection, it is subcloned by limiting dilution assay.By being subcloned three times,
Screening obtains 6 strain of hybridoma strains (1G3,2C5,2A6,4F7,6B8,7D1).
Embodiment 7: a large amount of preparations and purifying of monoclonal antibody
The Balb/c healthy mice of 6-8 week old is taken, atoleine is injected intraperitoneally, 500 μ L/ only, are injected intraperitoneally miscellaneous after 4-6 days
Hand over oncocyte (about 1 × 106A/only), after 7 days, mouse web portion is heaved, collection ascites, 12000rpm centrifugation 3min, in collection
Clearly, 60mM pH4.5 sodium-acetate buffer is added in 1:2.5 ratio, the ratio of 25 μ L caprylic acids is added with every milliliter of ascites supernatant
Example is slowly added to caprylic acid under the conditions of magnetic stirrer, and room temperature continues after stirring 30min, 4 DEG C of 5000rpm centrifugations
30min takes supernatant.Supernatant is pre-chilled under the conditions of 4 DEG C, the ratio of 0.227g ammonium sulfate is added with 1mL volume supernatant, slowly
Ammonium sulfate powder is added, stirring while adding, room temperature continues after stirring 30min, and 5000rpm is centrifuged 15min, and precipitating is dissolved in 1/10 body
In long-pending 10mM PBS (pH7.4) buffer, dialysis to no ammonium sulfate.By the ProteinA affinity column of the monoclonal antibody after dialysis
Purifying collects eluting peak to get the monoclonal antibody arrived after purification after washing.
Embodiment 8: the preparation of label typhoid bacillus H protein monoclonal antibody fluorescent microsphere pad
The fluorescent microsphere of diameter 210nm (is purchased from the limited public affairs of Nanjing micrometering biotechnology with 50mM pH4.5MES buffer
Department) concentration is adjusted to 1%, it will be screened early period by the way of carbodiimide (EDC) and succinimide (NHS) covalent coupling
To 6 plants of labeling of monoclonal antibodies to fluorescent microsphere on, antibody concentration 0.2mg/ml.It is fixed that the fluorescent microsphere prepared is used
Amount spray film instrument is sprayed on fluorescent microsphere pad with the amount of 4 μ l/cm, and it is spare to be placed in dry environment by 25 DEG C of 1~2h of vacuum drying.
Embodiment 9: the preparation of nitrocellulose filter (NC film)
It is adjusted respectively with the PBS (phosphate buffer, wherein including 5% sucrose and 0.05%Tween-20) of 10mM pH7.4
Saving 6 plants of monoclonal antibodies (1G3,2C5,2A6,4F7,6B8,7D1) to its concentration screened early period is 0.4mg/mL, by institute
It obtains solution and is sprayed on formation detection zone (T line) on NC film respectively；With the PBS (phosphate buffer, wherein including of 10mM pH7.4
5% sucrose and 0.05%Tween-20) adjusting sheep anti-mouse igg concentration be 0.5mg/mL, acquired solution is sprayed on shape on NC film
At quality control region (C line).The spray film amount in twoth area is 1 μ L/cm, and twoth area are separated by 5mm, quality control region distance NC film one end 2mm, 37 DEG C of bakings
It is dry to stay overnight, it is spare to be placed in drying at room temperature environment.
Embodiment 10: the preparation of fluorescent microsphere immunochromatographydetection detection card
Assemble test strips: successively overlap joint is pasted on PVC bottom plate: (1) filter paper and sample pad, and sample pad is a kind of process
The glass fibre membrane of 5%Tween-20 processing；(2) be coated with fluorescent microsphere label typhoid bacillus H protein monoclonal antibody (1G3,
2C5,2A6,4F7,6B8,7D1) fluorescent microsphere pad；(3) spray typhoid bacillus H protein monoclonal antibody (1G3,2C5,2A6,
4F7,6B8,7D1) NC film as detection zone and sheep anti-mouse igg as quality control region；(4) blotting paper is cut into after being completed
The width of 4mm is loaded onto reagent strip shell and is compressed to get fluorescent microsphere immunochromatographydetection detection card.
Embodiment 11: pairing monoclonal antibody screening
Typhoid patients clinical serum sample, 100 hole μ L/ loadings, after being placed at room temperature for 15min, (is purchased from by fluorescence analyser
With biotech inc advanced in years) T, C line signal on NC film are read respectively and are calculated measured value T/ (T+C), see Table 1 for details.
Table 1 matches monoclonal antibody measured value T/ (T+C) statistics
By upper table it is found that it is optimal combination that 4F7 monoclonal antibody coating, which detects typhoid bacillus with the label pairing of 2A6 fluorescent microsphere,.
Embodiment 12: monoclonal antibody specificity identification
By Salmonella paratyphi A (ATCC 9150), Salmonella choleraesuls (ATCC 10708), paratyphoid B
Salmonella (CICC 21495), Salmonella anatis (CICC 21498), Bacterium enteritidis (CICC24119) and typhoid bacillus
6 kinds of bacterial cultures such as (CMCC 50071) are diluted to 10 with the sterile PBS buffer of 10mM pH7.47CFU/mL, it is mono- with 4F7
Anti- coating marks fluorescent microsphere immunochromatographydetection detection card obtained with 2A6 fluorescent microsphere to detect its specificity.Respectively take 100 μ L bacterium
Liquid adds in detection card well, and T line signal value is read after 10min, and control group is the sterile PBS buffer of 10mM pH7.4.
In triplicate.As a result (table 2) shows only typhoid bacillus test positive, other are feminine gender.
2 specificity experiments result of table
Note: " ++ " indicates strong positive, and "-" indicates negative.
SEQ ID NO1: the amino acid sequence of typhoid bacillus recombinant protein；
SEQ ID NO2: the amino acid sequence of typhoid bacillus H protein A dominant antigen epitope；
SEQ ID NO3: the amino acid sequence of typhoid bacillus H protein B dominant antigen epitope；
SEQ ID NO4: the nucleotide sequence of coding typhoid bacillus recombinant protein.
Claims (6)
1. a kind of typhoid bacillus recombinant protein, amino acid sequence is as shown in SEQIDNo:1.
2. a kind of typhoid bacillus recombinant protein, it includes the amino acid sequences as shown in SEQIDNo:2 and SEQIDNo:3.
3. a kind of nucleotide sequence, it is characterised in that the nucleotide sequence is as shown in SEQIDNo:4, codified claim 1-2
The recombinant protein.
4. a plasmid vector, it is characterised in that the plasmid vector contains nucleotide sequence as claimed in claim 3.
5. a kind of bacterial strain converted using plasmid vector described in claim 4.
6. the preparation that typhoid bacillus recombinant protein claimed in claims 1-2 can be used for monoclonal antibody, it is characterised in that include
Following steps:
(a) engineer and be aided with the Dominant Epitopes of computer simulation typhoid bacillus H antigen, chemical synthesis include BamHI and
The nucleotide sequence of EcoRI restriction enzyme site；
(b) pET- of same BamHI and EcoRI double digestion will be connected to after chemosynthesis product BamHI and EcoRI double digestion
In 28a (+) carrier, recombinant plasmid is obtained；
(c) recombinant plasmid transformed inducing expression into Escherichia coli, purifying are dialysed up to amino acid sequence shown in SEQIDNo:1
Typhoid bacillus recombinant protein；
(d) it after repeatedly Balb/c mouse is immunized in recombinant protein, takes its spleen cell to merge with sp2/0 myeloma cell, is taken turns more
Screening obtains hybridoma cell strain；
(e) monoclonal antibody purification and respectively mark fluorescent microballoon, orthogonal experiment determines optimum monoclonal antibody combinations of pairs.
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