CN102060920A - Construction of abrin A chain mutant and application as candidate vaccine antigen - Google Patents
Construction of abrin A chain mutant and application as candidate vaccine antigen Download PDFInfo
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Abstract
The invention discloses an abrin vaccine related protein and a coding gene and application thereof. The protein provided by the invention is named as mATA, and one of the following 1) and 2) is the protein: 1) protein consisting of amino acid residue sequences of a sequence 2 in a sequence table; and 2) protein consisting of amino acid residue sequences of the sequence 2 in the sequence table by substitution and/or deletion and/or addition of one or more amino acid residues, having the same functions and derived from the 1). Experiments prove that the mutant mATA obtained by point mutation is abrin A chain mutant protein obtained by recombinant expression; and by detection, the protein has the characteristics of low toxicity, no vascular leakage, good immunogenicity and the like, and can be used as a candidate vaccine antigen.
Description
Technical field
The present invention relates to a kind of structure of abrin A chain mutant and, relate in particular to a kind of and abrin vaccine associated protein and encoding gene and application as candidate vaccine antigens.
Background technology
Along with the fast development of biotechnology and the development and use of biotoxin, the importance of toxin in mass destruction weapon shows especially out, and the threat of biotoxin and bio-terrorism thereof grows with each passing day.Abrin is made up of A, B chain, and the A chain is the effect chain, has RNA N-glycosidase activity, and the B chain is a marriage chain, and mediation A chain enters performance toxic action in the cell.The Semen Abri Precatorii in the different places of production contains discrepant slightly abrin on primary structure, and it is variant to show as its toxicity size, the abrin mouse peritoneal LD of bibliographical information
50Between 4-20 μ g/kg.Raw materials for production yearning between lovers seed wide material sources obtain easily, can how to be extracted the detail file of preparation abrin again easily on many disclosed professional magazines and internet, so the threat of terrorism of abrin can not be ignored.Poison at abrin, still do not have vaccine, specificity toxinicide and toxicide at present.By analyzing existing progress and achievement, the biological and ecological methods to prevent plant disease, pests, and erosion expert of U.S. army thinks that vaccine research is effective preventive means of its poisoning of antagonism.Therefore, carry out the research of abrin vaccine, have crucial military affairs and social effect.
Chinese scholar is at the early-stage to research fields such as the detection of abrin biological warfare agent and medical protections, still do not have supporting diagnostic reagent and effective immunotherapy and preventive means at the attack of terrorism of abrin, therefore press for detection and protective articles that foundation and development have independent intellectual property right.In order to prevent that hostile force and terroristic organization from using the attack of biological warfare agents such as abrin, should set up and improve the detection and the medical protection system of biological warfare agents such as abrin as early as possible, guarantee in time to take emergency measures and countermeasure, bio-terrorism is attacked the destruction of causing be reduced to bottom line.
Summary of the invention
An object of the present invention is to provide a kind of and abrin vaccine associated protein and encoding gene thereof.
Protein provided by the invention, called after mATA is following 1) or 2) in arbitrary described protein:
1) protein of forming by the amino acid residue sequence of the sequence in the sequence table 2;
2) with 2 amino acid residue sequences of the sequence in the sequence table through the replacement of one or several amino-acid residue and/or disappearance and/or interpolation and have identical function by 1) deutero-protein.
The encoding gene mATA of mATA also is a scope of protection of the invention, is following 1)-3) in arbitrary described dna molecular:
1) dna molecular shown in the sequence 1 in the sequence table;
2) under stringent condition with 1) dna sequence dna hybridization that limits and dna molecular with identical function;
3) with 1) dna sequence dna that limits has 70% at least, have 75% at least, have 80% at least, have 85% at least, have 90% at least, have 95% at least, have 96% at least, have 97% at least, have 98% or have 99% homology at least and have the dna molecular of identical function at least.
The recombinant vectors, reorganization bacterium, transgenic cell line or the expression cassette that contain described proteinic encoding gene also are the scope of protection of the invention.
Described recombinant vectors is the recombinant vectors that obtains between the EcoR I of described encoding gene insertion carrier pET-His and Nhe I recognition site.
Described reorganization bacterium is for importing the reorganization bacterium that the host bacterium obtains with described recombinant vectors.
The described encoding gene total length that increases or arbitrary segmental primer are to also being the scope of protection of the invention, and a right primer of described primer is the dna molecular shown in the sequence 3, and another primer is the dna molecular shown in the sequence 4.
Another object of the present invention provides a kind of vaccine, and its activeconstituents is following 1)-3) in any: 1) described protein; 2) described recombinant vectors; 3) described reorganization bacterium.
The application of described protein in the preparation vaccine also is the scope of protection of the invention; The application of described encoding gene in the preparation vaccine also is the scope of protection of the invention; The application of described recombinant vectors in the preparation vaccine also is the scope of protection of the invention; The application of described reorganization bacterium in the preparation vaccine also is the scope of protection of the invention.
Described vaccine is the abrin vaccine, is specially abrin A chain mutant vaccine.
Of the present invention experimental results show that; mutant mATA through the point mutation acquisition; it is the abrin A chain mutant protein that obtains by recombinant expressed; by multiple different immunization protocols such as the immunogenic immunization route of A chain mutant, dosage, number of times, adjuvants; analyze the immune protective effect of abrin A chain mutant; the result can be used as candidate vaccine antigens for this mutant has hypotoxicity, do not have vascular leakage, characteristics such as good immunogenicity are arranged.
Description of drawings
Fig. 1 is abrin A chain mutant purifying figure
Fig. 2 is the SDS-PAGE electrophorogram of abrin A chain mutant
Fig. 3 measures the protein concentration typical curve for the BCA method
Fig. 4 is the lethal effect curve of abrin A chain mutant to human liver cancer cell SMMC-7721
Fig. 5 is the lethal effect curve of abrin A chain mutant to myeloma cell Sp2/0
Fig. 6 is a neutralization experiment body weight change in the mATA body
Fig. 7 is the external neutralization experiment of a mATA body weight change
Embodiment
Employed experimental technique is ordinary method if no special instructions among the following embodiment.
Used material, reagent etc. if no special instructions, all can obtain from commercial channels among the following embodiment.
The acquisition of embodiment 1, mutant mATA
1, the acquisition of mutant mATA
(the rATA nucleotides sequence is classified sequence 5 as according to A chain gene (rATA) sequence of existing abrin, the rATA aminoacid sequence is a sequence 6), adopt Quickchange Lighting Site-Directed Mutagenesis Kit (Stratagene company, Catalog#210518) carry out rite-directed mutagenesis and obtain mutant mATA-E164AR167L (plasmid), through order-checking, the unnamed gene that mATA-E164AR167L contains is mATA, its nucleotides sequence is classified the sequence 1 in the sequence table as, the coding region be in the sequence table sequence 1 from 5 ' terminal 1-753 position nucleotide sequence; The aminoacid sequence of mATA encoded protein mATA is the sequence shown in the sequence 2 in the sequence table.Considerable change does not take place in the antigenicity of using the constructed mutant of Lasergene software prediction.
Sequence verification sudden change result, specific as follows:
The 491st base A of sequence 5 is sported the base C of the 491st of sequence 1, and the bases G that sequence 5 is the 500th sports the base T of the 500th of sequence 1.
The 164th amino acids L-glutamic acid (E) of sequence 6 is sported the 164th amino acids L-Ala (A) of sequence 2, and the 167th amino acids arginine (R) of sequence 6 sports the 167th amino acids leucine (L) of sequence 2.
The design primer:
1.ABRaA?Upper：5’-G
GAATTCGAAGATCGCCCGATCAAATTTTCCACCGAAGGTGCCAC-3’
(EcoR I) (sequence 3)
2.ABRaA?Lower：5’-G
GCTAGCATTCGGCGGATTGCAGACAAACAGCATCAGTGCCAGAACTGC-3’
(Nhe I) (sequence 4)
With the mutant plasmid mATA-E164AR167L of above-mentioned acquisition as template, increase with primer ABRaA Upper and ABRaA Lower, the PCR product that obtains is connected to cloning vector pMD18-T (precious biotechnology (Dalian) company limited, catalog number D101B) (but also artificial synthesized sequence 1 is connected to pMD18-T and obtains pMD18T-mATA to obtain pMD18T-mATA.), use EcoR I (NEB company again, R0101V) and Nhe I (NEB company, R0131V) reclaim small segment behind the double digestion pMD18T-mATA, with the expression vector pET-His (Sun Simei that cuts through same enzyme, Wangjinglin etc. the proteic solubility expression of reorganization ricin A chain, purifying and antigenicity analysis. Chinese biological engineering magazine .2005,25 (4): the 47-51. public can obtain from Inst. of Epidemiology and Microbiology, Academy of Military Medical Sciences, PL.) carrier segments connect, the connection product that obtains changes e. coli bl21 (DE3) pLysS (Beijing Quanshijin Biotechnology Co., Ltd over to, catalog number D30619), positive bacteria after screening, check order after extracting plasmid, the plasmid of result for obtaining between the recognition site of this plasmid for the EcoR I of sequence in the sequence table 1 being inserted expression vector pET-His and Nhe I, with this plasmid called after pET-His-mATA, positive bacteria called after BL21 (DE3) pLysS/pET-His-mATA that it is corresponding.
2. purifying mATA
BL21 (DE3) pLysS/pET-His-mATA of above-mentioned acquisition is inoculated in the LB substratum that 5ml contains Amp (100 μ g/ml) and Glu (0.5%) in 1: 100 ratio, and 37 ℃, 200rpm is cultured to A
600=0.6 o'clock, be forwarded to the same LB substratum of 500ml (and establishing negative control) in 1: 100 ratio again, the same condition shaking culture is to A
600=0.6 o'clock, induce group to add IPTG to final concentration 1mM and add Amp, continue shaking culture then, inductive condition changes 30 ℃ into, after 150rpm induces 5h, and 10000rpm, 4 ℃ of centrifugal 10min collect the thalline of expression strain, full bacterium precipitation are washed three times with PBS, again with lysis buffer (0.02M PB, 500mM NaCl, the 50mM imidazoles is pH7.4) after the resuspended ultrasonication, 10000rpm, 4 ℃ of centrifugal 10min collect supernatant liquor and precipitation, and supernatant liquor directly adds 2 * SDS-PAGE gel sample-loading buffer (H
2O, glycerine, 1.5mM pH6.8Tris, 10% tetrabromophenol sulfonphthalein, beta-mercaptoethanol), and add 2 * SDS-PAGE gel sample-loading buffer after the urea dissolving of precipitation with 8M, all boil 5min at last at 100 ℃, and of short duration centrifugal.Draw 20 μ l samples, carry out the SDS-PAGE electrophoresis.
Through the 15%SDS-PAGE electrophoretic analysis, abrin A chain mutant (mATA) has specific protein expression at supernatant liquor, and the albumen relative molecular weight is about 30kDa, conforms to the albumen size of expection.
The supernatant liquor of above-mentioned acquisition is carried out affinity chromatography, chromatography column is Ni Sepharose High performance affinity column (GE Healthcare, 17-5248-01), elutriant (is made up of PB, NaCl, imidazoles and water for containing 500mM imidazoles elutriant, wherein PB is that 0.02M, NaCl are that 500mM, imidazoles are 500mM at the final concentration of elutriant at the final concentration of elutriant at the final concentration of elutriant, PH is 7.4), elution flow rate is 2ml/min, when imidazole concentration is 300mM, collect the mATA that elution peak is purifying, the result as shown in Figure 1.
With the mATA of the purifying collected through the SDS-PAGE electrophoretic analysis, result such as Fig. 2, wherein M is low molecular weight protein (LMWP) marker, 1-3 is mATA behind ni-sepharose purification, as can be seen, the albumen relative molecular weight is about 30kDa, the purity of eluted protein is higher than 98.5%.
Adopting uses the same method changes empty carrier pET-His over to e. coli bl21 (DE3) pLysS, bacterium BL21 (DE3) pLysS/pET-His that obtains recombinating, and the albumen that abduction delivering obtains is albumen in contrast, does not have target protein to express through the checking of SDS-PAGE electrophoresis.
Adopting uses the same method imports the dna molecular shown in the sequence 5 among the carrier pET-His, change e. coli bl21 (DE3) pLysS bacterium BL21 (DE3) pLysS/pET-His-rATA that obtains recombinating again over to, the albumen that abduction delivering obtains is rATA (reorganization abrin A catenin (sudden change)).
The biological activity assay of embodiment 2, mutant mATA
1. mutant protein is quantitative
(purchase the hundred Tyke Bioisystech Co., Ltd in Beijing, Lot#20120613) mATA of the purifying that embodiment 1 is obtained carries out quantitatively, tests triplicate, results averaged to adopt BCA method protein quantification test kit.
Concrete steps:
Test kit is formed: protein standard (5mg/mlBSA), Solution A, Solution B.
When (1) using Solution A is rocked mixing, quantity adds an amount of BCA working fluid of 1 volume Solution B (50: 1) preparation, fully mixing by 50 volume Solution A per sample.
(2) complete soluble protein standard substance (5mg/mlBSA) are diluted to 1.5mg/ml with PBS with it, 1.0mg/ml, and 0.8mg/ml, 0.4mg/ml, 0.2mg/ml is as standard substance.
(3) standard substance and the testing sample with above-mentioned each concentration is added to respectively in 96 orifice plates.
(4) each hole adds the 200ulBCA working fluid, with application of sample rifle piping and druming mixing, places 30-60 minute for 37 ℃ gently.
(5) behind the cool to room temperature, measure A562 with microplate reader.
(6) calculate protein concentration in the testing sample according to typical curve.
The result as shown in Figure 3, the concentration of the mATA of purifying is 1.46mg/ml (target protein that obtains after Ni affinity chromatography column purification), 500ml bacterium liquid gained protein content is 23.5mg.
2, abrin A chain mutant (mATA) antigenicity analysis
The mATA of the purifying that will be obtained by embodiment 1 carries out the Western engram analysis: mATA is transferred on the cellulose acetate film behind the SDS-PAGE electrophoresis, with anti-natural abrin rabbit polyclonal antibody (preparation method sees below and states) is one anti-, with horseradish enzyme labelling goat antirabbit lgG (U.S. Santa Cruz company, ZB-2301) be two anti-, the negative contrast of reference protein that obtains with embodiment 1.
The result is presented at that to have near the 30KDa special colour developing to take out of existing, illustrates that resulting recombination mutation body protein can be discerned by anti-natural abrin rabbit polyclonal antibody, has good antigenicity.Specific antigen antibody reaction does not take place in negative control.
MATA behind the purifying that will be obtained by embodiment 1 carry out serial dilution with coating buffer, wrap quilt then, and the 100ul/ hole is one anti-with anti-natural abrin rabbit polyclonal antibody, be two anti-with horseradish enzyme labelling goat antirabbit lgG, carry out ELISA and detect.With the positive contrast of natural abrin, the negative contrast of reference protein that embodiment 1 obtains.The experiment triplicate, results averaged.
The result is as follows: (the natural abrin of purifying) compares with positive control, the mATA bag of the above concentration purifying of 2ug/ml is strong positive by the hole, illustrate mutant protein mATA can with specific antigen antibody reaction takes place with anti-natural abrin rabbit polyclonal antibody, further verified the exactness of the mutant that obtains.Specific antigen antibody reaction does not take place in negative control.
Natural abrin, its genbank number is X76720, also can obtain by the following method: yearning between lovers seed (Yunnan Semen Abri Precatorii (Abrus precatorius L.), available from Kunming Inst. of Botany, Chinese Academy of Sciences) homogenate, suction filtration, lixiviate, saturated ammonium sulphate, slightly carry, affinity chromatography, gel-filtration, obtain the natural abrin of purifying.(reference: Nie Cong etc. abrin antigen and Antibody Preparation. Chinese Frontier Sanitary Quarantine magazine .2009.32 (4): 281-284).
Anti-natural abrin rabbit polyclonal antibody preparation method: natural abrin attenuation treatment is prepared toxoid with the phosphate buffered saline buffer that contains 1% formaldehyde, with the toxoid is that immunizing antigen divides the purebred large ear rabbit of immune New Zealand 4 times, ear edge vein exploitating blood, with natural abrin is antigen, tire with indirect ELISA mensuration, tiring reaches 1: 10
6Back heart blood sampling.Rabbit anteserum filters through filter membrane, add the acetate buffer solution dilution, under acidic conditions, n-caprylic acid precipitates non-IgG albumen, centrifuging and taking supernatant liquor, saturated ammonium sulfate in neutrallty condition underlying ice bath, with volume fraction is 45% saturation ratio precipitation IgG albumen, collecting precipitation protein, dialysis, centrifugal, get supernatant liquor, filter through filter membrane, by specification is through HiTrapTM rProtein A FF affinity column antibody purification.
3, the activation analysis of mutant protein
By the MTS method measure mutant protein mATA to human liver cancer cell SMMC-7721 (the emerging aokang bio tech ltd in Beijing, AK169711) and myeloma cell Sp2/0 (the true Bioisystech Co., Ltd in Shanghai, lethal effect GS-183), specific as follows:
(1) with human liver cancer cell SMMC-7721 (or myeloma cell Sp2/0) counting, regulates cell count to 10
5/ ml, preparation 10ml cell suspension adds 96 orifice plates, 100 μ l/ holes, promptly cell count is 10
4/ hole.Blank well does not add the cell suspension.
(2) 5%CO
2, cultivate 24h under 37 ℃ of conditions.
(3) every hole adds 100 μ l (Sai Mo flies your biological chemistry goods (Beijing) of generation company limited, SH30809.01B) mATA of the purifying that is obtained by embodiment 1 of 10 times of serial dilutions with 1640 substratum that contain 10% new-born calf serum.Negative control hole does not add toxin.
(4) 5%CO
2, cultivate 72h under 37 ℃ of conditions.
(5) absorb nutrient solution, PBS washes plate three times, adds 100 μ l serum free mediums, and (CellTiter 96 for 20 μ l MTS
AQueous One Solution Cell Proliferation Assay, 3h G3580), cultivates under 37 ℃ of conditions in Sigma company.
(6) measure the A490 value on the micro-pore plate type microplate reader.
(7) calculate cell survival rate:
Cell survival rate (%)=(test group A490/ negative control group A490) * 100%.
RATA that obtains with embodiment 1 and natural abrin are in contrast.The experiment triplicate, results averaged.
The result as shown in Figure 4 and Figure 5, Fig. 4 is the lethal effect curve of abrin A chain mutant to human liver cancer cell SMMC-7721, Fig. 5 is the lethal effect curve of abrin A chain mutant to myeloma cell Sp2/0.
Abrin A chain mutant is specially the lethal effect result of human liver cancer cell SMMC-7721:
MATA IC
50Be about 1.5 * 10
-7M, natural abrin IC
50Be about 1 * 10
-11M, rATA IC
50Be about 5 * 10
-11M.
Abrin A chain mutant is specially the lethal effect result of myeloma cell Sp2/0:
MATA IC
50Be about 1.2 * 10
-7M, natural abrin IC
50Be about 1 * 10
-11M, rATA IC
50Be about 6 * 10
-11M.
As can be seen, mutant protein (mATA) is than preceding its IC that do not suddenly change
50Obviously raise, illustrate that its toxicity obviously weakens, reach the sudden change purpose of expection.
4, the mutant protein immunogenicity is analyzed
(1) immunization: laboratory animal is adopted BALB/c mouse, and is female, and 5-6 age in week, the about 18-20 of body weight restrains (Military Medical Science Institute's Experimental Animal Center, SCXK-(army) 2007-004), and every group with 20 mouse.The immune group mouse is carried out initial immunity, be about to the mATA (250 μ g/ml, every mouse 100 μ l) and equivalent aluminium adjuvant Imject of the purifying that obtains by embodiment 1 of PBS dilution
Behind Alum (Thermo Scientific company, the 77161) thorough mixing, every injected in mice 200 μ l (adopting subcutaneous respectively and two kinds of immunization wayses of muscle), one week back tail vein get blood, 37 ℃ hatch 2h after, it is standby that the centrifugal 15min of 6000r/min leaves and takes serum.After just exempting from for two weeks, carry out twice booster immunization again, two weeks at interval, each immunity one week of back all gets blood by preceding method and stays serum to carry out indirect elisa method to detect and tire.
Immune mouse serum is tired and adopted indirect elisa method to measure, and concrete grammar is as follows: the mATA of the purifying after quantitative is diluted to 5ug/ml with coating buffer, and 4 ℃ of bags that spend the night are by elisa plate; Outwell coating buffer next day, wash plate 3 times with washings, 2min/ time; Add the sealing fluid-tight then and close 1h; Wash plate behind the 1h again 3 times, (one is anti-, uses diluent according to 1: 10,1: 10 to add immune mouse serum
2, 1: 10
3, 1: 10
4, 1: 10
5, 1: 10
6, 1: 10
7, 1: 10
8Dilution), effect 1h; Wash plate behind the 1h earlier, add again horseradish enzyme labelling goat anti-mouse lgG (U.S. santa Cruz company, ZB-2305) (two is anti-, with diluent by dilution in 1: 5000) is behind the effect 1h; Add colour developing liquid A, B successively, add stop buffer (2MH behind the colour developing 5min
2SO
4), detect OD450 at enzyme connection instrument.
(every 1L solution contains Na for 0.01mol/L, pH7.2 with PBS solution
2HPO
412H
2O 2.86g, NaH
2PO
42H
2O0.312g, NaCl 8.5g) inject in contrast.The experiment triplicate, results averaged.
Serum titer reached 1: 10 after mATA three exempted from
6
After PBS solution control group three is exempted from ELISA detect negative, no specific antibody generation.
(2) neutralizing effect in the body of immune serum:
The improvement karber's method is measured natural abrin mouse LD
50BALB/c mouse, female, 20g/ is only, be divided into 5 groups, 8 every group, wherein one group of injection PBS is as negative control, all the other each groups are respectively through abdominal injection 0.1 μ g/ml, 0.2 μ g/ml, 0.4 μ g/ml, 0.8 the natural abrin of μ g/ml observed 7 days, record is respectively organized mortality ratio and is respectively 0,0,0,87.5%, 100%, application of formula log LD
50=Xm-i (∑ P-0.5) calculates LD
50Be 7.7 μ g/kg.Wherein: Xm: the logarithm P of maximal dose group dosage: each organizes mortality ratio i: the logarithm n of dosage ratio between group: every treated animal number.
20 ELISA antibody titers reach 1: 10
6Immunity back mouse, be divided into four groups, 5 every group, the PBS immune mouse is a control group.
The 1-4 group is injected 1LD respectively
50, 4LD
50, 8LD
50, 10LD
50The natural abrin of purifying (the natural abrin of purifying is to the abdominal injection LD of mouse
50Be 7.7 μ g/kg, the body weight of mouse is only calculated by 22g/), (0.01mol/L, pH7.2) dilution is 0.5ml/ to volume injected, injects the back and observes 10 days, writes down mouse body weight change and death condition every day with PBS.
Attack poison back one all tail veins and get blood, adopt above-mentioned indirect elisa method to measure antibody titer and change, the result is as follows: attack poison one week of back, measure antibody titer, raise before 1-4 group serum titer ratio is attacked poison, by 1: 10
6Reach 1: 10
7
Attacked poison back 10 days, record mouse body weight, concrete outcome such as following table 1 and shown in Figure 6:
Neutralization experiment body weight change in the table 1 mATA body
Attacked poison back 10 days, the mouse survival condition sees Table 2:
Mouse survival condition after the neutralizing effect in the immune mouse-anti mATA of table 2 body
From as can be seen above-mentioned, to attack after the poison, animal has produced secondary immune response, and the serum titer ratio raises before attacking poison, and the mouse body weight slightly alleviates, and recovers normal in 10 days gradually, does not see dead mouse.Control group loses weight greater than 20%, and all dead in back 2 days of immunity.
(3) the external neutralizing effect of immune serum:
Collect the ELISA antibody titer and reach 1: 10
6Immune mouse serum (20 ℃ of preservations).The natural abrin of purifying is mixed with 110 μ g/ml stostes, and standby (the natural abrin of purifying is to the abdominal injection LD of mouse
50=7.7 μ g/kg, the mouse body weight is only calculated by 20g/), implement external neutralization test with following proposal.
A) first group: 7 μ l stostes+1243 μ l PBS solution+1250 μ l immune mouse serums mix, 37 ℃ of incubation 30min, 500 μ l/ then, abdominal injection.The consumption of abrin is 0.154 μ g/, is equivalent to 1 times of LD
50
B) second group: 28 μ l stostes+1222 μ l PBS solution+1250 μ l immune mouse serums mix, 37 ℃ of incubation 30min, 500 μ l/ then, abdominal injection.The consumption of abrin is 0.616 μ g/, is equivalent to 4 times of LD
50
C) the 3rd group: 56 μ l stostes+1194 μ l PBS solution+1250 μ l immune mouse serums mix, 37 ℃ of incubation 30min, 500 μ l/ then, abdominal injection.The consumption of abrin is 1.232 μ g/, is equivalent to 8 times of LD
50
D) the 4th group: 70 μ l stostes+1180 μ l PBS solution+1250 μ l immune mouse serums mix, 37 ℃ of incubation 30min, 500 μ l/ then, abdominal injection.The consumption of abrin is 1.54 μ g/, is equivalent to 10 times of LD
50
E) the 5th group: control group, 70 μ l stostes+1180 μ l PBS solution+1250 μ l normal mouse serums mix, 37 ℃ of incubation 30min, 500 μ l/ then, abdominal injection.The consumption of abrin is 1.54 μ g/, is equivalent to 10 times of LD
50
The injection back was observed 10 days, record mouse body weight change and death condition.
The result:
Attacked poison back 10 days, record mouse body weight, concrete outcome such as following table 3 and shown in Figure 7:
The external neutralization experiment of table 3 mATA body weight change
Attack the poison back 10 days after survival condition see Table 4.
Mouse survival condition after the external neutralizing effect of the immune mouse-anti mATA of table 4
As can be seen, the mouse body weight is uninfluenced, and increases by the normal growth rule, and all mouse normally survive; Control group loses weight greater than 20%, and all dead in back 2 days of immunity.
Show, the mATA immune mouse serum can be effectively in and 10LD
50The natural abrin of dosage (natural abrin) illustrates that the mutant mATA that is obtained can be used as a kind of good immunogen.
Conclusion: the present invention adopts rite-directed mutagenesis, gene clone splicing and recombinant expressed equimolecular biology techniques, selects and reduces toxicity, reservation or improve immunogenic abrin A chain mutant mATA.By recombinant expressed abrin A chain mutant protein; set up Recombinant Protein Expression and method for quickly purifying; then by multiple different immunization protocols such as the immunogenic immunization route of A chain mutant, dosage, number of times, adjuvants; analyze the immune protective effect of abrin A chain mutant, determine that finally abrin A chain mutant mATA can be used as a kind of suitable, effective candidate vaccine antigens.
Claims (10)
1. a protein is following 1) or 2) in arbitrary described protein:
1) protein of forming by the amino acid residue sequence of the sequence in the sequence table 2;
2) with 2 amino acid residue sequences of the sequence in the sequence table through the replacement of one or several amino-acid residue and/or disappearance and/or interpolation and have identical function by 1) deutero-protein.
2. the described proteinic encoding gene of claim 1.
3. encoding gene according to claim 2 is characterized in that: described encoding gene is following 1)-3) in arbitrary described dna molecular:
1) dna molecular shown in the sequence 1 in the sequence table;
2) under stringent condition with 1) dna sequence dna hybridization that limits and dna molecular with identical function;
3) with 1) dna sequence dna that limits has 70% at least, have 75% at least, have 80% at least, have 85% at least, have 90% at least, have 95% at least, have 96% at least, have 97% at least, have 98% or have 99% homology at least and have the dna molecular of identical function at least.
4. the recombinant vectors, reorganization bacterium, transgenic cell line or the expression cassette that contain claim 2 or 3 described proteinic encoding genes.
5. recombinant vectors according to claim 4 is characterized in that: described recombinant vectors is for inserting the recombinant vectors that obtains between the multiple clone site of carrier pET-His with claim 2 or 3 described encoding genes.
6. reorganization bacterium according to claim 4 is characterized in that: described reorganization bacterium is for importing the reorganization bacterium that the host bacterium obtains with claim 4 or 5 described recombinant vectorss.
7. amplification claim 2 or 3 described encoding gene total lengths or arbitrary segmental primer are right, and a right primer of described primer is the dna molecular shown in the sequence 3, and another primer is the dna molecular shown in the sequence 4.
8. vaccine, its activeconstituents is following 1)-3) in any: 1) the described protein of claim 1; 2) claim 4 or 5 described recombinant vectorss; 3) claim 4 or 6 described reorganization bacterium.
9. the application of protein described in the claim 1 in the preparation vaccine; The application in the preparation vaccine of claim 2 or 3 described encoding genes; The application in the preparation vaccine of claim 4 or 5 described recombinant vectorss; Claim 4 or 6 application of described reorganization bacterium in the preparation vaccine.
10. application according to claim 9 is characterized in that: described vaccine is an abrin A chain mutant vaccine.
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| CN 201010552967 Active CN102060920B (en) | 2010-11-19 | 2010-11-19 | Construction of abrin A chain mutant and application as candidate vaccine antigen |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103694326A (en) * | 2013-12-11 | 2014-04-02 | 中国人民解放军军事医学科学院微生物流行病研究所 | Truncated-expressed protein antigen of chain A of abrin and application thereof |
| CN114437209A (en) * | 2022-01-25 | 2022-05-06 | 北京弘进久安生物科技有限公司 | Monoclonal antibody specifically binding to abrin and application thereof |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101561438A (en) * | 2009-06-05 | 2009-10-21 | 中国检验检疫科学研究院 | Colloidal gold detection test paper for abrin, preparation method thereof and application thereof |
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Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101561438A (en) * | 2009-06-05 | 2009-10-21 | 中国检验检疫科学研究院 | Colloidal gold detection test paper for abrin, preparation method thereof and application thereof |
Non-Patent Citations (2)
| Title |
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| WANG ET AL.: "Abrin-aA chain expressed as soluble form in Escherichia coli from a PCR-synthesized gene is catalytically and functionally active.", 《BIOCHIMIE》 * |
| 聂聪 等: "相思子毒素抗原和抗体制备", 《中国国境卫生检疫杂志》 * |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103694326A (en) * | 2013-12-11 | 2014-04-02 | 中国人民解放军军事医学科学院微生物流行病研究所 | Truncated-expressed protein antigen of chain A of abrin and application thereof |
| CN103694326B (en) * | 2013-12-11 | 2016-06-08 | 中国人民解放军军事医学科学院微生物流行病研究所 | The abrin A catenin antigen of shorten expression and application thereof |
| CN114437209A (en) * | 2022-01-25 | 2022-05-06 | 北京弘进久安生物科技有限公司 | Monoclonal antibody specifically binding to abrin and application thereof |
| CN114437209B (en) * | 2022-01-25 | 2022-09-20 | 北京弘进久安生物科技有限公司 | Monoclonal antibody specifically binding to abrin and application thereof |
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| CN102060920B (en) | 2013-07-10 |
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