CN105481956B - Recombinate ricin toxin B chain truncated protein and its expression and application - Google Patents
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Abstract
Ricin toxin B chain truncated protein and its expression and application are recombinated, is related to bioengineering field, solves the technical problems such as low, the recombinant protein stability difference of existing recombination ricin toxin B chain protein renaturation rate.The recombination ricin toxin B chain truncated protein, nucleotide sequence is as shown in the SEQ ID NO:1 in sequence table, and amino acid sequence is as shown in the SEQ ID NO:2 in sequence table.The present invention provides a kind of expressions for recombinating ricin toxin B chain truncated protein, express recombination ricin toxin B chain truncated protein by escherichia expression system.Recombination ricin toxin B chain renaturation yield of the invention improves, stability enhancing, immunogenicity is good, enhance immune effect of vaccine, reduce vaccine dosage, can be used as the adjuvant of a variety of vaccines such as inactivated vaccine, attenuated live vaccine, split vaccine, DNA vaccination, recombinant vaccine, subunit vaccine, polypeptide protein vaccine.
Description
Technical field
The present invention relates to technical field of bioengineering, and in particular to a kind of recombination ricin toxin B chain truncated protein and its table
Up to methods and applications.
Background technique
Ricin (WA) (ricin) is a kind of heterodimer glycoprotein, is made of A chain (RTA) and B chain (RTB), two chains
It is connected by disulfide bond.RTA is a kind of ribosome inactivating protein, can inhibit protein synthesis in mammalian cell.RTB without
Poison has activity of lectin, and A chain is assisted to enter intracellular performance biological function.RTB is the molecule of two leaf structures,
It is made of the topology globular domain of two identical foldings, each region includes three subprovinces (α, beta, gamma), only 1 α again
There is apparent gala Sugar-binding activitv with 2 β, RTB can reconcile various bioprocess, including cell in conjunction with different sugared structures
With host, pathogen interaction and innate immune response.
Summary of the invention
In order to solve the technical problems such as low, the recombinant protein stability difference of existing recombination ricin toxin B chain protein renaturation rate, this
Invention provides a kind of recombination ricin toxin B chain truncated protein and its expression and application.
Used technical solution is as follows in order to solve the technical problem by the present invention:
The present invention provides a kind of recombination ricin toxin B chain truncated protein, the SEQ ID in nucleotide sequence such as sequence table
Shown in NO:1, amino acid sequence is as shown in the SEQ ID NO:2 in sequence table.
Further, there are 9 cysteines, by the amino of ricin toxin B chain albumen in ricin toxin B chain protein gene
First five amino acids truncates in acid sequence, obtains ricin toxin B chain truncated gene sequence, and truncate base with the ricin toxin B chain
Because sequence carries out protein recombinant expression, to obtain recombination ricin toxin B chain truncated protein.
The present invention also provides the expressions of above-mentioned recombination ricin toxin B chain truncated protein, comprising the following steps:
(1) Bacillus coli expression engineering bacteria BL21 (DE3)/PET28a-RTB acquisition
Under conditions of amino acid sequence is constant, codon optimization is carried out according to existing ricin toxin B chain sequence, and lead to
Complete sequence after crossing artificial synthesized ricin toxin B chain codon optimization, nucleotides sequence are classified as the sequence 1 in sequence table, ammonia
Base acid sequence is the sequence 2 in sequence table；
It is arranged with the total order after ricin toxin B chain codon optimization as template, with rRTB sense primer and rRTB
Anti-sense primer carries out PCR amplification, and the PCR product of acquisition is connected to pMD18-T cloning vector, obtains pMD18T-
RTB；
Double digestion pMD18T-RTB and PET-28a expression vector, digestion products are linked and are transferred to E. coli competent
Cell BL21 (DE3) obtains positive bacteria BL21 (DE3)/PET28a-RTB through screening, extracts plasmid PET28a-RTB and is sequenced；
(2) inducing expression and purifying of ricin toxin B chain truncated protein are recombinated
It is 100ug/mL that positive bacteria BL21 (DE3)/PET28a-RTB, which is inoculated in 5mL containing concentration in the ratio of 1:100v/v,
In the LB culture medium of Kan, at 37 DEG C, 180rpm constant-temperature shaking culture to OD600When=0.6, then in the ratio switching of 1:100v/v
Into the same LB culture medium of 500mL, and Kan to final concentration of 100ug/mL is added, at 37 DEG C, 180rpm constant-temperature shaking culture
To OD600=0.6；Induction group adds IPTG to final concentration of 1mM, at 37 DEG C, 180rpm constant temperature oscillation induce 16h；Through inducing
After 16h, at 4 DEG C, 8000rpm centrifugation obtain bacterial sediment；
Bacterial sediment is cracked with E. coli bacteria lytic reagent box, centrifugation obtains inclusion body precipitating；Inclusion body is precipitated
With 2X SDS-PAGE sample-loading buffer is added after the dissolution of 8M urea, 5min is boiled at 100 DEG C, pipette samples carry out SDS-PAGE electricity
Swimming；Through 12%SDS-PAGE electrophoretic analysis, the expression that ricin toxin B chain truncated protein has specificity in inclusion body, and its are recombinated
Molecular weight of albumen is 38Kd, is consistent with expected albumen size.
Further, in step (1), PCR amplification condition are as follows: initial denaturation 94 DEG C: 5min, denaturation: 94 DEG C of 30s, annealing: 60
DEG C 30s extends: 72 DEG C of 45s coamplifications 25 recycle.
Further, in step (2), the 2X SDS-PAGE sample-loading buffer includes: the 100mmol/L of pH6.8
TrisCl, 200mmol/mL DTT, 4%SDS, 0.2% bromophenol blue and 20% glycerol.
Further, further include step (3): the solubilization of inclusion bodies liquid that step (2) are obtained carries out affinity chromatography, affine layer
Analysis column is cOmplete His-Tag Purification Resin Ni2+, eluent is the 8M urea of the imidazoles containing 500mM, when
When imidazole concentration is 300mM, collecting eluting peak is recombination ricin toxin B chain truncated protein after purification.
Application the present invention provides recombination ricin toxin B chain albumen as vaccine adjuvant.
The beneficial effects of the present invention are: recombination ricin toxin B chain renaturation yield of the invention improves, stability enhancing is immunized
Originality is good, and enhancing immune effect of vaccine reduces vaccine dosage, can be used as inactivated vaccine, attenuated live vaccine, split vaccine, DNA epidemic disease
The adjuvant of a variety of vaccines such as seedling, recombinant vaccine, subunit vaccine, polypeptide protein vaccine.
Detailed description of the invention
Fig. 1 is that immune rear rabies neutralizing antibody is horizontal.
Specific embodiment
The present invention provides a kind of recombination ricin toxin B chain truncated protein, the SEQ ID in nucleotide sequence such as sequence table
Shown in NO:1, amino acid sequence is as shown in the SEQ ID NO:2 in sequence table.
There are 9 cysteines in ricin toxin B chain protein gene, it will be in the amino acid sequence of ricin toxin B chain albumen
First five amino acids truncates, and obtains ricin toxin B chain truncated gene sequence, and with the ricin toxin B chain truncated gene sequence into
Row protein recombinant expression, to obtain recombination ricin toxin B chain truncated protein.
The present invention provides a kind of expressions for recombinating ricin toxin B chain truncated protein, pass through Bacillus coli expression system
System expression recombination ricin toxin B chain truncated protein.
Through SDS-PAGE electrophoretic analysis, the recombination ricin toxin B chain truncated protein of two methods expression has spy in inclusion body
Anisotropic expression, and its molecular weight of albumen is 38Kd, is consistent with expected albumen size.
Application the present invention provides recombination ricin toxin B chain albumen as vaccine adjuvant, by recombinating ricin toxin B chain
Truncated protein is as vaccine adjuvant enhancing rabies vaccine immune effect test and recombination ricin toxin B chain truncated protein as epidemic disease
Seedling adjuvant, which enhances the test of influenza virus vaccine immune effect, to be proved: recombination ricin toxin B chain albumen of the invention is helped as vaccine
Immune effect of vaccine can be enhanced in agent.
1 escherichia expression system of embodiment expression recombination ricin toxin B chain truncated protein
1, Bacillus coli expression engineering bacteria BL21 (DE3)/PET28a-RTB acquisition
Under conditions of amino acid sequence is constant, it is excellent that codon is carried out according to existing ricin toxin B chain (rRTB) sequence
Change, particularly: there are 9 cysteines, by first five amino acids in its amino acid sequence in ricin toxin B chain protein gene
It truncates, obtains ricin toxin B chain truncated gene sequence, and pass through the total order after artificial synthesized ricin toxin B chain codon optimization
Column, nucleotides sequence are classified as the sequence 1 in sequence table, and amino acid sequence is the sequence 2 in sequence table.
According to the complete sequence after above-mentioned ricin toxin B chain codon optimization as template, with rRTB sense primer
(sequence 3) and rRTB anti-sense primer (sequence 4) carry out PCR amplification, and PCR amplification condition is as follows: 94 DEG C of initial denaturation:
5min, denaturation: 94 DEG C of 30s, annealing: 60 DEG C of 30s extend: 72 DEG C of 45s coamplifications 25 recycle.The PCR product of acquisition is connected to
PMD18-T cloning vector (precious bioengineering (Dalian) Co., Ltd) obtains pMD18T-RTB.
Small fragment is recycled with EcoR I and Hind III double digestion pMD18T-RTB, with the same EcoR I and Hind of process
The PET-28a expression vector of III double digestion links, and obtained link product is transferred to competent escherichia coli cell BL21 (DE3),
Positive bacteria is obtained through screening, is sequenced after extracting plasmid to positive bacteria, as a result the plasmid is that sequence 1 is inserted into sequence table
The plasmid obtained between the restriction enzyme site of EcoR I and the Hind III of PET28a expression vector, is named as PET28a- for the plasmid
RTB, corresponding positive bacteria are named as BL21 (DE3)/PET28a-RTB.
2, the inducing expression and purifying of ricin toxin B chain truncated protein are recombinated
Positive bacteria BL21 (DE3)/PET28a-RTB of above-mentioned acquisition is inoculated in 5mL containing Kan in the ratio of 1:100v/v
In the LB culture medium (formula: peptone 10g/L, yeast extract 5g/L, NaCl 10g/L) of (concentration 100ug/mL), 37
At DEG C, 180rpm constant-temperature shaking culture to OD600When=0.6, then the same LB of 500mL is forwarded in the ratio of 1:100v/v and is trained
It supports in base (formula: peptone 10g/L, yeast extract 5g/L, NaCl 10g/L), and adds Kan to final concentration of 100ug/
ML, with above-mentioned condition of culture i.e. at 37 DEG C, 180rpm constant-temperature shaking culture to OD600=0.6；Induction group adds IPTG to end
Concentration is 1mM, and with above-mentioned condition of culture i.e. at 37 DEG C, 180rpm constant temperature oscillation induces 16h；After inducing 16h, at 4 DEG C,
8000rpm centrifugation obtains bacterial sediment.
The bacterial sediment of acquisition is cracked with E. coli bacteria lytic reagent box, and centrifugation obtains inclusion body precipitating；It will forgive
Add after 8M urea (formula: Tris 50mmol/mL, urea 8mol/L, NaCl 0.5mol/mL, the pH=8.0) dissolution of body precipitating
2X SDS-PAGE sample-loading buffer (formula: 100mmol/L TrisCl (pH6.8), 200mmol/mL DTT, 4%SDS,
0.2% bromophenol blue, 20% glycerol), 5min is boiled at 100 DEG C, draws 20ul sample, carries out SDS-PAGE electrophoresis.
Through 12%SDS-PAGE electrophoretic analysis, the expression that ricin toxin B chain truncated protein has specificity in inclusion body is recombinated,
And its molecular weight of albumen is 38Kd, is consistent with expected albumen size.
The solubilization of inclusion bodies liquid of above-mentioned acquisition is subjected to affinity chromatography, affinity column is cOmplete His-Tag
Purification Resin Ni2+, eluent is the 8M urea (formula: Tris50mmol/mL, urea of the imidazoles containing 500mM
8mol/L, NaCl 0.5mol/mL, pH=8.0).When imidazole concentration is 300mM, collecting eluting peak is recombination after purification
Ricin toxin B chain truncated protein.
3, the renaturation of ricin toxin B chain truncated protein is recombinated
Recombination ricin toxin B chain truncated protein after purification adds glycerol to 100mL/L, and sucrose to 5g/L, GSH is extremely
0.9mmol/L, GSSG add a little Brij 35 to 0.1mmol/L, and recombination ricin toxin B chain truncated protein concentration is adjusted
To 0.6mg/mL, pipette in the dialysis band that interception is 3Kd.Prepare protein renaturation liquid (formula: Tris50mM, glycerol
100mL/L, sucrose 5g/L, GSH.9mmol/L, GSSG 0.1mmol/L, a little Brij 35, pH=8.0) carry out dialysis gradient
Renaturation.PEG20000 protein concentrate is used after renaturation, BCA method measures protein concentration, with protecting after 0.22um filter filtration sterilization
It is stored in -80 DEG C.
Embodiment 2 recombinates ricin toxin B chain truncated protein as vaccine adjuvant enhancing rabies vaccine immune effect test
1, experimental animal and grouping
50 7 week old Kunming mouses are randomly divided into 5 groups, and every group 10, no adjuvant viral control group, aluminium hydroxide are helped
Vaccinating agent group, rabies vaccine+20ug recombination ricin toxin B chain truncated protein (RTB) vaccine group, rabies vaccine+40ug recombinate castor
Numb toxin B chain truncated protein (RTB) vaccine group, rabies vaccine+80ug recombinate ricin toxin B chain truncated protein (RTB) vaccine group.
2, experimental procedure
Cultivating titre is 106TCID50JX08-45CC plants of the hydrophobin (the 120th generation) of/mL, inactivates through beta-propiolactone
It afterwards, will be without adjuvant viral control group, aluminum hydroxide adjuvant vaccine group, rabies vaccine+recombination ricin toxin B chain truncated protein vaccine
Mouse is immunized in group after mixing respectively with virus liquid, 50 μ L/ of hindlimb muscle injection only, 3 days and 7 days after being immunized, adopt respectively by tail vein
Blood, after separating serum, for rabies neutralizing antibody detection (FAVN method).
3, result
Shown in result figure 1, the rabies neutralizing antibody of recombination ricin toxin B chain truncated protein Adjuvanted vaccines group induction generation
Average level is significantly higher than common aluminum hydroxide adjuvant vaccine group, this shows: recombination ricin toxin B chain truncated protein is to mad dog epidemic disease
Seedling has synergistic effect.
Embodiment 3 recombinates ricin toxin B chain truncated protein as vaccine adjuvant enhancing influenza virus vaccine immune effect examination
It tests
1, experimental animal and grouping
30 7 week old Kunming mouses, are randomly divided into 3 groups, and every group 10, Normal group (PBS), H1N1VLP vaccine
Group, H1N1VLP+40ug recombinate ricin toxin B chain truncated protein (RTB) vaccine group.
2, experimental procedure
It is immunized 0 week, 3 weeks, 50 μ L/ of hindlimb muscle injection only, with Influenza B virus -6-E2 collunarium modeling, are attacked on the 5th week
Toxic dose is 5LD50, and 50 μ L/ are only.
3, result
1) survival rate is as shown in table 1, and 1~4 week, 3 groups of survival rate was 100%, and 5~14 weeks, H1N1VLP vaccine is immunized
The survival rate of group and immune H1N1VLP+40ug recombination ricin toxin B chain truncated protein vaccine group is 100%, and the 5th week, just
The survival rate of normal control group is reduced to 80%, and by the 6th week, the survival rate of Normal group was reduced to 60%, and 7~14 weeks, normal control
The survival rate of group is 0.This explanation: after using recombination ricin toxin B chain as adjuvant, the immunogene of influenza virus vaccine is not influenced
Property.
Table 1
2) changes of weight is as shown in table 2, and the 1st week, 3 groups of weight was unchanged, 2-6 weeks, the weight of Normal group by
94% is reduced to 77%, and the weight of immune H1N1VLP vaccine group is reduced to 91% by 96%, and H1N1VLP+40ug is immunized and recombinates castor-oil plant
The weight of toxin B chain truncated protein vaccine group is reduced to 93%, 7-14 weeks by 98%, be immunized H1N1VLP vaccine group weight by
94% is upgraded to 103%, and the weight of immune H1N1VLP+40ug recombination ricin toxin B chain truncated protein vaccine group is upgraded to by 96%
104%.This explanation: after using recombination ricin toxin B chain as adjuvant, infected by influenza vaccine has synergistic effect.
Table 2
3) as shown in table 3, H1N1VLP vaccine union and recombination ricin toxin B chain truncated protein is immune can for neutralizing antibody variation
Induction generates the specific antibody of higher level, can more effectively improve the protecting effect of H1N1 split vaccine.
Table 3
Claims (4)
1. recombinating ricin toxin B chain truncated protein, which is characterized in that the SEQ ID NO:1 in its nucleotide sequence such as sequence table
Shown, amino acid sequence is as shown in the SEQ ID NO:2 in sequence table.
2. recombination ricin toxin B chain truncated protein according to claim 1, which is characterized in that ricin toxin B chain albumen base
There are 9 cysteines because in, first five amino acids in the amino acid sequence of ricin toxin B chain albumen are truncated, castor-oil plant is obtained
Toxin B chain truncated gene sequence, and protein recombinant expression is carried out with the ricin toxin B chain truncated gene sequence, to obtain
Recombinate ricin toxin B chain truncated protein.
3. the expression of recombination ricin toxin B chain truncated protein as described in claim 1, which is characterized in that including following
Step:
(1) Bacillus coli expression engineering bacteria BL21 (DE3)/PET28a-RTB acquisition
Under conditions of amino acid sequence is constant, codon optimization is carried out according to existing ricin toxin B chain sequence, and pass through people
Work synthesizes the complete sequence after ricin toxin B chain codon optimization, and nucleotides sequence is classified as the sequence 1 in sequence table, amino acid
Sequence is the sequence 2 in sequence table；
It is arranged with the total order after ricin toxin B chain codon optimization as template, with rRTB sense primer and rRTB anti-
Sense primer carries out PCR amplification, and the PCR product of acquisition is connected to pMD18-T cloning vector, obtains pMD18T-RTB；
The sequence of the rRTB sense primer is shown in the sequence 3 in sequence table, the rRTB anti-sense primer's
Sequence is shown in the sequence 4 in sequence table；
Double digestion pMD18T-RTB and PET-28a expression vector, digestion products are linked and are transferred to competent escherichia coli cell
BL21 (DE3) obtains positive bacteria BL21 (DE3)/PET28a-RTB through screening, extracts plasmid PET28a-RTB and is sequenced；The matter
Grain is that the sequence 1 in sequence table is inserted into the matter obtained between the restriction enzyme site of EcoR I and the Hind III of PET28a expression vector
Grain, is named as PET28a-RTB for the plasmid；
In step (1), PCR amplification condition are as follows: initial denaturation 94 DEG C: 5min, denaturation: 94 DEG C of 30s, annealing: 60 DEG C of 30s extend: 72
DEG C 45s coamplification 25 recycles；
(2) inducing expression and purifying of ricin toxin B chain truncated protein are recombinated
It is 100ug/mL Kan that positive bacteria BL21 (DE3)/PET28a-RTB, which is inoculated in 5mL containing concentration in the ratio of 1:100v/v,
LB culture medium in, at 37 DEG C, 180rpm constant-temperature shaking culture to OD600When=0.6, then it is forwarded in the ratio of 1:100v/v
In the same LB culture medium of 500mL, and add Kan to final concentration of 100ug/mL, at 37 DEG C, 180rpm constant-temperature shaking culture extremely
OD600=0.6；Induction group adds IPTG to final concentration of 1mM, at 37 DEG C, 180rpm constant temperature oscillation induce 16h；Through inducing 16h
Afterwards, at 4 DEG C, 8000rpm centrifugation obtains bacterial sediment；
Bacterial sediment is cracked with E. coli bacteria lytic reagent box, centrifugation obtains inclusion body precipitating；Inclusion body precipitating is used into 8M
Add 2X SDS-PAGE sample-loading buffer after urea dissolution, 5min is boiled at 100 DEG C, pipette samples carry out SDS-PAGE electrophoresis；Through
12%SDS-PAGE electrophoretic analysis, recombination ricin toxin B chain truncated protein have the expression of specificity, and its albumen point in inclusion body
Son amount is 38Kd, is consistent with expected albumen size；
In step (2), the 2X SDS-PAGE sample-loading buffer include: pH6.8 100mmol/L TrisCl,
200mmol/mL DTT, 4%SDS, 0.2% bromophenol blue and 20% glycerol；
Step (3): the solubilization of inclusion bodies liquid that step (2) are obtained carries out affinity chromatography, and affinity column is cOmplete His-
Tag Purification ResinNi2+, eluent is the 8M urea of the imidazoles containing 500mM, when imidazole concentration rises to 300mM,
Collecting eluting peak is recombination ricin toxin B chain truncated protein after purification.
4. recombination ricin toxin B chain albumen is as the application prepared in vaccine adjuvant as described in claim 1.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0156633B1 (en) * | 1984-03-22 | 1988-09-28 | Eisai Co., Ltd. | Composition for prevention and treatment of mycoplasmal pneumonia |
| CN102552876A (en) * | 2012-02-17 | 2012-07-11 | 中国人民解放军军事医学科学院军事兽医研究所 | Novel application of ricin B chain protein in immunoloregulation |
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2015
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0156633B1 (en) * | 1984-03-22 | 1988-09-28 | Eisai Co., Ltd. | Composition for prevention and treatment of mycoplasmal pneumonia |
| CN102552876A (en) * | 2012-02-17 | 2012-07-11 | 中国人民解放军军事医学科学院军事兽医研究所 | Novel application of ricin B chain protein in immunoloregulation |
Non-Patent Citations (3)
| Title |
|---|
| Ricin B chain fragments expressed in Escherichia coli are able to bind free galactose in contrast to the full length polypeptide;Richard Wales et al;《Glycoconjugate Journal》;19941231;274-281 * |
| Ricin E B，RRF:1303208A;GenPept;《GenPept》;19960729;全文 * |
| 重叠PCR合成蓖麻毒素B链基因及其在大肠杆菌中表达和抗原性分析;王俊虹等;《军事医学科学院院刊》;20060228;29-33 * |
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