CN101199843B - Whooping cough genetic engineering blending second unit vaccine and preparing method thereof - Google Patents
Whooping cough genetic engineering blending second unit vaccine and preparing method thereof Download PDFInfo
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Abstract
The invention relates to a pertussis genetic-engineering fused subunit vaccine, which is fused protein produced by connecting pertussis toxin S1 subunit mutant with a peptide Fs. The invention also relates to a preparation method of bordetella pertussis double-subunit genetic engineering vaccine. The invention, which overcomes a variety of deficiencies of the traditional vaccine and the traditional preparation method, has the advantages of mass production potential, easy operation, high security, small side effect and no reverse mutation.
Description
Technical field
The present invention relates to a kind of vaccine and preparation method thereof, particularly relate to a kind of pertussis genetic engineering and merge subunit vaccine and preparation method thereof.
Background technology
Pertussis is a kind of respiratory system disease that to have strong communicable breathing be property that is caused by the gram negative bacilli bordetella pertussis; Its popular cycle is the regional disease in 2～5 years; The mankind are unique hosts of bordetella pertussis; Main through spittle air-borne transmission, ill object is mainly infant below 5 years old.Pertussis has extremely strong infectiousness, and in not immune member that the pertussis patient contacts closely, the people of 90 %～100% will develop into pertussis; Because the restriction of vaccine immunity guard time is contacting among patient's the completion inoculation person closely, subclinical infection may take place in the people more than 50%.Though the application of vaccine is to controlling the pertussal popular significant effect that played, the pertussis prevalence still demonstrates tangible ascendant trend in recent years, so-called " resume combustion " phenomenon occurred.And adult's pertussis patient proportion constantly increases; Because this type patient's atypical symptom; Pertussal Clinics and Practices is incured loss through delay and made them become the topmost source of infection (Crowcroft NS; Stein C, et al. Lancet Infect Dis.2003,3:413 – 418).According to World Health Organization's statistics, all there is the pertussis patient above 4,000 ten thousand in the whole world every year in recent years, and wherein dead person is about 300,000, and the overwhelming majority is the child, and the dead ratio of adult and teenager rises to some extent.
Vaccine is the popular the most effectively means of prevention and control infectious disease.Traditional pertussis vaccine mainly comprises whole cell pertussis vaccine (WPV) and acellular pertussis vaccine (APV), and all exists with the form of PertussisDiphtheriaTetanus triple vaccine.Wherein, though that WPV early is applied to is clinical because it is the whole cell vaccine of deactivation, during inoculation side reaction big, cause inoculating the decline and the rising of pertussis sickness rate in recent years of coverage rate; APV directly mentions various antigenic components from antibacterial, after the physics detoxification, combine by different kinds and dosage.These antigenic components mainly contain pertussis toxin, PT (PT), filamentous hemagglutinin (FHA), bordetella pertussis adhesion plain (Prn) and agglutinogen (AGGs) etc., and wherein PT is a composition total among the various APV.Clinical research confirmation, these acellular vaccines have good immunogenicity, and side reaction is few.But following two factors have restricted its development: (1) is high to purifying process, equipment requirements, and the purification expense is high, has increased the development and production cost; (2) some antigenic component of producing of antibacterial itself very little, especially toxin antigen such as PT is unfavorable for the large-scale production of vaccine.
The development of recombinant vaccine at first also the most important thing is to select antigen safely and effectively; Along with genomics; Proteomics, the continuous development of molecular immunology and reverse vaccinology, bioinformatics has brought facility for antigenic extensive prediction or screening.At present, about bordetella pertussis antigenic component research maximum mainly be PT, FHA and Prn.Because these antigenic component molecular weight are all very big, directly recombinant expressed impossible basically, existing mainly is to study that therefrom screening has immunogenic fragment and to its protection.
FHA is by bordetella pertussis fhaB gene code, is a kind of main adhesion molecule of the synthetic justacrine of bordetella pertussis, adheres to and settle down at airway epithelial cell relevant with bordetella pertussis, do not have toxicity basically.The FHA molecular weight is big, and precursor protein is 370-kDa, and the adult molecular weight of albumen after the protease effect is 220-kDa, is difficult to direct heterogenous expression (Sylvie A, Nathalie R, infection and immunity, 2002,70 (8): 4142 – 4147).Discover: FHA has two main immunocompetence sites; Difference called after type I, II domain; Research shows, 456 the amino acid whose type I domains (below be designated as FHA ') that comprise that are positioned at the C-terminal of FHA have better immunogenicity, contain most of active epi-position.As far back as 1997, (Elizabeth L, Steven B, et al. The Journal of Infectious Diseases 1997 such as Elizabeth; 175:1423 – 31) just the mode through section of synthesized peptide confirms this segmental immunogenicity.(Knight JB such as Knight; Huang YY, et al.Clinical and experimental immunology.2006,144 (3): 543-550) with behind itself and maltose-binding protein (MBP) amalgamation and expression; In serum and saliva, all detected high antibody of tiring; Animal experiment confirms that also its bronchus bordetella pertussis clearance rate is higher than matched group, points out it to have good immunogenicity.The same problem of expressing difficulty that exists of natural FHA ', in the early-stage Study, the applicant is building up to PET-22b with FHA ', in PET-28a and the PQE30 carrier, but does not all see the expression of destination protein.
PT is the A-B type toxin that is made up of 6 subunits (S1-S5); Wherein, the A substance is made up of the S1 of a part, and the B oligomer is that bimolecular S4 combines formed pentamer respectively with after S2, the S3 of a part combine again with S5; The mediation toxin is adsorbed on the host cell membrane and auxiliary A substance gets into target cell (Tania DP; Mark JW. Vaccine, 1997, I5 (9): 966-975).Natural PT has the various biological activity, and like ADP-ribose transferase active, histamine sensitivity, insulin increase activity and short leukocytosis activity etc.Research shows; These BAs mediate by the S1 subunit; And the main protectiveness determinant of PT is positioned on the S1 subunit, and the test of ADP-ribose transferring enzyme shows that the S1 subunit of all native sequences all has enzymatic activity in addition; The S1 subunit of meristic variation then demonstrates enzymatic activity (Castro MG, McNamara U. Cell Microbiol 2001 in various degree; 3 (1): 45 – 54.), therefore, the S1 of sudden change possibly be good candidate antigens.Through the point mutation technology, obtained the mutant of a plurality of S1 subunits now, wherein S1-9K/129G is of greatest concern; This double-mutant has lost natural S1 BA; Good immune protection active (Pizza M, Covacci A, Bartoloni A have but been kept; Et al. Science, 1989; 246 (4929): 497 – 500).The S1 subunit contains 235 amino acid residues; Research shows that its main epitope is positioned at 180 amino acid residues of N end, and two mutational sites of above-mentioned S1 mutant are just in time in this zone; Therefore this fragment can be used as good immunogen, below is designated as S1 '.(Lee SF, Scott AH, Danny F such as LEE; Et al. Infection and immunity, 2003,71 (4): 2272 – 2275) construction expression a S1 ' S1 ' CTB fusion rotein; Wherein, CTB is a choleratoxin B subunit, is a kind of good mucosal adjuvants, can effectively induce humoral immunization and mucosal sIgA to reply.Behind this albumen intranasal immune mouse, detected IgG and sIgA antibody in serum and the saliva, and the external natural PT that neutralizes of these antibody.These researchs confirm that all S1 ' has good immunogenicity.But research all confirms the extremely difficult heterogenous expression of S1 ' both at home and abroad.
Summary of the invention
For reaching above purpose, the technical scheme that the present invention adopts is: the two subunit genetic engineering vaccines of a kind of pertussis born of the same parents Te Shi bacillus, this vaccine is to connect the fusion rotein that peptide section Fs is constituted by known pertussis toxin, PT S1 subunit mutant.
The C end of described fusion rotein has the 6-His purification tag, and has the aminoacid sequence shown in the SEQ ID NO:1.
The coding DNA of described fusion rotein is the sequence that has the genetic code degeneracy with it of the nucleotide sequence shown in the SEQ ID NO:2 or the same protein of encoding.
Described pertussis toxin, PT S1 subunit mutant is 9K/129G, and peptide section Fs is 138 the amino acid whose peptide sections that comprise from filamentous hemagglutinin, has the albumen that the aminoacid sequence shown in the SEQ ID NO:3 is formed.
The coding DNA of described peptide section Fs is the sequence that has the genetic code degeneracy with it of the nucleotide sequence shown in the SEQ ID NO:4 or the same protein of encoding, and peptide section Fs is positioned at the aminoterminal of pertussis toxin, PT S1 subunit mutant or is positioned at the c-terminus of pertussis toxin, PT S1 subunit mutant.
One section nucleotide sequence through the coding connexon between described peptide section Fs and the pertussis toxin, PT S1 subunit mutant is connected, and one section nucleotides sequence of its coding connexon is classified PQDPP as.
Another object of the present invention provides the method for preparing of the two subunit genetic engineering vaccines of pertussis born of the same parents Te Shi bacillus, comprises the steps: to make up the S1 mutant; Fs fragment among prediction and the evaluation FHA '; Make up recombination engineering pET-22b (+)-FsS1 '/BL21; Measure the gene order of recombiant plasmid; Induce recombination engineering, express obtaining vaccine candidate albumen FsS1 '; The purifies and separates destination protein adopts affinity column that destination protein is carried out purification; Identify the purity of destination protein; Identify the immunogenicity of fusion rotein; Process the two subunit genetic engineering vaccines of pertussis born of the same parents Te Shi bacillus.
In the structure of fusion rotein, the present invention adopts following connected mode and connexon:
(1) connected mode of fusion protein F sS1 ' is: the encoding gene of Fs is connected with S1 ' encoding gene through one section nucleotide sequence of coding connexon, thereby obtains fusion protein F sS1 '.Wherein, Fs is positioned at 5 ' end; S1 ' is through coding connexon (CCTCAAGATCCGCCA; Proline-glutamine-aspartic acid-proline-proline, one section nucleotide sequence PQDPP) are connected in 3 ' end of Fs gene, connect 6 polyhistidyl coded sequences at FsS1 ' fusion gene 3 ' end.Fig. 1 is recombiant plasmid pET-22b (+)-FsS1 ' construction strategy figure.
(2) connected mode of fusion protein F sS1 ' S1 ' is: above-mentioned fusion protein F sS1 ' encoding gene is connected the encoding gene that obtains fusion protein F sS1 ' S1 ' through one section nucleotide sequence of coding connexon with another S1 ' thereby encoding gene.Wherein, FsS1 ' fusion gene is positioned at 5 ' end; S1 ' encoding gene is through coding connexon (TACGCGCCGCAGGATCCT; Tyrosine-alanine-proline-glutamine-aspartic acid-proline, one section nucleotide sequence YAPQDP) are connected in 3 ' end of fusion gene, connect 6 polyhistidyl coded sequences at FsS1 ' S1 ' fusion gene 3 ' end.Fig. 2 is recombiant plasmid pET-22b (+)-FsS1 ' S1 ' construction strategy figure.The structure of described fusion rotein is the aminoacid sequence shown in the SEQ ID NO:5; The gene order of encoding said fusion protein is the sequence that has the genetic code degeneracy with it of the nucleotide sequence shown in the SEQ ID NO:6 or the same protein of encoding.
The fundamental characteristics of gene engineering recombinant bacterium of the present invention and recombination fusion protein FsS1 ': 1. the host bacterium is BL21, and expression vector is pET-22b; 2. recombiant protein is with the inclusion body formal representation, and FsS1 ' molecular weight is 38kDa; 3. the expression of recombiant protein FsS1 ' is more than 30%; 4. the proteic isoelectric point, IP of FsS1 ' is 6.2.
Compared with present technology, advantage of the present invention is following:
Because the present invention passes through genetic engineering means; Active fragment construction of fusion protein in a plurality of protective antigens of screening bordetella pertussis is as the candidate albumen of recombinant vaccine; Traditional directly various virulence factors of extraction and follow-up chemical attenuation and physics amalgamation mode from bordetella pertussis have been broken through; Inventor of the present invention has done a preliminary research through a series of experiments to BA and the immunologic competence of FsS1 ', and the result shows, FsS1 ' has lost various biology of the toxicity of natural S1 ' basically; And has a good immunologic competence; Can stimulate body to produce high antibody of tiring, suitable with the traditional vaccine immune effect, explain that this albumen can be used as the candidate antigens of pertussis recombinant vaccine.Therefore; Compare with traditional vaccine; The two subunit genetic engineering vaccines of a kind of pertussis born of the same parents Te Shi of the present invention bacillus have can large-scale production, easy and simple to handle, safe, side effect is little, do not have advantage such as back mutation; Therefore have good market prospect, have huge social benefit and economic benefit.
Because the present invention is from FHA the '; Adopt the bioinformatics means that its protein structure and antigenicity are predicted, successfully doped 138 amino acid fragment Fs, immunoblotting confirms that it has good immunoreactivity; What is more important; This expressing quantity has successfully been realized antigenic efficiently expressing more than 50%, has overcome bordetella pertussis antigenic component technical barrier beyond expression of words.
Because the present invention adopts the Fs fragment of above-mentioned evaluation and pertussis toxin, PT S1 subunit mutant (in description of the present invention, being called for short S1 ') to merge; Successfully made up recombiant plasmid pET-22b (+)-FsS1 '; Made up recombiant plasmid pET-22b (+)-FsS1 ' S1 ' on this basis, the two has all obtained efficiently expressing in e. coli bl21, and latter's expression is lower than the former relatively; These two albumen have fundamentally reduced various biology of the toxicity of natural S1; And kept good immunogenicity, and successfully break through single S 1 ' and be difficult to this technical bottleneck of heterogenous expression, make the research of pertussis recombinant vaccine become possibility.
Owing to generally acknowledge that at present spendable ideal whooping cough subunit vaccine (DTaP) candidate antigens only has diphtheria toxin, diphtherotoxin (Diphtheria Toxin; DT) A fragment mutant and tetanus toxin (Tetanus Toxin; TT) heavy chain C end (Hc); The successful structure of the two subunit genetic engineering vaccine candidate albumens of pertussis born of the same parents Te Shi bacillus of the present invention will be established solid foundation because of the development of engineered vaccine for whooping cough three symbasis.
For let above and other objects of the present invention, feature and advantage can be more obviously understandable, below the special embodiment that lifts, and cooperate Figure of description, elaborate as follows.
Fig. 1: recombiant plasmid pET-22b (+)-FsS1 ' construction strategy figure.
Fig. 2: recombiant plasmid pET-22b (+)-FsS1 ' S1 ' construction strategy figure.
Description of drawings
Fig. 3: the enzyme action of 1 mutant 9K/129G recombinant expression plasmid is identified.
Fig. 4: pMD18-T-S1 9K/129G sequencing result.
Proteic structure of Fig. 5: FHA ' and antigenicity predict the outcome.
Fig. 6 A:Fs PCR enzyme action qualification result.
Fig. 6 B:pET-22b (+)-Fs enzyme action qualification result.
Fig. 7: pET-22b (+)-Fs sequencing result.
Fig. 8: pET-22b (+)-Fs abduction delivering and expression-form qualification result.
Swimming lane M is low molecular protein Marker;
Swimming lane 4-7 is respectively that IPTG induces 0,1,3, the 5h sampling.
Fig. 9: west-blotting identifies Fs
Swimming lane M is low molecular protein Marker;
Swimming lane 1-5 is followed successively by the pET-22b empty carrier, and PET-22b (+)-Fs induces sampling in 0,1,3,5 hour.
Figure 10: pET-22b (+)-FsS1 ' enzyme action qualification result.
Swimming lane M is nucleic acid (DNA) molecular weight standard (Marker)
Figure 11: pET-22b (+)-Fs S1 ' sequencing result.
Figure 12: pET-22b (+)-FsS1 ' abduction delivering and expression-form qualification result.
Swimming lane M is low molecular protein Marker;
Swimming lane 4-7 is respectively that IPTG induces 0,1,3, the 5h sampling.
Figure 13: albumen FsS1 ' purification result.
Figure 14: leukocytosis experimental result.
Figure 15: pET-22b (+)-FsS1 ' S1 ' enzyme action qualification result.
Swimming lane M is nucleic acid (DNA) molecular weight standard (Marker)
Figure 16: pET-22b (+)-Fs S1 ' S1 ' sequencing result.
Figure 17: pET-22b (+)-FsS1 ' S1 ' abduction delivering and expression-form qualification result.
Swimming lane M is low molecular protein Marker;
Swimming lane 4-7 is respectively that IPTG induces 0,1,3, the 5h sampling.
Below in conjunction with accompanying drawing and embodiment the present invention is further described.
Material therefor and reagent
Tryptone, yeast extract Britain Oxoid company
EB U.S. Sigma company
BamH I, Nde I, Xho I Dalian Takara company
Agarose Promega company
Ex-Taq archaeal dna polymerase Dalian Takara company
DNA Marker days is Time Inc.
Glue reclaims test kit Omega company
Plasmid extraction test kit Omega company
The homemade analytical pure of Ampicillin
T4 DNA ligase Fermentas company
Na
2EDTA2H
2The homemade analytical pure of O
Small-molecular weight standard protein Shanghai rises positive biotech firm
DTT Sjgma company
CaCl
2Homemade analytical pure
The import packing of the magnificent company of Tris
The import packing of the magnificent company of SDS
Acrylamide, the import packing of the magnificent company of methylene bisacrylamide
The homemade analytical pure of Coomassie brilliant blue R-250
The homemade analytical pure of Ammonium persulfate.
Worker's import packing is given birth in X-Gal Shanghai
Worker's import packing is given birth in IPTG Shanghai
Worker's import packing is given birth in TEMED Shanghai
The homemade analytical pure of bromophenol blue
The homemade analytical pure of Ponceau S
The homemade analytical pure of Tween-20
Worker's import packing is given birth in BSA Shanghai
Worker's import packing is given birth in DAB Shanghai
H
2O
2Homemade analytical pure
Mouse-anti B.pertus FHA, S1 monoclonal antibody
The Chinese biological drug inspection office
Pvdf membrane Roche company
HRP labelling sheep anti-mouse igg antibody Wuhan doctor's moral company
Expression vector pET22b preserves this chamber
The simple T carrier of pMD18 Dalian Takara company
The structure of embodiment 1. S1 mutant 9K/129G
1. design of primers
AY879289 gene order according to the GenBank registration; Design is through the overlap extension principle; Utilization sudden change test kit MutanBEST Kit (TaKaRa) design two pairs of primers
and II obtain to contain the recon that dibit point (9K/129G) suddenlys change.Other designs primer
, introduces the restriction enzyme site of Bam H I and Pst I respectively.Primer is given birth to worker bio-engineering corporation by Shanghai and is synthesized.
5’-CTGGCACACCGGCGCATTCCG-3’
II 5’-CGCCCGCCGGAGGACGTTTTCCAG-3’
5’-GGAGTCATACTTGTATACGGTGGCCAT-3’
5’-
CTGCAGCTAGAACGAATACGCGATG-3’?
PstⅠ
2. the amplification of target gene fragment
Being converted into the extractive plasmid of DH5 α bacterium liquid with PT-PGEM is template; Carry out the PT fragment that overlap extension obtains sudden change with primer
and II, method is pressed MutanBEST Kit (TaKaRa) description.With above-mentioned product is template; Utilization primer
carries out the amplification of S1 subunit mutant (rS1), about 700bp, has obtained the purpose band.Above PCR reaction condition is 94 ℃ of preparatory degeneration 5min, and 94 ℃ of 30s → 56 ℃ 30s → 72 ℃ of 30s carry out 30 circulations, and last 72 ℃ are extended 10min.
3. the clone of PCR product and order-checking
The S1 mutant S1 ' gene fragment clone that obtains is gone into pMD18-T vector; Transformed E. coli DH5 α; Ammonia benzyl resistance screening positive recombinant; The extracting plasmid adopts Bam H I and Pst I double digestion to identify correct (Fig. 3), and enzyme action is identified that correct plasmid serves the sea and give birth to worker bio-engineering corporation and check order.Sequencing result is as shown in Figure 4: (cgc → aaa, gaa-ggc), the AY879289 gene order of all the other base sequences and GenBank registration is in full accord, the success of prompting mutation construction except two mutational sites successfully suddenly change.
Segmental prediction of embodiment 2. Fs and evaluation
Adopt DNAstar software to predict; Inventor of the present invention does integrated forecasting to proteic structure of FHA ' and antigenicity; The result is as shown in Figure 5: from figure, can find; The 320th later aminoacid of FHA ' is rich in a spiral, and hydrophilic, pliability and surperficial accessibility are all higher, and antigenicity analysis is apparently higher than other sections.Can judge that in view of the above this section (containing 138 aminoacid) has good antigenicity, therefore confirm this section is carried out clonal expression.
1. design of primers
The bacterial strain that adopts among the present invention is a pertussis CS bacterial strain, and it does not accomplish genome sequencing as yet, but according to reports in the pertussis bacterial strain homology of FHA gene than higher.Therefore, inventor of the present invention is a template with general in the world Tohama bacterial strain fhaB gene order, adopts software Primer Premier5.0 design primer (primer is given birth to worker bio-engineering corporation by Shanghai and synthesized) as follows:
P1?5’-
CATATGCTGAAGAACCTCGACCTGGGC-3’
Nde1
P2?5’-
CTCGAGCTGCAGCCGCACCGC-3’
Xho1
2. the pcr amplification of genes of interest
The genome that employing is extracted from pertussis CS bacterial strain is as template; The PCR condition is: 94 ℃ of preparatory degeneration 5min, and 94 ℃ of 30s → 56 ℃ 30s → 72 ℃ of 30s carry out 30 circulations, and last 72 ℃ are extended 10min; Amplified the target gene fragment about 400bp, shown in Fig. 6-A.
3. the clone identifies and expresses
(1) the Fs gene fragment clone that obtains is gone into pMD18-T vector, transform
E. coliDH5 α, ammonia benzyl resistance screening positive recombinant, the extracting plasmid is used
NdeIWith
XhoIDo double digestion and identify, identify correct back recovery target gene fragment.
(2) the Fs genetic fragment that reclaims in (1) is implemented in expression vector pET-22b, uses
NdeIWith
XhoIDo double digestion and identify (the enzyme action result is shown in Fig. 6-B).
(3) enzyme action is identified that errorless gene recombination plasmid is converted into the host bacterium
E.coliBL21 (DE3) obtains recombination engineering.And recombiant plasmid pET-22b (+)-Fs is served the sea give birth to the order-checking of worker bio-engineering corporation.Sequencing result (Fig. 7) shows: except two codon generation nonsense mutations were arranged, the Tohama bacterial strain corresponding sequence that other sequences and GenBank announce was in full accord.
4. the abduction delivering of recombination engineering pET-22b (+)-Fs/BL21 and form are identified
Recombination engineering is cultivated in the LB fluid medium that contains ampicillin (50 μ g/ml), to bacterium liquid OD
600Add during ≈ 0.6 IPTG to final concentration be 1.0mmol/L; Induce different time to collect bacterium liquid; Ultrasound wave breaks bacterium, and differential centrifugation is left and taken cleer and peaceful deposition respectively, carries out gel electrophoresis after the processing and identifies expression-form and expression (abduction delivering and form are identified as shown in Figure 8).The result shows: albumen Fs has obtained efficiently expressing, and expression induces 3 hours expressions the highest more than 50%, and how with the inclusion body formal representation.
5. Western Blot identifies
Because the multi-resistance of the not anti-FHA total length of inventor of the present invention; What adopt in this test is an anti-FHA monoclonal antibody of Chinese biological goods calibrating serum chamber director Zhang Shuming of institute present, and Western Blot result shows that this monoclonal antibody can combine with the albumen that the inventor is predicted; As shown in Figure 9: expression product has a tangible band at relative molecular weight 15000 places; The no band of empty carrier contrast explain to have obtained destination protein, and this albumen has good immunogenicity.
The structure of embodiment 3. recombination engineering pET-22b (+)-FsS1 '/BL21
1. design of primers and pcr amplification
The recombiant plasmid pMD18-T-S1 ' and the pMD18-T-Fs that make up among the above embodiment 1,2 are template, adopt software Primer Premier5.0 to carry out primer design and analysis.With linker (
CCTCAAGATCCGCCA) sequential design is at 5 ' end of the 3' of Fs encoding gene end and S1 ' encoding gene, utilizes the overlap extension PCR method that Fs encoding gene and S1 ' encoding gene are coupled together through linker.Primer is given birth to worker bio-engineering corporation by Shanghai and is synthesized, and primer sequence is following:
Fs forward primer P1:5-CATATGCTGAAGAACCTCGACCTGGGC-3 Nde1
Downstream primer P2:5-TCTGGCGGATCTTGAGGCTGCAGTCGCACCG-3 linker
S1 ' forward primer P3:5-CAGCCTCAAGATCCGCCAGACGATCCTCCCGC-3 linker
Downstream primer P4:5-CTCGAGTGTGTAGGGGTTGG-3 Xho1
2. the pcr amplification of genes of interest
Be template with recombiant plasmid pMD18-T-S1 ' and the pMD18-T-Fs that makes up in the step 1,2 respectively, with the upstream and downstream primer of Fs and S1 ' increase respectively Fs and S1 ' genetic fragment.The PCR reaction condition is: 94 ℃ of preparatory degeneration 5min, and 94 ℃ of 30s → 60 ℃ 30s → 72 ℃ of 30s carry out 30 circulations, and last 72 ℃ are extended 10min.Fs and S1 ' amplified production are reclaimed and adopt the two as template, are primer with P1, P4, and 94 ℃ of preparatory degeneration 5min carry out 30 circulations according to 94 ℃ of 50s → 60 ℃ 40s → 72 ℃ of 1min, and last 72 ℃ are extended 10min, amplify FsS1 ' fusion gene.
3. the clone identifies and expresses
(1) FsS1 ' the fusion gene fragment cloning that obtains is gone into pMD18-T vector construction recombination plasmid pMD18-T-FsS1 ', transform
E. coliDH5 α, ammonia benzyl resistance screening positive recombinant, the extracting plasmid is used
Nde1With
XhoIDo double digestion and identify, identify correct back recovery genetic fragment.
(2) FsS1 ' genetic fragment that reclaims in (1) is implemented in expression vector pET-22b, uses
NdeIWith
XhoIDoing double digestion identifies.(the enzyme action result is shown in figure 10)
(3) enzyme action is identified that errorless gene recombination plasmid is converted into the host bacterium
E.coliBL21 (DE3) obtains recombination engineering pET-22b (+)-FsS1 '/BL21, and plasmid pET-22b (+)-FsS1 ' is served the sea give birth to the order-checking of worker bio-engineering corporation.Sequencing result (Figure 11) shows: FSS1 is totally 1011 bases, and 336 aminoacid of encoding have three codons that sudden change has taken place, and the corresponding sequence that other sequences and GenBank announce is in full accord.The sudden change codon is respectively:
GCC------GCT (nonsense mutation)
CGG-----CGA (nonsense mutation)
GCA------GGA (alanine---glycine)
The base concordance is: 999/1002=99.7%
The aminoacid concordance is: 332/333=99.7%
4. the abduction delivering of recombination engineering pET-22b (+)-FsS1 '/BL21 and form are identified
Recombination engineering is cultivated in the LB fluid medium that contains ampicillin (50 μ g/ml), to bacterium liquid OD
600Add during ≈ 0.6 IPTG to final concentration be 1.0mmol/L; Induce different time to collect bacterium liquid; Ultrasound wave breaks bacterium, and differential centrifugation is left and taken cleer and peaceful deposition respectively, carries out gel electrophoresis after the processing and identifies expression-form and expression (abduction delivering and form are identified shown in figure 12).The result shows: albumen Fs has obtained efficiently expressing, and expression induces 3 hours expressions the highest more than 30%, and how with the inclusion body formal representation.
5. the purification of fusion protein F sS1 '
German B.Braun 10L fermentation tank is adopted in the fermentation of reorganization bacterium, carries out according to this laboratory engineering bacterium fermentation common process.Centrifugal collection antibacterial after the fermentation ends, the back of weighing is frozen subsequent use.The resuspended antibacterial of TE buffer with pH value 7.5; Break bacterium through the high-pressure homogenization appearance; Differential centrifugation is collected inclusion body, uses the TE buffer that contains 1%Triton X100 respectively and contains 1M, 2M carbamide TE buffer washing inclusion body, with 8M carbamide dissolving inclusion body; Utilize after the dialysis albumen with 6 His labels, adopt the method for affinity chromatograph to carry out protein purification.Purified target protein is carried out SDS-PAGE, examines and determine its purity, and visible by Figure 13: destination protein purity reaches more than 90%.
The BA of embodiment 4. fusion rotein detects
1. Chinese hamster ovary celI toxicity detects test
(1) with the DMEM culture medium culturing Chinese hamster ovary celI that contains 10% hyclone, cultivation makes and is paved into monolayer under 37 ℃ of 5%CO2.
(2) cell cleans once with PBS liquid, and 0.1% trypsinization 5min is transferred to centrifuge tube from culture bottle then.
(3) after centrifugal, clean cell once with PBS liquid.
(4) a centrifugal back part adds cryopreserving liquid and contains and be suspended from liquid nitrogen container in frozen pipe and make a slip of the tongue night, is sunken in the liquid nitrogen in second day to preserve; Another part is with the resuspended subsequent experimental of carrying out of complete medium.
(5) cell (every hole 200 μ l cell suspension contain 104 cells) with same quantity is added on 48 orifice plates.
(6) treat to blot liquid in the hole behind the cell adhesion 4h, add 200 μ l toxin dilutions and continue and in the environment of 37 ℃ of 5%CO2, cultivate 24h, each concentration of toxin hole of writing in reply is detected.
(7) after cell washes with PBS liquid, fix the dyeing of reuse 0.04% trypan blue with methanol.
(8) cell is cleaned in the dyeing back, dries, and microscopically is observed and taken a picture.
The specific embodiment
The Chinese hamster ovary celI toxicity test shows: PT can make the Chinese hamster ovary celI distortion surpass 50% as long as concentration reaches 20ng/ml; And FsS1 ' has only the few cell distortion even arrived 100ng/ml; Not obvious with matched group difference, think that FsS1 ' has lost the toxicity to Chinese hamster ovary celI basically.
2. murine interleukin increases (LP) test
Every group 10 5 to 8 the week age female BALB/c mouse; Be inoculated in mouse web portion and groin is subcutaneous; Injections of antigens albumen (PT 0.5 μ g mixes in PBS, and all the other antigen amounts are 3 μ g) back 3 days in injection, every group every mice is adopted tail vein 20 μ l and measures leukocyte count.
| Group number | Immunizing antigen | Every treated animal number |
| Ⅰ | S1 | 10/group |
| Ⅱ | FsS1’ | 10/group |
| Ⅲ | PT | 10/group |
| Ⅳ | PBS | 10/group |
Experimental result shows, FsS1 ' group and the no significant difference of PBS group (P>0.05), and there were significant differences (P < 0.01) with PT and S1 group, and S1 group and PBS organize that there were significant differences (P < 0.01), organize no significant difference with PT.Can think that FsS1 ' has lost basically and induce leukocytotic activity (Figure 14).
Like this; Antigen FsS1 ' the substantial loss that obtains through biotechnology bad toxicity biology that natural S1 had; And compare with the chemical detoxication mode with traditional physics, its maximum advantage is not exist back mutation, and character is relatively stable.
The immunogenicity of embodiment 5. fusion rotein is identified
1 immune mouse
Destination protein FsS1 ' is through the Balb/c mice in 4～5 ages in week of immunity behind the purification, and 100 μ g//inferior, 100 μ L antigens mix with equivalent Fu Shi Freund's complete adjuvant, the subcutaneous immunity of mouse web portion and groin.Immune programme for children is: 0,1,2 weeks, and totally 3 times, add the Fu Shi Freund's complete adjuvant the 1st, 2 time, do not add adjuvant the 3rd time, be inoculated in mouse web portion and groin is subcutaneous, the amount of injections of antigens and adjuvant is about 0.2ml.The 3rd immunity docking blood sampling in back 6 days, ELISA detects the change of serological specificity antibody titer.Animal divides into groups as follows:
| Group number | Immunizing antigen | Every treated animal number |
| I | FsS1 ' | 22/group |
| II | The whole | 22/ |
| III | PBS | |
| 22/group |
Vaccine group converts by body weight, the antigen amount be 100 μ g/ only/inferior, matched group PBS concentration is 50mmol/L.
2. the detection of specific immune response
(1) preparation of ELISA antigen coated microplate:
PT antigen is diluted to 5 μ g/ml with coating buffer, and 100 μ l/ holes encapsulate elisa plate, and 4 ℃ are spent the night.Cleaning mixture is washed 4 times.Every hole adds 300 μ l 1%BSA, 37 ℃ of sealing 2h, and after cleaning mixture was washed 4 times, 4 ℃ of preservations were subsequent use.
(2) collection of mice serum BIAO and BEN:
Serum is gathered: respectively at before the immunity and after the immunity 2,3 times 7 days, will every about 100 μ l of mice docking blood sampling, and the aseptic EP pipe of 1.5ml is collected, and room temperature is placed 1h, 5000g * 10min, collection serum, packing ,-70 ℃ of preservations
(3) detection of serological specificity IgG antibody:
The detection of serum antigen specific IgG: begin from 1:5000, with antibody diluent doubling dilution serum to be checked, 1:100 dilutes negative serum, 100 μ l/ holes; Hatch 60min for 37 ℃, cleaning mixture is washed 4 times, adds the sheep anti-mouse igg (1:20 000) of HRP labelling, 100 μ l/ holes; Hatch 30min for 37 ℃, cleaning mixture is washed 4 times, adds OPD substrate colour developing liquid, room temperature lucifuge reaction 30min; 50 μ l stop buffer cessation reactions are made blank with the PBS hole, and 492nm measures each hole OD value.
3. result
| ? | Groups | IgG |
| Ⅰ | FsS1 ' group | 0.756±0.056 |
| Ⅱ | The whole cell pertussis vaccine | 0.873±0.132 |
| Ⅲ | PBS | 0.143±0.0184 |
As above shown in the table: the mice serum specific IgG level of all immune group all has rising in various degree; Wherein FsS1 ' group relatively differs significantly (p < 0.01) with the PBS group; Vaccine immunity group and PBS group relatively differs significantly (p < 0.01), explains that used immune group all can bring out systemic immunity to a certain extent and reply; The anti-PT antibody response of FsS1 ' immune group is similar.Above results suggest FsS1 ' can effectively stimulate body to produce stronger immunne response, has reached the immune effect suitable with traditional vaccine basically, is a kind of good vaccine candidate antigen.Though the antibody response of vaccine group is slightly strong, the antigenic advantage of FsS1 ' is that it can large-scale production, and is safe, and side effect is little, do not have the back mutation of traditional vaccine etc., so it has good market prospect.
The structure of embodiment 6. recombination engineering pET-22b (+)-FsS1 ' S1 '/BL21
1. design of primers and pcr amplification
Recombiant plasmid pMD18-T-S1 ' and pMD18-T-FsS1 ' to make up among the embodiment 1,3 are template, adopt software Primer Premier5.0 to carry out primer design and analysis.With linker (
TACGCGCCGCAGGATCCT) sequential design is at 5 ' end of the 3' of Fs encoding gene end and S1 ' encoding gene, introduce among the linker
BamH1Restriction enzyme site, utilize enzyme to connect method FsS1 ' encoding gene and S1 ' encoding gene coupled together through linker.Primer is given birth to worker bio-engineering corporation by Shanghai and is synthesized, and primer sequence is following:
Fs S1 ' forward primer P1:5 '-
CATATGCTGAAGAACCTCGACCTGGGC-3 '
Nde1
Downstream primer P2:5 '-TC
AGGATCCTGCGGCGCGTACTGCAGTCGCACCG-3 ' linker
S1 ' forward primer P3:5 '-CAG
TACGCGCCGCAGGATCCTGACGATCCTCCCGC-3 ' linker
Downstream primer P4:5 '-
CTCGAGTGTGTAGGGGTTGG-3 '
Xho1
2. the pcr amplification of genes of interest
Be template with recombiant plasmid pMD18-T-S1 ' and the pMD18-T-FsS1 ' that makes up among the embodiment 1,3 respectively, with the upstream and downstream primer of S1 ' and the FsS1 ' S1 ' that increases respectively
BAnd FsS1 '
BGenetic fragment.The PCR reaction condition is: 94 ℃ of preparatory degeneration 5min, and 94 ℃ of 50s → 60 ℃ 40s → 72 ℃ of 1min carry out 30 circulations, and last 72 ℃ are extended 10min.
3. the clone identifies and expresses
(1) with the S1 ' that obtains
BAnd FsS1 '
BGenetic fragment is cloned into pMD18-T vector construction recombination plasmid pMD18-T-S1 ' respectively
BAnd pMD18-T-FsS1 '
B, transform
E. coliDH5 α, ammonia benzyl resistance screening positive recombinant, the extracting plasmid, the former adopts
BamH1 and Xho1Make enzyme action and identify that the latter adopts
Nde1With
BamH1Make enzyme action and identify, identify correct back recovery genetic fragment.
(2) with the FsS1 ' that reclaims in (1)
BGenetic fragment is implemented in expression vector pET-22b, uses
NdeIWith
BamH1Do double digestion and identify, identify correct after with plasmid pET-22b (+)-FsS1 '
BAdopt
BamH1 and Xho1Behind the double digestion with (1) in S1 ' of reclaiming
BFragment connects construction recombination plasmid pET-22b (+)-FsS1 ' S1 ', uses
NdeIWith
XhoIDoing double digestion identifies.(the enzyme action result is shown in figure 15)
(3) enzyme action is identified that errorless gene recombination plasmid is converted into the host bacterium
E.coliBL21 (DE3) obtains recombination engineering pET-22b (+)-FsS1 ' S1 '/BL21, and plasmid pET-22b (+)-FsS1 ' S1 ' is served the sea give birth to the order-checking of worker bio-engineering corporation.Sequencing result (Figure 16) shows: 1566 bases of FSS1S1 total length, and 521 aminoacid of encoding have four codons that sudden change has taken place, and the corresponding sequence that other sequences and GenBank announce is in full accord.The codon of sudden change is respectively:
CAC------CTC (histidine---leucine)
GCC------GCT (nonsense mutation)
CGG------CGA (nonsense mutation)
CGC------TGC (cysteine---arginine)
The base concordance is: 1562/1566=99.7%
The aminoacid concordance is: 519/521=99.6%
4. the abduction delivering of recombination engineering pET-22b (+)-FsS1 ' S1 '/BL21 and form are identified
Recombination engineering is cultivated in the LB fluid medium that contains ampicillin (50 μ g/ml), to bacterium liquid OD
600Add during ≈ 0.6 IPTG to final concentration be 1.0mmol/L; Induce different time to collect bacterium liquid; Ultrasound wave breaks bacterium, and differential centrifugation is left and taken cleer and peaceful deposition respectively, carries out gel electrophoresis after the processing and identifies expression-form and expression (abduction delivering and form are identified shown in figure 17).The result shows: albumen Fs has obtained efficiently expressing, and expression induces 3 hours expressions the highest about 15%, and how with the inclusion body formal representation.
Certainly; The present invention also can have other embodiment; Under the situation of spirit that does not deviate from the present invention and essence; The person of ordinary skill in the field works as can make various corresponding changes and distortion according to the present invention, but these corresponding changes and distortion all should belong to the protection domain of claim of the present invention.
Sequence table
< 110>Military Medical Univ No.3, P.L.A
< 120>two subunit genetic engineering vaccines of a kind of pertussis born of the same parents Te Shi bacillus and preparation method thereof
<130>
<160>6
<170>PatentIn?version3.2
<210>1
<211>333
<212>PRT
< 213>FsS1 ' protein sequence
<400>1
<210>2
<211>1002
<212>DNA
< 213>FsS1 ' gene order
<400>2
<210>3
<211>138
<212>PRT
< 213>Fs protein sequence
<400>3
<210>4
<211>540
<212>DNA
< 213>Fs gene order
<400>4
<210>5
<211>521
<212>PRT
< 213>Fs S1 ' S1 ' protein sequence
<400>5
<210>6
<211>1566
<212>DNA
< 213>FsS1 ' S1 ' gene order
<400>6
Claims (1)
1. a fusion rotein that is used to prepare the two subunit genetic engineering vaccines of pertussis born of the same parents Te Shi bacillus is characterized in that the aminoacid sequence of this fusion rotein is shown in SEQ ID NO:1.
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| CN113512598B (en) * | 2020-12-07 | 2024-03-22 | 上海仁度生物科技股份有限公司 | Real-time fluorescent nucleic acid isothermal amplification detection kit for bordetella pertussis and special primer and probe thereof |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| FR2768928A1 (en) * | 1998-10-06 | 1999-04-02 | Pasteur Institut | Bordetella pertussis filamentous haemagglutinin immunostimulant |
| US7232671B2 (en) * | 1989-02-15 | 2007-06-19 | The United States Of America As Represented By The Secretary, Department Of Health And Human Services | Pertussis toxin gene: cloning and expression of protective antigen |
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| US7232671B2 (en) * | 1989-02-15 | 2007-06-19 | The United States Of America As Represented By The Secretary, Department Of Health And Human Services | Pertussis toxin gene: cloning and expression of protective antigen |
| FR2768928A1 (en) * | 1998-10-06 | 1999-04-02 | Pasteur Institut | Bordetella pertussis filamentous haemagglutinin immunostimulant |
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