CN101845084A - Recombination polypeptide serving as group A streptococcic tetravalent vaccine protective antigen - Google Patents
Recombination polypeptide serving as group A streptococcic tetravalent vaccine protective antigen Download PDFInfo
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Abstract
The invention provides recombination polypeptide, which is prepared by gene recombination technology. The recombination polypeptide is prepared by splicing type-specific amino acid sequences of M1, M3, M6 and M18 serological-type M proteins of rheumatic fever immunogenic group A streptococcus prevailing in the Guangdong province of China, and a common section of conservative amino acid sequence of the C ends of the four serological-type M proteins, wherein the N ends of the four serological-type M proteins comprise protective antigen epitopes and do not comprise human cross reaction epitopes. The recombination polypeptide is experimentally proved to be capable of inducing the generation of bactericidins for the type specificity of the M1, M3 and M6 proteins of the A-group streptococcus so as to be used for preparing recombination polypeptide vaccines for the A-group streptococcus.
Description
Technical field
The present invention relates to DNA recombinant technology and vaccine pharmaceutical field; more specifically say; the present invention relates to a kind of recombinant polypeptide; this recombinant polypeptide can be used as and is primarily aimed at the protective antigen of tetravalent vaccine that China's Guangdong Province's popular can cause four serotypes of A group streptococcus of rheumatic fever; the encode dna sequence dna of this recombinant polypeptide; the carrier that contains this dna sequence dna; the host cell that contains this carrier; the DNA recombinant technology prepares the method for this recombinant polypeptide, and the application of this recombinant polypeptide in A group streptococcus vaccine development.
Background technology
The A group streptococcus is one of modal clinically pathogenic leather Lan Shi positive bacteria.The A group streptococcus is propagated by modes such as the air spittle, skin contact can cause multiple infective disease.In the disease that all A group streptococcus cause, the harm of acute rheumatic fever that causes with A group streptococcus upper respiratory tract infection and the rheumatic heart disease of bringing out thus is the most serious, studies show that untreated children's pharyngitis of about 3% may develop into rheumatic fever, and finally causes rheumatic heart disease.Cause this sick reason to be because there are common antigen in the A group streptococcus and the human body cardiac muscular tissue of part serotype, the A group streptococcus can cause autoimmune disorder after infecting.According to the report of the World Health Organization, annual 517000 people in the whole world die from because of the various diseases due to the streptococcal infection, wherein 2/3 diseases such as rheumatic fever, rheumatic heart disease, acute glomerulonephritis due to dying from after the streptococcal infection.In recent years, though antibiotic is extensive use of, the A group streptococcus infects still very serious in some developing countries.The epidemiology survey of the A group streptococcus that China carries out during the State's Eighth Five-Year Plan period finds that China school-ager A group streptococcus upper respiratory tract annual infection rate reaches 50%, and part Rural areas A group streptococcus infection rate is up to 70%～80%.Conservative estimation, the rheumatic heart patient that China causes because of the A group streptococcus infects is more than 2,500,000, and the operation of great number and lifelong medical expense have caused serious economical load for patient family and society.Therefore, one of development is primarily aimed at very necessity of A group streptococcus polyvalent vaccine.
A group streptococcus serotype is numerous, according to the difference of its cell walls M proteantigen, can be divided into about more than 150 serotypes, and lack cross protection between each serotype, therefore is difficult to a kind of vaccine at all epidemic isolates of research and development.Simultaneously, there are cross-reacting antigen in the A group streptococcus M proteantigen and the human tissue albumen of part serotype.Therefore, traditional thalline vaccine or the subunit vaccine autoimmune response that may cause human body.Up to the present, still none A group streptococcus vaccine listing safely and effectively of the whole world.
M albumen is the main virulence factor on A group streptococcus thalline surface, and antiphagocytosis is arranged, and the proteic antibody of anti-M that produces behind the organism infection A group streptococcus is protection antibody, can continue after the natural infection to have more than 30 year.Discovering in recent years, M protein gene (emm) encoded polypeptides C-terminal is a high conservative, the variation of N-terminal height is the basis of A group streptococcus type specificity; The N end is made up of A, B, three repeated fragments of C, and the antigenic determinant that produces the protection antibody of type specificity is positioned at the A district, and generation is positioned at the flank in B district, B-C district, the flank in A-B district with the antigenic determinant of tissue cross reaction.(Alan L.Bison, Fran A.Rubin, P.Patrick Cleary and James B.Dale Clinical InfectiousDiseases 2005; 41:11501156) therefore; with M albumin A district is basic design had both been avoided human body generation cross reaction at the vaccine of A group streptococcus different serotypes zone; can make body produce protection antibody again, be a feasible method of A group streptococcus vaccine development.
Studies show that the M albumen of A group streptococcus is the heterodimeric protein that is made of two polypeptide chains that are entirely α-Luo Xuanjiegou, space structure is a linear molecule.Therefore, its epitope should be linear epitope, does not have complicated space conformation.The protective epitope is contained in the district with different serotypes A group streptococcus M albumin A; the be connected in series polypeptide of the artificial design that the back forms of the aminoacid sequence that does not contain human body cross reaction epi-position should have similar space structure with natural M albumen, and can not destroy original epitope.
The A group streptococcus M somatotype that American-European developed country has confirmed to cause rheumatic fever originality through long term studies mainly concentrates on M1,3,5,6,14,18,19,24,10 serotypes such as 27 and 29.Guangdong Prov. Cardiovascular disease Inst. in 2004 at home reported first in the result of the M albumen emm gene type of the isolating 104 strain A group streptococcus in Guangdong Province and other some areas of China, leading serotype is emm1,18,12,69, types such as 110, wherein the emm1 type accounts for 29.8%, and the emm18 type accounts for 9.6%.These two serotypes be rheumatic fever originality the A group streptococcus (Chen Zhihong, Gong Shoufang, Dong Taiming etc. use the emm gene sequencing to 104 strain Lancefield groups. Chinese microbiology and Journal of Immunology, 2004,24 (6): 489-491.).The emm gene type of area, Guangzhou childhood infection A group streptococcus 87 strains of reports such as the Chen Zhao of Guangzhou Children's Hospital letter in 2008 has also confirmed above result (Chen Zhaohong, thank to Guoqiang, Deng Qiulian etc. the emm gene type of area, Guangzhou childhood infection A group streptococcus 87 strains. Guangdong medical science, 2008,29 (4) 583-584.).
Summary of the invention
First purpose of the present invention provides a kind of protective antigen of tetravalent vaccine of the A group streptococcus that is primarily aimed at China Guangdong Province popular rheumatic fever originality; it is that M albumen n end by the A group streptococcus M1 of Guangdong Province's popular rheumatic fever originality, M3, M6, four serotypes of M18 contains protective epitope, does not contain the aminoacid sequence of type specificity of human body cross reaction epi-position and the recombinant polypeptide that one section total conserved amino acid sequence of this four serotype M PROTEIN C ends is formed by connecting.
Another object of the present invention provide DNA, this dna sequence dna of this recombinant polypeptide of coding preparation method, contain the carrier of this dna sequence dna and contain the host cell of this carrier.
The 3rd purpose of the present invention provides the method that obtains this recombinant polypeptide.
In a first aspect of the present invention, a kind of recombinant polypeptide is provided, it comprises: 1-50 the amino acid (SEQ ID NO 3) of A group streptococcus M1 type M protein type specific amino acid; 21-70 the amino acid (SEQ ID NO 4) of A group streptococcus M3 type M protein type specific sequence; 1-35 the amino acid (SEQ ID NO 5) of A group streptococcus M6 type M protein type specific sequence; 1-45 the amino acid (SEQ ID NO 6) of A group streptococcus M18 type M protein type distinguished sequence; Total one of A group streptococcus M1, M3, M6, four serotype M of M18 PROTEIN C end contains 14 amino acid whose sequences (SEQ ID NO:7).
The order of connection of 5 sections aminoacid sequences of above-described formation recombinant polypeptide of the present invention is from left side NH
2End to right side COOH end is followed successively by: 1-50 amino acid of A group streptococcus M1 type M protein type specific amino acid, 21-70 amino acid of A group streptococcus M3 type M protein type specific sequence, 1-35 amino acid of A group streptococcus M6 type M protein type specific sequence, 1-45 amino acid of A group streptococcus M18 type M protein type distinguished sequence, total one of A group streptococcus M1, M3, M6, four serotype M of M18 PROTEIN C end contains 14 amino acid whose sequences.See Fig. 1.
Recombinant polypeptide provided by the invention has the aminoacid sequence shown in the sequence 1 in the sequence table.
In a second aspect of the present invention, provide the preparation method of a kind of isolated DNA molecule and this dna molecular.Recombinant polypeptide shown in its coding SEQ ID NO:1, described dna molecular comprises the nucleotide sequence shown in the SEQ ID NO:3.
The method of obtaining above-mentioned dna molecular comprises: the recombinant polypeptide aminoacid sequence is selected suitable genetic codon direct chemical composite coding recombinant polypeptide dna molecular of the present invention according to the present invention; The method of employing PCR amplifies the goal gene of each serotype from A group streptococcus genome, again by the method splicing of SOE-PCR, form the dna molecular of code book invention recombinant polypeptide; The recombinant protein aminoacid sequence is selected suitable genetic codon according to the present invention, synthesizes a series of oligonucleotide fragments, passes through the dna molecular of overlap-PCR composite coding recombinant polypeptide of the present invention then.May be different with its genetic codon of dna molecular that these methods obtain, but final translation product necessarily contains the aminoacid sequence of recombinant polypeptide of the present invention.
In a third aspect of the present invention, a kind of carrier is provided, this carrier is characterised in that it contains the dna molecular of the aminoacid sequence that includes code book invention recombinant polypeptide that obtains by above-mentioned any method.
Aspect the 4th of the present invention, the host cell that contains above-mentioned carrier is provided, behind the abduction delivering, the recombinant protein of generation necessarily contains the aminoacid sequence of recombinant polypeptide of the present invention to this host cell under appropriate condition.
Aspect the 5th of the present invention, provide a kind of a kind of method that produces recombinant polypeptide of the present invention.It comprises step: be fit to express under the described recombinant polypeptide condition, cultivate above-mentioned host cell, thereby give expression to recombinant polypeptide or contain the recombinant protein of recombinant polypeptide aminoacid sequence, by suitable method separation and purification recombinant polypeptide or contain the recombinant protein of recombinant polypeptide aminoacid sequence.
Aspect the 6th of the present invention, the vaccine that provides a kind of A of prevention group streptococcus M1, M3, M6, M18 type to infect, this vaccine contain described recombinant polypeptide of claim 1 and acceptable adjuvant pharmaceutically, also can contain some immunostimulants.Adjuvant is as aluminium hydroxide, aluminum phosphate, oil-in-water emulsion, nucleic acid class adjuvant etc., but immunostimulant comprises protein, polysaccharide, lipid or the micromolecular compound of human body, plant or the microorganism of known enhance immunity.
Definition
As used herein, term " aminoacid sequence of the proteic type specificity of M " is meant the at present disclosed aminoacid sequence that can make a variation from the different serotypes A group streptococcus M protein N terminal height of U.S.'s disease and the download of prevention and control central database, the serotype of the antigen-specific decision A group streptococcus of this section sequence.
Download address: http://www.cdc.gov/ncidod/biotech/strep/strepindex.htm.
Somatotype as used herein, that term " M1, M3, M6, M18 etc. " expression A group streptococcus carries out with M protein antigenicity difference; The somatotype that term " emm1, emm3, emm6, emm18 etc. " expression A group streptococcus carries out with the difference of M protein gene.Pass between the two is that the M1 type is equal to the emm1 type, and the M3 type is equal to the emm3 type, and the rest may be inferred.
As used herein; term " contains protective epitope; the aminoacid sequence that does not contain the type specificity of human body cross reaction epi-position is meant in the aminoacid sequence of type specificity of A group streptococcus M protein N terminal can induce body to produce protection antibody, can not cause the section of autoimmune response simultaneously again.
As used herein, one section of being meant that the most of serotype C end of the disclosed A group streptococcus of document M albumen conserved regions all has of term " C end total one section conserved amino acid sequence " and tissue albumen do not have section (the Hayman WA of cross-reacting antigen, Brandt ER, Relf WA, et al.Int Immunol, 1997,9 (11): 1723-1733).
As used herein, term " carrier " comprises plasmid, clay, expression vector, cloning vector etc.Can select various carrier known in the art such as commercially available carrier in the present invention for use, then the nucleotide sequence of code book invention recombinant protein be cloned after the expression regulation sequence that into carrier provides, can form protein expression vector.
As used herein, term " host cell " mainly refers to prokaryotic cell prokaryocyte, comprises intestinal bacteria, Bacillus subtilus etc.
In an example of the present invention, the gene Strep4 of coding recombinant polypeptide is cloned into prokaryotic expression carrier PQE30 plasmid, and change recombinant plasmid over to intestinal bacteria M15, but obtained the intestinal bacteria reorganization bacterium M15/PQE30-Strep4 of solubility express recombinant polypeptide.
Can produce bactericidin behind the recombinant immunogenic polypeptide rabbit of the present invention at the type specificity of A group streptococcus M1 type, M3 type, M6 type.In an example, rabbit anteserum behind the recombinant immunogenic polypeptide is carried out indirect immunofluorescence experiment with the thalline of A group streptococcus M1 type, M3 type, M6 type, M5 type respectively, found that, the rabbit immune serum only and comprise A group streptococcus M1 type, M3 type, the reaction of M6 type of serotype on the recombinant polypeptide, the irrelevant M5 type A group streptococcus of discord reacts.This shows, is type specificity antibody by the antibody that produces behind the recombinant immunogenic polypeptide.In another example, recombinant immunogenic polypeptide rabbit anteserum behind the deactivation complement is mixed with A group streptococcus M1 type, M3 type, M6 type, M5 type and the normal adults anticoagulation that do not contain at these serotype A group streptococcus antibody respectively, carry out the extracorporeal disinfecting experiment, found that, immune serum only has higher sterilizing rate to A group streptococcus M1 type, M3 type, the M6 type that comprises serotype on the recombinant polypeptide, and irrelevant M5 type A group streptococcus is not had germicidal action.This shows, is the bactericidin of type specificity by the antibody that produces behind the recombinant immunogenic polypeptide.
In sum; the present invention passes through gene recombination technology; make up one and contained A group streptococcus M1 type, M3 type, M6 type, four serotype M of M18 type albumen n end that can cause rheumatic fever and contain protective epitope, do not contained the recombinant polypeptide of the type specificity aminoacid sequence of human body cross reaction epi-position.Experiment shows that this recombinant polypeptide can be induced the specific bactericidin of generation at A group streptococcus M1 type, M3 type, M6 type, thereby can be used as the protective antigen of the A group streptococcus tetravalent vaccine of prevention China Guangdong Province main popular rheumatic fever originality.
Description of drawings
Fig. 1 is a recombinant polypeptide structural representation of the present invention.
Fig. 2 is the electrophoretogram of the recombinant polypeptide gene of overlap-PCR amplification, and 1 is reacted product of the overlap-PCR first round among the figure; 2,3 take turns reacted product (recombinant polypeptide gene) for overlap-PCR second; 4 is blank; 5:DL2000marker.
Fig. 3 is the escherichia coli cloning carrier PUC18-Strep4 that contains the recombinant polypeptide gene.
Fig. 4 is the coli expression carrier PQE30-Strep4 that contains the recombinant polypeptide gene.
Fig. 5 is a SDS-PAGE electrophoretogram behind the M15/PQE30-Strep4 engineering bacteria abduction delivering, and 1 is protein molecular weight standard among the figure; 2,4,6 is M15/PQE30-Strep4 engineering bacteria IPTG abduction delivering; 3,5,7 is not abduction delivering of M15/PQE30-Strep4 engineering bacteria; 8 for to have the intestinal bacteria M15 of PQE30 plasmid through the IPTG abduction delivering; 9 are the intestinal bacteria M15 that has PQE30 plasmid abduction delivering not.
Fig. 6 is the SDS-PAGE electrophoretogram of reorganization expression of polypeptides purifying, and 1 is protein molecular weight standard among the figure; 2 is not abduction delivering of M15/PQE30-Strep4 engineering bacteria; 3 is M15/PQE30-Strep4 engineering bacteria abduction delivering; 4 is ultrasonic degradation supernatant behind the M15/PQE30-Strep4 engineering bacteria abduction delivering; 5 for adsorbing the back supernatant with the Ni-NTA gel; 6 is with supernatant behind elution buffer 1 wash-out; 7 is with supernatant (purification of Recombinant polypeptide) behind elution buffer 2 wash-outs.
Fig. 7 is the IgG antibody titers change curve of anti-recombinant polypeptide in the serum after the rabbit immunity;
Fig. 8 immune serum and preimmune serum respectively with the indirect immunofluorescence reaction result of A group streptococcus M1, M3, M6, M5 type, wherein A is the Immunofluorescence Reactions of immune serum and M1 type A group streptococcus; B is the Immunofluorescence Reactions of preimmune serum and M1 type A group streptococcus; C is the Immunofluorescence Reactions of immune serum and M3 type A group streptococcus; D is the Immunofluorescence Reactions of preimmune serum and M3 type A group streptococcus; E is the Immunofluorescence Reactions of immune serum and M6 type A group streptococcus; F is the Immunofluorescence Reactions of preimmune serum and M6 type A group streptococcus; G is the Immunofluorescence Reactions of immune serum and M5 type A group streptococcus; H is the Immunofluorescence Reactions of preimmune serum and M5 type A group streptococcus.
Fig. 9 is the bar shaped synoptic diagram of immune serum at different serotypes A group streptococcus sterilizing rate.
Embodiment
Further set forth the present invention below in conjunction with specific examples.These examples only are used to illustrate the present invention, rather than limit the scope of the invention, the experimental technique of unreceipted concrete actual conditions in the following example, usually according to normal condition, people's such as sanbrook molecular cloning for example: the condition described in the laboratory manual, or the condition of advising according to manufacturer.
The structure of the synthetic and escherichia coli cloning carrier of recombinant polypeptide gene order
Download A group streptococcus emm1, emm3, emm6, four type M of emm18 albumen n end type specificity sequence from U.S.'s disease and prevention and control central database, obtain these four type corresponding amino acid sequence in recombinant polypeptide; From the report document, obtain the consensus sequence of these four type M PROTEIN C ends.By the order of holding the total conserved sequence of C end 1-3-6-18-from N, whole aminoacid sequences (SEQ ID NO:1) is spliced in series connection with 5 sections aminoacid sequences.
Aminoacid sequence is imported DNAWORK 2.0 online softwares, obtain a nucleotide sequence (SEQ ID NO:2) that has the coding recombinant polypeptide of the inclined to one side preferendum codon of intestinal bacteria after the operation, and a cover oligonucleotide sequence (SEQ ID NO:8-17) and a corresponding techniques parameter that adopts synthetic this nucleotide sequence of overlap-PCR method.Introduced the restriction enzyme site of BamHI and HindIII respectively at 5 ' end of article one and the last item oligonucleotide sequence.
Synthetic this cover oligonucleotide sequence is by two-wheeled PCR reaction amplifying target genes sequence.
Table 1. first round PCR reaction system
| Reaction system | ??μl |
| Oligonucleotide sequence 1-10 (5 μ M) | ??2×10 |
| ??5×PCR?buffer | ??10 |
| ??dNTP?Mix(10μM) | ??4 |
| ??primeSTAR TMHS?DNA?polymerase(2.5U/ml) | ??0.5 |
| ??ddH2O | ??15.5 |
| The reaction cumulative volume | ??50 |
Reaction conditions is 95 ℃ of pre-sex change in 5 minutes, 95 ℃, 1 minute, 64 ℃ of annealing 1 minute, 72 ℃ were extended 1 minute, after totally 15 circulations again 72 ℃ extended 2 minutes.
Table 2. second is taken turns the PCR reaction system
| Reaction system | ??μl |
| First round PCR product | ??2 |
| |
??2 |
| Reaction system | ?? |
| Oligonucleotide sequence | |
| 10 | ??2 |
| ??5×PCRbuffer | ??10 |
| ??dNTP?Mix(10μM) | ??4 |
| ??primeSTAR TMHS?DNA?polymerase(2.5U/ml) | ??0.5 |
| ??ddH2O | ??29.5 |
| The reaction cumulative volume | ??50 |
Reaction conditions is 95 ℃ of pre-sex change in 5 minutes, 95 ℃, 1 minute, 64 ℃ of annealing 1 minute, 72 ℃ were extended 1 minute, after totally 30 circulations again 72 ℃ extended 2 minutes.
By two-step pcr, successfully amplify a dna fragmentation about 600bp, identical with expected design.(Fig. 2)
Rear clone makes up the recombinant clone plasmid, called after PUC18-Strep4 (Fig. 3) behind purified and BamH I of PCR product and the HindIII double digestion in escherichia coli cloning carrier PUC18.Blue hickie method screening positive clone, in full accord through nucleotide sequence and the sequence among the SEQ ID NO:3 that sequencing analysis inserts.
The structure of recombinant polypeptide expression vector
BamH I and HindIII be double digestion recombinant clone plasmid PUC18-Strep4 respectively, and escherichia coli prokaryotic expression plasmid PQE30.Agarose gel electrophoresis separates endonuclease bamhi, reclaims the dna fragmentation of purifying coding recombinant polypeptide vaccine and the endonuclease bamhi of PQE30 plasmid, connects to make up recombinant expression plasmid, called after PQE30-Strep4 (Fig. 4).Transformed into escherichia coli M15, penbritin, the two resistance screening positive colonies of kantlex, in full accord through nucleotide sequence and the sequence among the SEQ ID NO:3 that sequencing analysis inserts.The engineering bacteria called after M15/PQE30-Strep4 of the express recombinant polypeptide that obtains.
The expression of recombinant polypeptide
The engineering bacteria M15/PQE30-Strep4 that example 2 is obtained is inoculated in 20ml and contains 100 μ g/ml penbritins, in the LB substratum of 25 μ g/ml kantlex, and 37 ℃ of thermal agitation overnight incubation.The second day bacterium liquid with 20ml cultivation overnight is inoculated in 1L and contains 100 μ g/ml penbritins, and in the LB substratum of 25 μ g/ml kantlex, 37 ℃ of thermal agitations are cultivated, to bacterium liquid OD
600Reach 0.6 back add IPTG to final concentration be 1mM.Continue to cultivate after 4 hours 4000 * g, 20 minutes centrifugal receipts bacterium.Bacterial sediment has the band (Fig. 5) of protein high expression level at the about 24KD of molecular weight place through the SDS-PAGE electrophoretic analysis.This molecular weight with the expression product of prediction is consistent.The expression product of prediction comprises 2 amino acid that contain 6 histidine-tagged 10 amino acid and BamH I restriction enzyme site coding on 194 amino acid, PQE 30 plasmids of recombinant polypeptide, and molecular weight is 23.8KD.
The Ni of recombinant polypeptide
2+Affinitive layer purification
With the bacterial sediment that example 3 obtains, resuspended with lysis buffer, washed twice is resuspended in thalline in 2 50ml centrifuge tubes with the 20ml lysis buffer at last, ultrasonic degradation thalline in the ice bath (200-300w ultrasonic 30 seconds, stopped 10 circulations 30 seconds)., 4000 * g, 4 ℃ 20 minutes centrifugal, collect ultrasonic supernatant, the 0.45um membrane filtration.The ultrasonic supernatant of every 5ml one pipe adds the Ni-NTA gel that 0.5ml anticipates, and the room temperature vibration mixed 1 hour in the 10ml centrifuge tube, 500rpm, and 5 minutes are centrifugal, remove supernatant.Add 10ml elution buffer 1, the room temperature vibration mixed 10 minutes, 500rpm, and 5 minutes are centrifugal, and repeated washing once removes supernatant, repeats this step once.Add 3ml elution buffer 2, the room temperature vibration mixed 30 minutes, 500rpm, and 5 minutes are centrifugal, collect the elution buffer that contains the purification of Recombinant polypeptide, repeat wash-out once.The recombinant polypeptide of purifying is collected in the dialysis tubing in 3.5KD aperture, dialyses as extracellular fluid dialysis, to remove residual imidazoles and Ni in the purifying protein with the phosphate buffered saline buffer of PH7.4
2+Change extracellular fluid dialysis altogether 10 times, 4 hours/time.With the purification of Recombinant polypeptide Sterile Filtration after the dialysis, frozen.Recombinant polypeptide behind the purifying shows that purity is higher after the SDS-PAGE electrophoresis is identified, see Fig. 6.
The detection of recombinant immunogenic polypeptide originality
Behind the recombinant polypeptide vaccine and the emulsification of isopyknic Freund's complete adjuvant thorough mixing of the purifying that example 4 is obtained, 3 of the healthy male rabbits about subcutaneous multi-point injection 2Kg, 2ml/.After 4 weeks of initial immunity and 8 weeks, with same doses of antigen, adopt incomplete Freund's adjuvant, subcutaneous multi-point injection is strengthened twice.Per two all tame rabbit ear veins blood samplings obtain immune serum before initial immunity and after the immunity for the first time, and to detect the variation of immunity back antibody titers, 12 weeks prepared immune serum in a large number with immunizing rabbit arteria carotis communis sacrificed by exsanguination after immunity.Enzyme linked immunosorbent assay (ELISA) detects the different time points of immunity, the titre changing conditions of anti-recombinant polypeptide vaccine I gG antibody in the rabbit anteserum.Titre is judged as with the OD value greater than the high dilution of 2 times of every the rabbit preimmune serum OD values titre as test serum.Detected result such as table 3:
The anti-recombinant polypeptide IgG of rabbit anteserum antibody titers behind table 3 recombinant immunogenic polypeptide
| Time (week) | The #1 rabbit | The #2 rabbit | The #3 rabbit |
| ??4 | ??1∶102400 | ??1∶25600 | ??1∶204800 |
| ??6 | ??1∶204800 | ??1∶51200 | ??1∶819200 |
| ??8 | ??1∶409600 | ??1∶204800 | ??1∶409600 |
| ??10 | ??1∶409600 | ??1∶204800 | ??1∶409600 |
| ??12 | ??1∶409600 | ??1∶204800 | ??1∶409600 |
The titre change curve is seen Fig. 7.
From detected result as can be seen, three rabbit have all produced the antibody at recombinant polypeptide of high titre behind recombinant immunogenic polypeptide, illustrate that recombinant polypeptide has better immunogenicity.
Antibodies specific detects behind the recombinant immunogenic polypeptide
Immune serum and the preimmune serum of 3 rabbit that obtain with example 5 carry out indirect immunofluorescence experiment with A group streptococcus M1 type, M3 type, M6 type, M18 type and the M5 type (bacterium numbering is respectively 32171,32172,32175,32180,32174) bought from the Chinese medicine strain library respectively, and wherein the M5 type is as the negative control sera type.With serum since 1: 50 two-fold dilution to 1: 6400, the dilution serum of difference is anti-ly reacted with the A group streptococcus of 5 serotypes respectively as one, 37 ℃, 1 hour, the phosphate buffered saline buffer of PH7.4 was given a baby a bath on the third day after its birth time; Drip the goat anti-rabbit igg of FITC mark, 37 ℃ 30 minutes, the phosphate buffered saline buffer of PH7.4 is given a baby a bath on the third day after its birth time; After the drying, drip one of buffering glycerine, add the cover glass mounting, under the fluorescent microscope, use 400 respectively *, 1000 * oily sem observation.With the high dilution of naked eyes visible fluorescence behind every part of serum sample and the bacterial reaction as the immunofluorescence titre of this serum sample at this serotype A group streptococcus.From detected result such as table 4 and shown in Figure 8.
Table 4 immune serum and preimmune serum and different serotypes A group streptococcus
The titre of indirect immunofluorescence reaction
"-" is the indirect immunofluorescence reaction negative.
From detected result as can be seen, three rabbit have all produced behind recombinant immunogenic polypeptide at A group streptococcus M1, M3, the proteic type specificity antibody of M6 type M.
The extracorporeal disinfecting antibody experiment of recombinant immunogenic polypeptide rabbit anteserum
M1 type used in the example 6, M3 type, M6 type, M18 type A group streptococcus are incubated in the 3ml THB substratum 37 ℃ of static spending the night respectively.The cultivation bacterium liquid 0.1ml overnight that gets each serotype A group streptococcus is diluted to 10 for continuous 10 times
-5With the bacterium liquid after the 50ul dilution, immune serum of 3 rabbit that 100ul example 5 obtains or preimmune serum (deactivation complement) and 350ul normal adults anticoagulated whole blood (preliminary experiment determines that this blood does not contain at experiment serotype A group streptococcus bactericidin) thorough mixing, 37 ℃ of vibrations were hatched 3 hours, the preimmune serum contrast is established in each experiment, after hatching 3 hours, get 50ul mixtures incubated coating rabbit blood agar plate, 37 ℃ of overnight incubation, add up each dull and stereotyped bacterium colony number that goes up next day, if the dull and stereotyped colony number of going up surpasses 1000 inconvenient accurate countings, by 1000 calculating.Calculate the sterilizing rate of immune serum to different serotypes A group streptococcus.Calculate sterilizing rate by following formula:
The flat-plate bacterial colony number * 100% detected result is as shown in table 5 before sterilizing rate=(preimmune serum flat-plate bacterial colony number-immune serum flat-plate bacterial colony number)/immunity:
Table 5 recombinant immunogenic polypeptide rabbit anteserum extracorporeal disinfecting antibody experiment antibody sterilizing rate
From detected result as can be seen, three rabbit have all produced the bactericidin at A group streptococcus M1, M3, M6 type behind recombinant immunogenic polypeptide.The bar shaped synoptic diagram of immune serum sterilizing rate is seen Fig. 9.
Below amino acid and the nucleotides sequence tabulation that relates to for patent application of the present invention, each sequence is followed successively by in the sequence table:
Sequence 1: the aminoacid sequence of recombinant polypeptide of the present invention
Sequence 2: recombinant polypeptide coding gene sequence of the present invention
1-50 amino acid of sequence 3:A group streptococcus M1 type M protein type specific amino acid
21-70 amino acid of sequence 4:A group streptococcus M3 type M protein type specific amino acid
1-35 amino acid of sequence 5:A group streptococcus M6 type M protein type specific amino acid
1-45 amino acid of sequence 6:A group streptococcus M18 type M protein type specific amino acid
Sequence 7:A group streptococcus M1 type, M3, M6, one section total conserved sequence of M18 type M PROTEIN C end
Sequence 8:overlap-PCR synthesizes the used oligonucleotide sequence 1 of recombinant polypeptide gene
Sequence 9:overlap-PCR synthesizes the used oligonucleotide sequence 2 of recombinant polypeptide gene
Sequence 10:overlap-PCR synthesizes the used oligonucleotide sequence 3 of recombinant polypeptide gene
Sequence 11:overlap-PCR synthesizes the used oligonucleotide sequence 4 of recombinant polypeptide gene
Sequence 12:overlap-PCR synthesizes the used oligonucleotide sequence 5 of recombinant polypeptide gene
Sequence 13:overlap-PCR synthesizes the used oligonucleotide sequence 6 of recombinant polypeptide gene
Sequence 14:overlap-PCR synthesizes the used oligonucleotide sequence 7 of recombinant polypeptide gene
Sequence 15:overlap-PCR synthesizes the used oligonucleotide sequence 8 of recombinant polypeptide gene
Sequence 16:overlap-PCR synthesizes the used oligonucleotide sequence 9 of recombinant polypeptide gene
Sequence 17:overlap-PCR synthesizes the used oligonucleotide sequence 10 of recombinant polypeptide gene
Sequence table SEQ UENCE LISTING
＜110〉Wuhan Biological Products Inst.
＜120〉a kind of recombinant polypeptide as A group streptococcus tetravalent vaccine protective antigen
<130>/
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<170>PatentIn?version?3.5
<210>1
<211>194
<212>PRT
＜213〉artificial
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Asn?Gly?Asp?Gly?Asn?Pro?Arg?Glu?Val?Ile?Glu?Asp?Leu?Ala?Ala?Asn
1????????????????5???????????????????10??????????????????15
Asn?Pro?Ala?Ile?Gln?Asn?Ile?Arg?Leu?Arg?His?Glu?Asn?Lys?Asp?Leu
20??????????????????25??????????????????30
Lys?Ala?Arg?Leu?Glu?Asn?Ala?Met?Glu?Val?Ala?Gly?Arg?Asp?Phe?Lys
35??????????????????40??????????????????45
Arg?Ala?Asn?Leu?Leu?Asp?Gln?Val?Thr?Gln?Leu?Tyr?Asn?Lys?His?Asn
50??????????????????55??????????????????60
Ser?Asn?Tyr?Gln?Gln?Tyr?Ser?Ala?Gln?Ala?Gly?Arg?Leu?Asp?Leu?Arg
65??????????????????70??????????????????75??????????????????80
Gln?Lys?Ala?Glu?Tyr?Leu?Lys?Gly?Leu?Asn?Asp?Trp?Ala?Glu?Arg?Leu
85??????????????????90??????????????????95
Leu?Gln?Glu?Leu?Arg?Val?Phe?Pro?Arg?Gly?Thr?Val?Glu?Asn?Pro?Asp
100?????????????????105?????????????????110
Lys?Ala?Arg?Glu?Leu?Leu?Asn?Lys?Tyr?Asp?Val?Glu?Asn?Ser?Met?Leu
115?????????????????120?????????????????125
Gln?Ala?Asn?Asn?Asp?Lys?Leu?Ala?Pro?Leu?Thr?Arg?Ala?Thr?Ala?Asp
130?????????????????135?????????????????140
Asn?Lys?Asp?Glu?Leu?Ile?Lys?Arg?Ala?Asn?Asp?Tyr?Glu?Ile?Gln?Asn
145?????????????????150?????????????????155?????????????????160
His?Gln?Leu?Thr?Val?Glu?Asn?Lys?Lys?Leu?Lys?Thr?Asp?Lys?Glu?Gln
165?????????????????170?????????????????175
Leu?Thr?Lys?Glu?Ala?Ser?Arg?Glu?Ala?Lys?Lys?Gln?Val?Glu?Lys?Ala
180?????????????????185?????????????????190
Leu?Glu
<210>2
<211>582
<212>DNA
＜213〉artificial
<400>2
aacggcgatg?gtaacccacg?cgaagtcatc?gaagacctgg?ccgccaataa?tccggccatc????60
cagaacattc?gtttacgcca?cgaaaacaaa?gatctgaaag?cacgtctgga?aaatgcaatg????120
gaggtcgcgg?gtcgcgattt?caaacgcgca?aatttactgg?atcaagtgac?gcaactgtac????180
aacaaacaca?atagcaatta?tcagcagtac?agcgcccagg?ccggtcgttt?agacctgcgc????240
caaaaagcag?aatacctgaa?aggcctgaat?gactgggcgg?agcgcttatt?acaggagtta????300
cgcgtgtttc?cacgcggcac?ggtcgaaaat?ccggacaaag?cgcgcgagtt?actgaataag????360
tacgatgtcg?agaactctat?gttacaggcc?aacaatgata?agctggcacc?gctgacccgt????420
gcaaccgccg?acaataagga?tgaattaatc?aaacgtgcca?acgattatga?gatccaaaac????480
caccagttaa?cggtggagaa?taagaagctg?aagacggaca?aggagcagct?gaccaaagag????540
gccagccgtg?aggcaaaaaa?acaagttgaa?aaggcgctgg?ag???????????????????????582
<210>3
<211>50
<212>PRT
＜213〉A group streptococcus M1 type
<400>3
Asn?Gly?Asp?Gly?Asn?Pro?Arg?Glu?Val?Ile?Glu?Asp?Leu?Ala?Ala?Asn
1???????????????5???????????????????10??????????????????15
Asn?Pro?Ala?Ile?Gln?Asn?Ile?Arg?Leu?Arg?His?Glu?Asn?Lys?Asp?Leu
20??????????????????25??????????????????30
Lys?Ala?Arg?Leu?Glu?Asn?Ala?Met?Glu?Val?Ala?Gly?Arg?Asp?Phe?Lys
35??????????????????40??????????????????45
Arg?Ala
50
<210>4
<211>50
<212>PRT
＜213〉A group streptococcus M3 type
<400>4
Asn?Leu?Leu?Asp?Gln?Val?Thr?Gln?Leu?Tyr?Asn?Lys?His?Asn?Ser?Asn
1???????????????5???????????????????10??????????????????15
Tyr?Gln?Gln?Tyr?Ser?Ala?Gln?Ala?Gly?Arg?Leu?Asp?Leu?Arg?Gln?Lys
20??????????????????25??????????????????30
Ala?Glu?Tyr?Leu?Lys?Gly?Leu?Asn?Asp?Trp?Ala?Glu?Arg?Leu?Leu?Gln
35??????????????????40??????????????????45
Glu?Leu
50
<210>5
<211>35
<212>PRT
＜213〉A group streptococcus M6 type
<400>5
Arg?Val?Phe?Pro?Arg?Gly?Thr?Val?Glu?Asn?Pro?Asp?Lys?Ala?Arg?Glu
1???????????????5???????????????????10??????????????????15
Leu?Leu?Asn?Lys?Tyr?Asp?Val?Glu?Asn?Ser?Met?Leu?Gln?Ala?Asn?Asn
20??????????????????25??????????????????30
Asp?Lys?Leu
35
<210>6
<211>45
<212>PRT
＜213〉A group streptococcus M18 type
<400>6
Ala?Pro?Leu?Thr?Arg?Ala?Thr?Ala?Asp?Asn?Lys?Asp?Glu?Leu?Ile?Lys
1???????????????5???????????????????10??????????????????15
Arg?Ala?Asn?Asp?Tyr?Glu?Ile?Gln?Asn?His?Gln?Leu?Thr?Val?Glu?Asn
20??????????????????25??????????????????30
Lys?Lys?Leu?Lys?Thr?Asp?Lys?Glu?Gln?Leu?Thr?Lys?Glu
35??????????????????40??????????????????45
<210>7
<211>14
<212>PRT
＜213〉A group streptococcus M1 type, M3, M6, M18 type
<400>7
Ala?Ser?Arg?Glu?Ala?Lys?Lys?Gln?Val?Glu?Lys?Ala?Leu?Glu
1???????????????5???????????????????10
<210>8
<211>56
<212>DNA
＜213〉artificial sequence
<400>8
ggcggatcca?acggtgacgg?taatccgcgc?gaggtgattg?aagatctggc?cgccaa????56
<210>9
<211>90
<212>DNA
＜213〉artificial sequence
<400>9
gcattctcta?agcgtgcctt?taaatcctta?ttttcgtgac?gcagacggat?attttggatc????60
gccgggttat?tggcggccag?atcttcaatc?????????????????????????????????????90
<210>10
<211>90
<212>DNA
＜213〉artificial sequence
<400>10
atttaaaggc?acgcttagag?aatgccatgg?aggtcgcagg?ccgcgacttt?aaacgtgcaa????60
atttactgga?ccaggttacg?caactgtaca?????????????????????????????????????90
<210>11
<211>90
<212>DNA
＜213〉artificial sequence
<400>11
ctgccttctg?acgtaaatcc?agacgacctg?cctgtgcaga?gtattgctgg?tagttagaat????60
tgtgcttatt?gtacagttgc?gtaacctggt?????????????????????????????????????90
<210>12
<211>90
<212>DNA
＜213〉artificial sequence
<400>12
tctggattta?cgtcagaagg?cagaatatct?gaagggcctg?aacgactggg?cggaacgcct????60
gttacaagag?ctgcgcgtgt?ttccacgcgg?????????????????????????????????????90
<210>13
<211>90
<212>DNA
＜213〉artificial sequence
<400>13
ttgcctgtaa?catagagttt?tcaacgtcgt?atttgttcag?cagttcacgt?gctttgtctg????60
ggttttcgac?cgtaccgcgt?ggaaacacgc?????????????????????????????????????90
<210>14
<211>90
<212>DNA
＜213〉artificial sequence
<400>14
acgttgaaaa?ctctatgtta?caggcaaata?atgataaact?ggcaccgctg?acgcgtgcga????60
cggcagacaa?caaggacgag?ctgattaagc?????????????????????????????????????90
<210>15
<211>90
<212>DNA
＜213〉artificial sequence
<400>15
tatcggtctt?cagcttcttg?ttctcaaccg?tcagttgatg?gttttgaatt?tcgtagtcat????60
tggcgcgctt?aatcagctcg?tccttgttgt?????????????????????????????????????90
<210>16
<211>90
<212>DNA
＜213〉artificial sequence
<400>16
gagaacaaga?agctgaagac?cgataaggag?cagctgacca?aagaagcatc?tcgcgaagcc????60
aagaaacagg?tggaaaaggc?attagaataa?????????????????????????????????????90
<210>17
<211>38
<212>DNA
＜213〉artificial sequence
<400>17
ccgaagcttt?tattctaatg?ccttttccac?ctgtttct????????????????????????????38
Claims (10)
1. a recombinant polypeptide is characterized in that, it has the aminoacid sequence shown in the sequence 1 in the sequence table.
2. an isolated DNA molecule is characterized in that, the described recombinant polypeptide of its coding claim 1.
3. isolated DNA molecule according to claim 2 is characterized in that, it has the nucleotide sequence shown in the sequence 2 in the sequence table.
4. the described polypeptide of claim 1 is being prepared at the application in the A group streptococcus vaccine as antigen.
5. method that produces claim 2 or 3 described dna moleculars, it is characterized in that, all can obtain the chemosynthesis of this dna molecular or the method for various pcr amplifications, can be different with its genetic codon of dna molecular that these methods obtain, but final translation product necessarily contains the aminoacid sequence of the described recombinant polypeptide of claim 1.
6. a carrier is characterized in that, it contains claim 2 or 3 described dna moleculars.
7. a host cell is characterized in that, it contains the described carrier of claim 8.
8. the preparation method of the described recombinant polypeptide of claim 1 is characterized in that, being fit to express under the described recombinant polypeptide condition, cultivates the described host cell of claim 8, separates and the described recombinant polypeptide of purifying.
9. a vaccine is characterized in that, it contains described recombinant polypeptide of claim 1 and acceptable adjuvant pharmaceutically.
10. vaccine according to claim 9 is characterized in that it also contains immunostimulant.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN200910061286A CN101845084B (en) | 2009-03-27 | 2009-03-27 | Recombination polypeptide serving as group A streptococcic tetravalent vaccine protective antigen |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN200910061286A CN101845084B (en) | 2009-03-27 | 2009-03-27 | Recombination polypeptide serving as group A streptococcic tetravalent vaccine protective antigen |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN101845084A true CN101845084A (en) | 2010-09-29 |
| CN101845084B CN101845084B (en) | 2012-10-10 |
Family
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Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN200910061286A Active CN101845084B (en) | 2009-03-27 | 2009-03-27 | Recombination polypeptide serving as group A streptococcic tetravalent vaccine protective antigen |
Country Status (1)
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|---|---|
| CN (1) | CN101845084B (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105223351A (en) * | 2014-08-18 | 2016-01-06 | 董俊 | Based on magnetic resolution and quantum dot-labeled people A group streptococcus method for quick and kit |
| CN107312075A (en) * | 2011-06-17 | 2017-11-03 | 田纳西大学研究基金会 | A group streptococcus polyvaccines |
-
2009
- 2009-03-27 CN CN200910061286A patent/CN101845084B/en active Active
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107312075A (en) * | 2011-06-17 | 2017-11-03 | 田纳西大学研究基金会 | A group streptococcus polyvaccines |
| CN107312075B (en) * | 2011-06-17 | 2021-01-12 | 田纳西大学研究基金会 | Group A streptococcus multivalent vaccine |
| CN105223351A (en) * | 2014-08-18 | 2016-01-06 | 董俊 | Based on magnetic resolution and quantum dot-labeled people A group streptococcus method for quick and kit |
| CN105223351B (en) * | 2014-08-18 | 2017-02-01 | 董俊 | Method and kit for rapidly detecting human group A streptococci based on magnetic separation and quantum dot labeling |
Also Published As
| Publication number | Publication date |
|---|---|
| CN101845084B (en) | 2012-10-10 |
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