CN101845084B - 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 dna sequence dna of this recombinant polypeptide of encoding; The carrier that contains this dna sequence dna; The host cell that contains this carrier, the method for this recombinant polypeptide of DNA recombinant technology preparation, 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 through modes such as the air spittle, skin exposure 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; Research shows that untreated children's pharyngitis of about 3% possibly 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 microbiotic is widely used, the A group streptococcus infects in some developing countries still very serious.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 has caused serious economical load for patient family and society with lifelong medical expense.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 to 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 possibly 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; 41:1150 1156) therefore; With M albumin A district is that vaccine that basic design is directed against A group streptococcus different serotypes had both been avoided the zone that human body produces cross reaction; Can make body produce protection antibody again, be a feasible method of A group streptococcus vaccine development.
Research shows, the M albumen of A group streptococcus is the heterodimeric protein that is made up of two polypeptied chains that are entirely α-Luo Xuanjiegou, and space structure is a linear molecule.Therefore, its epitope should be linear epitope, does not have the complex spatial 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 manual work 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 Guangdong Province and other some areas of China the result of the M albumen emm gene type of isolating 104 strain A group streptococcus; 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 aminoacid sequence and the recombinant polypeptide that one section total conserved amino acid sequence of this four serotype M PROTEIN C ends is formed by connecting of the type specificity of human body cross reaction epi-position.
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 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, the NH from the left side
2The COOH end is followed successively by end: 1-50 amino acid of A group streptococcus M1 type M protein type specific amino acid to the right side; 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 second aspect of the present invention, the preparation method of a kind of isolated DNA molecule and this dna molecular is provided.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 through 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.Its genetic codon of dna molecular with these methods obtain maybe be different, but the final translated product product necessarily contains the aminoacid sequence of recombinant polypeptide of the present invention.
In the third aspect of the 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, a kind of a kind of method that produces recombinant polypeptide of the present invention is provided.It comprises step: be fit to express under the said recombinant polypeptide condition; Cultivate above-mentioned host cell; Thereby give expression to recombinant polypeptide or contain the recombinant protein of recombinant polypeptide aminoacid sequence, through 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 said recombinant polypeptide of claim 1 and acceptable adjuvant pharmaceutically, also can contain some immunostimulants.Adjuvant is like white lake, phosphagel phosphaljel, 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 mikrobe 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.Relation between the two is equal to the emm1 type for the M1 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 a1.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, the nucleotide sequence of then code book being invented recombinant protein is cloned after the expression regulation sequence that into carrier provides, and 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 instance 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 to 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; The result finds; The rabbit immune serum only with on the recombinant polypeptide comprises A group streptococcus M1 type, M3 type, the reaction of M6 type of serotype, and 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 to these serotypes A group streptococcus antibody respectively; Carry out the extracorporeal disinfecting experiment; The result finds 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 recombinant gene; 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 that produces to 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:DL2000 marker.
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 the intestinal bacteria M15 that has the 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 to different serotypes A group streptococcus sterilizing rate.
Embodiment
Further set forth the present invention below in conjunction with specific examples.These instances only are used to explain the present invention; Rather than limit scope of the present invention; The experimental technique of unreceipted concrete actual conditions in the following instance; 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.
The synthetic cover oligonucleotide sequence that is somebody's turn to do is through 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× |
10 |
| dNTP Mix(10μM) | 4 |
| primeSTAR TMHS DNA polymerase(2.5U/ml) | 0.5 |
| ddH2O | 15.5 |
| The reaction TV | 50 |
Reaction conditions is 95 ℃ of preparatory 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 |
2 |
| |
2 |
| |
2 |
| 5× |
10 |
| dNTP Mix(10μM) | 4 |
| primeSTAR TMHS DNA polymerase(2.5U/ml) | 0.5 |
| ddH2O | 29.5 |
| The reaction TV | 50 |
Reaction conditions is 95 ℃ of preparatory 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.
Through two-step pcr, successfully amplify a dna fragmentation about 600bp, identical with expected design.(Fig. 2)
The PCR product purified with BamH I and HindIII double digestion after rear clone in escherichia coli cloning carrier PUC18, structure recombinant clone plasmid, called after PUC18-Strep4 (Fig. 3).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 instance 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 cultures.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, PQE30 plasmid of recombinant polypeptide, and molecular weight is 23.8KD.
The Ni of recombinant polypeptide
2+Affinitive layer purification
With the bacterial sediment that instance 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 purified recombinant polypeptide 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/inferior.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 purified recombinant polypeptide vaccine and the emulsification of isopyknic Freund's complete adjuvant thorough mixing with instance 4 acquisitions, 3 of the healthy male rabbit about subcutaneous multi-point injection 2Kg, 2ml/ is only.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.Before initial immunity, with immunity back per two all tame rabbit ear vein blood samplings for the first time, obtain immune serum, to detect the variation of immunity back antibody titers, 12 weeks prepared immune serum in a large number with immunizing rabbit carotid atery sacrificed by exsanguination after immunity.EUSA (ELISA) detects the different time points of immunity, the titre changing conditions of anti-recombinant polypeptide vaccine IgG 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 |
| 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.
Can find out that from detected result three rabbit have all produced the antibody to recombinant polypeptide of high titre behind recombinant immunogenic polypeptide, explain that recombinant polypeptide has better immunogenicity.
Antibodies specific detects behind the recombinant immunogenic polypeptide
The immune serum of 3 rabbit that obtain with instance 5 and preimmune serum carry out indirect immunofluorescence experiment with A group streptococcus M1 type, M3 type, M6 type, M18 type and M5 type (bacterium numbering is respectively 32171,32172,32175,32180,32174) from the purchase of 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 deckglass 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 to this serotypes 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.
Can find out that from detected result three rabbit have all produced to A group streptococcus M1, M3, the proteic type specificity antibody of M6 type M behind recombinant immunogenic polypeptide.
The extracorporeal disinfecting antibody experiment of recombinant immunogenic polypeptide rabbit anteserum
M1 type used in the instance 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 culture bacteria liquid 0.1ml overnight that gets each serotypes A group streptococcus is diluted to 10 for continuous 10 times
-5, with the bacterium liquid after the 50ul dilution, immune serum of 3 rabbit that 100ul instance 5 obtains or preimmune serum (deactivation complement) and 350ul normal adults anticoagulated whole blood (preliminary experiment confirms that this blood does not contain to experiment with serotypes A group streptococcus bactericidin) thorough mixing; 37 ℃ of vibrations were hatched 3 hours; The preimmune serum contrast is established in each experiment, hatch 3 hours after, get 50ul mixtures incubated coating rabbit blood agar plate; 37 ℃ of incubated overnight; Add up each dull and stereotyped bacterium colony number that goes up next day, if the dull and stereotyped colony count 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
Can find out that from detected result three rabbit have all produced the bactericidin to 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.
The amino acid and the nucleotides sequence tabulation that below relate to for patented claim 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>/
<160>17
<170>PatentIn version 3.5
<210>1
<211>194
<212>PRT
< 213>manual work
<400>1
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>manual work
<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 said polypeptide of claim 1 is directed against the application in the A group streptococcus vaccine as antigen in preparation.
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; Its genetic codon of dna molecular with these methods obtain can be different, but the final translated product 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 6.
8. the preparation method of the said recombinant polypeptide of claim 1 is characterized in that, being fit to express under the said recombinant polypeptide condition, cultivates the described host cell of claim 7, separates and the described recombinant polypeptide of purifying.
9. a vaccine is characterized in that, it contains said recombinant polypeptide of claim 1 and acceptable adjuvant pharmaceutically.
10. vaccine according to claim 9 is characterized in that it also contains immunostimulant.
Priority Applications (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 |
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 |
|---|---|
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| CN101845084B true CN101845084B (en) | 2012-10-10 |
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| Application Number | Title | Priority Date | Filing Date |
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| CN200910061286A Active CN101845084B (en) | 2009-03-27 | 2009-03-27 | Recombination polypeptide serving as group A streptococcic tetravalent vaccine protective antigen |
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| CN (1) | CN101845084B (en) |
Families Citing this family (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US8697085B2 (en) * | 2011-06-17 | 2014-04-15 | University Of Tennessee Research Foundation | Group A Streptococcus multivalent vaccine |
| 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 |
-
2009
- 2009-03-27 CN CN200910061286A patent/CN101845084B/en active Active
Non-Patent Citations (7)
| Title |
|---|
| .《A群与B群链球菌疫苗研制近展》.《国外医学.预防.诊断.治疗用生物制品分册》.2004, |
| .《A群链球菌的研究进展》.《国外医学(微生物学分册) 》.1999, |
| 方平楚 |
| 罗海波 |
| 罗海波;方平楚;.《A群链球菌的研究进展》.《国外医学(微生物学分册) 》.1999, * |
| 黄雪萍 |
| 黄雪萍;.《A群与B群链球菌疫苗研制近展》.《国外医学.预防.诊断.治疗用生物制品分册》.2004, * |
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|---|---|
| CN101845084A (en) | 2010-09-29 |
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