CN101880675B - High-level expression of tetanus toxin receptor binding domain Hc in Escherichia coli and application - Google Patents
High-level expression of tetanus toxin receptor binding domain Hc in Escherichia coli and application Download PDFInfo
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Abstract
The invention relates to a method for a tetanus toxin receptor binding domain Hc to be subjected to high-level soluble expression in Escherichia coli through nucleotide sequence optimization. According to the sequencing result of a domestic C.Tetani virulent strain CMCC64008, the tetanus toxin receptor binding domain Hc sequence is analyzed and optimized, the optimized sequence is SEQ ID No.1 and the coded protein sequence is SEQ ID No.2. The synthesized Hc gene is linked into an expression vector pET32a(+) after undergoing double enzyme digestion, the recombinant Hc is subjected to high soluble expression in Escherichia coli and the target protein accounts for about 46% of the total protein in the supernatant undergoing bacteriociasis. After QFF column purification, phenyl hydrophobic column purification and SP column purification, the purity of the target protein can be more than 95% and the yield thereof is more than 300mg/L. The recombinant protein prepared by the method of the invention has good immunogenicity, can induce the mice to produce high-titre protective antibodies and can resist attack of high-dose lethal toxins. The method has extensive application prospect in large-scale high-level preparation of the tetanus toxin recombinant subunit vaccine Hc.
Description
Technical field:
The invention belongs to gene engineering technology field, relate in particular to a tetanus toxin receptor combination region Hc and proteins encoded thereof and as subunit vaccine in the application aspect the tetanus prevention.
Technical background:
Tetanus is the healthy disease of a kind of serious harm people's life; It is a kind of acute specific infection that is caused by Gram-positive, anaerobic clostridium-tetanus bacillus intrusion human body wound, growth and breeding, generation tetanus toxin; Tetanus toxin invasion neural system can cause the muscle spasm of people's general, and then general depletion or death by suffocation.All open injuries all have tetanic possibility take place.The toxicity of tetanus toxin is very strong, is only second to botulinus toxin, and the tetanus toxin of about 100ng just can the causing death.Tetanus is very serious in the third world countries and the area of vast backward.According to estimates, have every year 1000000 cases to take place in the world approximately, mortality ratio is about 50%.In developing country, the tetanus neonatorum mortality ratio is up to 90%.Seeking efficient, safe tetanus prevention and medicine is the direction that various countries endeavour.
1315 amino acid of tetanus toxin total length, molecular weight is 150kD, is made up of A, B, C three parts altogether, every part molecular weight is 50kD.The C fragment is the C end of heavy chain, has the effect that combines neurocyte.The B fragment is the N end of heavy chain, has the importing effect.The A fragment is a light chain, and protease activity is arranged, and can suppress the release of transmitter substance, has the paralysis effect.Traditional toxoid vaccine is that tetanus toxin forms through formaldehyde detoxification, aluminium adjuvant refining with adsorbents, though its immune effect is better, but still has some problems: have certain side reaction to take place behind the inoculation toxoid; Clostridium tetani can form the gemma form, and toxicity is high, and producing vaccine has certain risk; The formaldehyde treated toxoid pollutes easily; Poison Reverse possibly take place in the toxoid after the chemical treatment; And importantly duration of immunity is short, with advancing age, tetanus immunological power is descended, and children need 4～6 years booster immunizations, and the adult needs once immunity of reinforcement in per 10 years.Therefore, existing toxoid vaccine also needs further to improve and development, and the recombinant vaccine of development of new is one of direction.Experiment has confirmed that it is not essential carrying out immunity with complete lps molecule.The natural C fragment of tetanus (Hc) has kept complete toxin and many character such as Sphingolipids,sialo combine; Immunizing potency and toxin are suitable; Nontoxicity; Allergenicity is low, is the candidate that develops subunit vaccine and recombinant vaccine in the future, has been used to study a lot of aspects such as subunit vaccine, bacterium and vector-viral vaccine, mucosal vaccine etc.
Subunit vaccine with escherichia coli expression tetanus toxin has great importance, and its technology is simple, and the Hc albumen of reorganization is not worried being polluted by complete toxin; Its security is higher; Shortcomings such as but the tetanus Hc fragment ubiquity expression amount of expressing at present is lower, is prone to form inclusion body, and immunizing potency is lower; This is that this has limited its expression to a certain extent because AT content is abundant and have a large amount of intestinal bacteria rare codons in the Hc sequence.Domestic and international many investigators improve the expression of tetanus Hc section, and expression amount has obtained certain raising, but still do not reach mass-produced demand, and the Hc immunogenicity for preparing is compared also lower with traditional toxoid vaccine.
In order to address this problem, means such as adopting sequence and carrier optimization are intended in this research, to domestic tetanus virulent strain CMCC64008 strain (GeneBank No:
AF154828) the Hc fragment sequence be optimized; The subunit vaccine that has native conformation through the preparation of the solubility high expression level in intestinal bacteria; And aspects such as the immunizing dose of this vaccine, immunizing potency, protection effect are studied and further explore Tet-Hc as the subunit vaccine feasibility, for deeply preparing tetanus Hc subunit vaccine, next step lays the foundation.
Summary of the invention:
The purpose of this invention is to provide a tetanus toxin receptor combination region Hc and the application of proteins encoded in the tetanus subunit vaccine thereof that to carry out in intestinal bacteria after optimizing that highly-soluble is expressed and can excitating organism tetanus toxin be produced immanoprotection action.
Tetanus toxin receptor combination region Hc provided by the present invention, name is called Tet-Hc, is one of following nucleotide sequence:
1) dna sequence dna of SEQ ID No.1 in the sequence table;
2) dna sequence dna of SEQ ID No.2 in the code sequence tabulation;
3) with sequence table in the dna sequence dna that limits of SEQ ID No.1 have 90% above homology and have the nucleotide sequence of excitating organism tetanus toxin generation immanoprotection action;
The nucleotide sequence of the dna sequence dna hybridization that 4) under the rigorous condition of height, can limit with SEQ ID No.1 in the sequence table.
The rigorous condition of said height be 0.1 * SSPE (or 0.1 * SSC), in the solution of 0.1%SDS, hybridization and wash film under 65 ℃ of conditions.
SEQ ID No.1 in the sequence table is by 1353 based compositions, and its encoding sequence is that coding has the protein of the amino acid residue sequence of SEQ ID No.2 in the sequence table from 5 ' end 1-1353 base.
Said tetanus toxin receptor combination region Hc encoded protein Tet-Hc is one of following amino acid residue sequences:
1) the SEQ ID No.2 in the sequence table;
2) with the amino acid residue sequence of SEQ ID No.2 in the sequence table through replacement, disappearance or the interpolation of one to ten amino-acid residue and have excitating organism and tetanus toxin is produced the protein of giving birth to immanoprotection action.
SEQ ID NO.2 in the sequence table is made up of 451 amino-acid residues.
One to ten amino-acid residue of said replacement, disappearance or interpolation can make the amino-acid residue in the non-structural domain, and its change can not exert an influence to this proteic function.
The transgenic cell line and the host bacterium that contain gene Tet-Hc of the present invention all belong to protection scope of the present invention.
With pET32a is carrier, and the recombinant prokaryotic expression vector that contains tetanus toxin receptor combination region Hc Tet-Hc and thioredoxin gene Trx of structure is pET32a-Tet-Hc.
The present invention also provides a kind of method of expressing above-mentioned tetanus toxin receptor combination region Hc proteins encoded Tet-Hc.
The method of the above-mentioned tetanus toxin receptor combination region Hc of expression provided by the present invention proteins encoded Tet-Hc is that the above-mentioned recombinant expression vector that contains tetanus toxin receptor combination region Hc Tet-Hc is imported host cell, expresses obtaining tetanus toxin receptor combination region Hc proteins encoded Tet-Hc.
Said host can be intestinal bacteria, yeast, mammalian cell, insect cell or Bacillus subtilus etc., is preferably intestinal bacteria.
Said intestinal bacteria can be E.coli DH5 α, E.coli BL21 (DE3), E.coli BL21 (DE3) pLysS or E.coliTop10 etc.
Above-mentioned reorganization all can make up according to ordinary method.
Cultivation contains the substratum and the culture condition of the host cell of tetanus toxin receptor land gene Tet-Hc, all can be substratum and the culture condition of cultivating the host that sets out.Wherein, need add inductor when cultivating said recombination bacillus coli, like IPTG etc., add IPTG concentration be 0.1-1.0mM, be preferably 0.2mM, inducing temperature is 16-37 ℃, is preferably 28 ℃, induction time is 2-8h, is preferably 6h.
The invention provides the purification process of a kind of tetanus toxin receptor combination region Hc proteins encoded Tet-Hc, can pass through the no label protein that QFF post, phenyl drainage column and three step of SP post purifying obtain higher degree.
The coded albumen of tetanus receptor combination region Hc of the present invention can be used for preparing the tetanus toxin subunit vaccine.
When needing, gene or the protein that can also incorporate one or more cytokines such as granular leucocyte-macrophage colony stimulant factor (GM-CSF), interferon-(IFN-γ), interleukin-22 (IL-2), TGF-β 4 with the tetanus subunit vaccine of tetanus toxin receptor combination region Hc proteins encoded preparation are as the molecular immune adjuvant.
Vaccine of the present invention can be processed various ways such as injection liquid, dry powder injection or sprays.The medicine of above-mentioned various formulations all can be according to the ordinary method preparation of pharmaceutical field.
The consumption of above-mentioned subunit vaccine can adopt the routine dose of subunit vaccine in the pharmaceutical field, like 1-10 μ g, and can adjust according to practical situation.
The invention provides and a kind ofly make tetanus toxin recombiant vaccine Hc in intestinal bacteria, obtain the method that highly-soluble is expressed through nucleotide sequence optimization.According to the sequencing result of domestic clostridium tetani C.Tetani virulent strain CMCC64008 strain (GeneBank No:
AF154828); Tetanus toxin Hc sequence to 451Aa is carried out analysis optimization, adopts intestinal bacteria codon replacement commonly used rare codon, and makes the AT content of optimizing the back sequence be reduced to 52.47% by 72.57%; Sequence after the optimization such as SEQ ID No.1, its encoded protein sequence such as SEQ ID No.2.Add EcoRI and XhoI restriction enzyme site respectively at the two ends of Hc sequence, carry out full gene and synthesize.Gene after synthetic connects into behind double digestion among the expression vector pET32a (+); Reorganization Hc has obtained the solubility high expression level in e. coli bl21 (DE3); Target protein accounts for the about 46% of broken bacterium supernatant total protein, and can take place specific the combination with tetanus horse toxinicide.Behind QFF post, phenyl drainage column and three step of SP post purifying, target protein purity can reach more than 95%, and yield is more than 300mg/L.Recombinant protein through method preparation of the present invention has good immunogenicity, can produce high titre protection antibody by inducing mouse, resists the attack of high dosage lethal toxin.The present invention has broad application prospects in the extensive efficient production of tetanus toxin recombinant subunit Hc.
Below in conjunction with specific embodiment the present invention is explained further details.
Description of drawings
Fig. 1 is that the SDS-PAGE protein electrophoresis figure and the western-blot of the solubility expression of tetanus toxin receptor land Hc albumen in e. coli bl21 (DE3) identifies figure.Wherein M is the molecular weight of albumen standard; 1 for expressing the broken bacterium supernatant of BL21 (DE3) of Hc; 2 is the broken bacterium supernatant of empty carrier.
Fig. 2 is for going on foot the SDS-PAGE protein electrophoresis figure of the Hc that obtains behind the purifying through QFF post, phenyl drainage column and SP post three.
Fig. 3 is the antibody titers that detects behind the Hc protein immunization BALB/c mouse of various dose in the serum.
Fig. 4 be in various degree the dilution the Hc immunity after serum to Tet-Hc and ganglioside GT1b bonded restraining effect.
Embodiment:
The sequence optimisation of embodiment one, tetanus toxin subunit vaccine Hc,
Sequencing result (GeneBank sequence number: AF154828) according to 64008 strains of domestic clostridium tetani C.Tetani virulent strain; The Tet-Hc sequence of 451Aa is carried out analysis optimization (sequence is following before and after optimizing); Adopt intestinal bacteria codon replacement commonly used rare codon, and make the AT content of optimizing the back sequence be reduced to 52.47% by 72.57%.Add EcoRI and XhoI restriction enzyme site respectively at the two ends of Tet-Hc sequence, and before initiator codon, add the TAA terminator codon, carry out full gene and synthesize to stop the upward proteic expression of Trx of carrier pET32a (+).
Compare before and after the Hc sequence optimisation:
AAAAATCTGGATTGTTGGGTTGATAATGAAGAAGATATAGATGTTATATTAAAAAA GAGT before optimizing
Optimize back AAAAACCTTGATTGTTGGGTCGACAACGAAGAAGACATCGATGTTATCCTGAAAAA GTCT
ACAATTTTAAATTTAGATATTAATAATGATATTATATCAGATATATCTGGGTTTAA TTCA before optimizing
Optimize back ACCATTCTGAACTTGGACATCAACAACGATATTATCTCCGACATCTCTGGTTTCAA CTCC
TCTGTAATAACATATCCAGATGCTCAATTGGTGCCCGGAATAAATGGCAAAGCAAT ACAT before optimizing
Optimize back TCTGTTATCACATATCCAGATGCTCAATTGGTGCCGGGCATCAACGGCAAAGCTAT CCAC
TTAGTAAACAATGAATCTTCTGAAGTTATAGTGCATAAAGCTATGGATATTGAATA TAAT before optimizing
Optimize back CTGGTTAACAACGAATCTTCTGAAGTTATCGTGCACAAGGCCATGGACATCGAATA CAAC
GATATGTTTAATAATTTTACCGTTAGCTTTTGGTTGAGGGTTCCTAAAGTATCTGC TAGT before optimizing
Optimize back GACATGTTCAACAACTTCACCGTTAGCTTCTGGCTGCGCGTTCCGAAAGTTTCTGC TTCC
CATTTAGAACAATATGACACAAATGAGTATTCAATAATTAGCTCTATGAAAAAATA TAGT before optimizing
Optimize back CACCTGGAACAGTACGACACTAACGAGTACTCCATCATCAGCTCTATGAAGAAATA CTCC
CTATCAATAGGATCTGGTTGGAGTGTATCACTTAAAGGTAATAACTTAATATGGAC TTTA before optimizing
Optimize back CTGTCCATCGGCTCTGGTTGGTCTGTTTCCCTGAAGGGTAACAACCTGATCTGGAC TCTG
AAAGATTCCGCGGGAGAAGTTAGACAAATAACTTTTAGGGATTTATCTGATAAATT TAAT before optimizing
Optimize back AAAGACTCCGCGGGCGAAGTTCGTCAGATCACTTTCCGCGACCTGTCTGACAAGTT CAAC
GCTTATTTAGCAAATAAATGGGTTTTTATAACTATTACTAATGATAGATTATCTTC TGCT before optimizing
Optimize back GCGTACCTGGCTAACAAATGGGTTTTCATCACTATCACTAACGATCGTCTGTCTTC TGCT
AATTTGTATATAAATGGAGTACTTATGGGAAGTGCAGAAATTACTGGTTTAGGAGC TATT before optimizing
Optimize back AACCTGTACATCAACGGCGTTCTGATGGGCTCCGCTGAAATCACTGGTCTGGGCGC TATC
AGAGAGGATAATAATATAACATTAAAACTAGATAGATGTAATAATAATAATCAATA CGTT before optimizing
Optimize back CGTGAGGACAACAACATCACTCTTAAGCTGGACCGTTGCAACAACAACAACCAGTA CGTA
TCTATTGATAAATTTAGGATATTTTGCAAAGCATTAAATCCAAAAGAGATTGAAAA ATTA before optimizing
Optimize back TCCATCGACAAGTTCCGTATCTTCTGCAAAGCACTGAACCCGAAAGAGATCGAAAA ACTG
TACACAAGTTATTTATCTATAACCTTTTTAAGAGACTTCTGGGGAAACCCTTTACG ATAT before optimizing
Optimize back TATACCAGCTACCTGTCTATCACCTTCCTGCGTGACTTCTGGGGTAACCCGCTGCG TTAC
GATACAGAATATTATTTAATACCAGTAGCTTATAGTTCTAAAGATGTTCAATTGAA AAAT before optimizing
Optimize back GACACCGAATATTACCTGATCCCGGTAGCTTACAGCTCTAAAGACGTTCAGCTGAA AAAC
ATAACAGATTATATGTATTTGACAAATGCGCCATCGTATACTAACGGAAAATTGAA TATA before optimizing
Optimize back ATCACTGACTACATGTACCTGACCAACGCGCCGTCCTACACTAACGGTAAACTGAA CATC
TATTATAGAAGGTTATATAGTGGACTAAAATTTATTATAAAAAGATATACACCTAA TAAT before optimizing
Optimize back TACTACCGACGTCTGTACAGCGGCCTGAAATTCATCATCAAACGCTACACTCCGAA CAAC
GAAATAGATTCTTTTGTTAGATCAGGTGATTTTATTAAATTATATGTATCATATAA CAAT before optimizing
Optimize back GAAATCGATTCTTTCGTTCGCTCTGGTGACTTCATCAAACTGTACGTTTCTTACAA CAAC
AATGAGCACATTGTAGGTTATCCGAAAGATGGAAATGCCTTTAATAATCTTGATAG AATT before optimizing
Optimize back AACGAACACATCGTTGGTTACCCGAAAGACGGTAACGCTTTCAACAACCTGGACAG AATC
CTAAGAGTAGGTTATAATGCCCCAGGTATCCCTCTTTATAAAAAAATGGAAGCAGT AAAA before optimizing
Optimize back CTAAGAGTAGGTTACAACGCTCCGGGTATCCCGCTGTACAAAAAAATGGAAGCTGT TAAA
TTGCGTGATTTAAAAACCTATTCTGTACAACTTAAATTATATGATGATAAAGATGC ATCT before optimizing
Optimize back CTGCGTGACCTGAAAACCTACTCTGTTCAGCTGAAACTGTACGACGACAAAGATGC TTCT
TTAGGATTAGTAGGTACCCATAATGGTCAAATAGGCAACGATCCAAATAGGGATAT ATTA before optimizing
Optimize back CTGGGTCTGGTTGGCACCCACAACGGTCAGATCGGTAACGACCCGAACCGTGACAT CCTG
ATTGCAAGCAACTGGTACTTTAATCATTTAAAAGATAAAACTTTAACATGTGATTG GTAC before optimizing
Optimize back ATCGCTTCTAACTGGTACTTCAACCACCTGAAAGACAAAACCCTGACCTGCGACTG GTAC
TTTGTACCTACAGATGAAGGATGGACAAATGAT before optimizing
Optimize back TTCGTTCCGACCGATGAAGGTTGGACCAACGAC
Embodiment two, tetanus toxin subunit vaccine Hc expression vector establishment
With optimize Tet-Hc gene after synthetic with EcoRI and XhoI double digestion after, connect into same with on the expression vector pET32a (+) behind EcoRI and the XhoI double digestion, transformed into escherichia coli competent cell DH5 α, 37 ℃ of overnight cultures.Next day, the picking mono-clonal was in 5mL LB (Amp+) substratum, and 37 ℃, 220rpm cultivates 12h, extracted plasmid, the insertion of EcoRI and XhoI double digestion evaluation goal gene, and send order-checking.The plasmid called after pET32a-Tet-Hc that checks order correct.
The escherichia coli expression of embodiment three, tetanus toxin recombinant subunit vaccine Hc and western-blot identify
Correctly connect into pET32a (+) carrier of Tet-Hc gene; Transformed into escherichia coli competent cell BL21 (DE3), picking mono-clonal in 5mL LB (Amp+) liquid nutrient medium, 37 ℃; When 220rpm is cultured to OD600nm ≌ 0.6; Adding final concentration is the IPTG of 0.2mM, and 28 ℃, 220rpm continues to cultivate 6h.5,000g, 4 ℃ of centrifugal collection thalline are with the ultrasonic broken bacterium in the resuspended back of PBS.12,000g, 4 ℃ of centrifuging and taking supernatants, row SDS-PAGE electrophoresis is contrast with the empty carrier expression product, identifies that size is the proteic expression of Hc of 50KD.
Western-blot analyzes Tet-Hc and combines with the specificity of mouse-anti tetanus monoclonal antibody.After will containing proteic ultrasonic broken bacterium supernatant of Tet-Hc and the capable SDS-PAGE protein electrophoresis of the broken bacterium supernatant of empty carrier; Albumen is transferred on the nitrocellulose filter; Behind the PBS room temperature sealing 1h that contains 3%BSA, add the mouse anti tetanus monoclonal antibody of dilution in 1: 1000,37 ℃ of reaction 1h.With film with PBST washing 4 times after, the sheep anti-mouse igg two of horseradish enzyme labelling that adds dilution in 1: 5000 is anti-, the PBST washing is 4 times behind 37 ℃ of reaction 1h, the colour developing of employing chemoluminescence method, compressing tablet, exposure.
Can see from the experimental result of Fig. 1; Compare with the empty carrier contrast; Conversion has the bacterial strain of pET32a-tet-Hc plasmid that tangible band is arranged about 50KD; Western-bolt result confirms that this albumen can take place specific the combination with mouse-anti tetanus monoclonal antibody, explains that this band is Hc purpose band.Account for 46% of broken bacterium supernatant total protein through thin layer scanning analysis purposes albumen, explain that Tet-Hc albumen has obtained the highly-soluble expression in intestinal bacteria.
Escherichia coli fermentation and the purifying of embodiment four, tetanus toxin subunit vaccine Hc
Express the proteic seed liquor 1L of Hc, switching is gone in the 30L fermentor tank, and when being cultured to OD600nm ≌ 0.6, adding final concentration is the IPTG of 0.2mM, and 28 ℃, 300rpm continues to cultivate 6h.10,000g, 4 ℃ of centrifugal collection thalline are with the broken bacterium of the resuspended back homogenate of 20mM Tris-HCl damping fluid (pH 8.5).20,000g, 4 ℃ of centrifugal collection supernatants.Supernatant is crossed the QFF post and is carried out anionresin, after 20mM Tris-HCl damping fluid (pH 8.5) balance, carries out gradient elution with the Tris-HCl damping fluid that contains 0-0.5M NaCl.The elutriant adding final concentration that contains target protein is the (NH of 0.5M
4)
2SO
4After, cross the phenyl drainage column and carry out next step purifying.Target protein is with (the NH that contains concentration 0.5-0M
4)
2SO
4The Tris-HCl damping fluid carry out gradient elution.The elutriant that contains target protein is crossed the SP post and is carried out cationic exchange after using desalting column displacement damping fluid as 20mM NaAc (pH4.0), and target protein carries out gradient elution with 20mM NaAc (pH 4.0) damping fluid that contains 0-0.5M NaCl.Behind three step purifying, the target protein Hc of no label has obtained good purifying, and purity can reach (Fig. 2) more than 95%, and yield is more than 300mg/L, and target protein finally is kept in the PBS damping fluid.
The immunogenicity research of embodiment five, Tet-Hc
Hc albumen 1 μ g, 5 μ g and the 10 μ g/ of various dose only; Immunity 6-8 Balb/c mouse in age in week; Per two all immunity once immune three times altogether, are adopted the Fu Shi Freund's complete adjuvant for the first time; Second adopts freund 's incomplete adjuvant for the third time, gets blood examination before each immunity two weeks of back, the immunity next time and surveys the antibody titers in the serum; The mensuration of antibody titers adopts the method for ELISA, Toxoid,tetanus 1 μ g/mL, and 100 μ L/ holes encapsulate 96 hole elisa plates, and 4 ℃ encapsulate and spend the night.PBST washing 4 times, 5min/ time.Antiserum(antisera) successively behind the doubling dilution, is added in the elisa plate from 1: 100 with PBST, 37 ℃ of reaction 1h, the serum of establishing simultaneously after the PBS immunity is contrast.PBST washing 4 times, 5min/ time.Add the anti-mouse mouse two of HRP-and resist 37 ℃ of reaction 30-40min.Add TMB colour developing liquid, 50 μ L/ holes, 2M H is used in the colour developing back
2SO
4Stop, ELIASA 450nm/ measures absorbance value.Colour developing value to add the negative control sera hole is contrast, with OD450nm greater than 0.1 and be higher than 2 times of positive dilution titers of negative hole.Can see that from experimental result the antibody titers of Hc group more than 000, all is higher than bibliographical information basically at 1: 200.And antigenic immunizing dose and antibody titers are irrelevant basically, but the Hc of 1 μ g just inducing mouse produce higher antibody titers.
The extracorporeal biology activity research of embodiment six, immunity back serum
Ganglioside GT1b is that tetanus toxin passes through one of Hc structural domain and neurocyte bonded acceptor, and our method through competitive ELISA has detected serum behind the Hc immune mouse that contains neutrality antibody to Tet-Hc and GT1b bonded restraining effect.With GT1b use methyl alcohol be diluted to final concentration be behind the 2 μ g/mL 4 ℃ encapsulate 96 hole elisa plates and spend the night.Mice serum after the Hc immunity done after the dilution in various degree to mix at 37 ℃ with the Tet-Hc of isopyknic 2 μ g/mL with PBST hatch 30min; Add and be coated with GT1b and washed 96 orifice plates; 37 ℃ of reaction 1h, with PBST and Tet-Hc mix aperture as contrast.After the PBST washing 4 times, add people's tetanus polyvalent antibody of dilution in 1: 100,100 μ L/ holes, 37 ℃ of reaction 1h.PBST washing 4 times, the goat anti-human igg two who adds the horseradish peroxidase-labeled of dilution in 1: 5000 resists, and behind 37 ℃ of reaction 40min, adds the TMB colour developing, and uses 2M H
2SO
4Stop, OD450nm reads light absorption value.Calculate inhibiting rate by following formula: inhibiting rate=(1-test holes OD450nm/Hc control wells OD450nm) * 100%
Can find out in various degree that from the experimental result of Fig. 4 the immune serum of dilution all can suppress combining of Tet-Hc and acceptor ganglioside GT1b to some extent, explain to contain in the mice serum after the Tet-Hc immunity to have the active neutrality antibody of extracorporeal biology.Along with the increase of serum diluting multiple, neutralizing antibody content reduces gradually, and restraining effect weakens gradually.
Embodiment seven, Hc albumen are as the protection effect of subunit vaccine to mouse
Carry out tetanus toxin after three immunity and attack experiment, with 2 * 10
3LD
50The tetanus toxin of dosage is dissolved in the borate buffer solution of 0.5mL, and abdominal injection is attacked poison, observes Hc albumen as the protection effect of subunit vaccine to mouse.Can find out the Hc immune mouse 3 times of various dose from the experimental result of table one after, all can protect mouse to resist 2 * 10 fully
3LD
50The attack of the tetanus toxin of dosage explains that Tet-Hc can bring out mouse in vivo as subunit vaccine and produce a large amount of neutrality antibodies, has preventive effect well.
Behind the Hc subunit vaccine of the table 1 various dose immunity BALB/c mouse to the protective effect in vivo of mouse
SEQ?ID?No.1
< 110>Microbiology and Epidemic Disease Inst., Academy of Military-Medical Sciences (C
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< 110>Microbiology and Epidemic Disease Inst., Academy of Military-Medical Sciences (C
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KNLDCWVDNE?EDIDVILKKS?TILNLDINND?IISDISGFNS?SVITYPDAQL?VPGINGKAIH 60
LVNNESSEVI?VHKAMDIEYN?DMFNNFTVSF?WLRVPKVSAS?HLEQYDTNEY?SIISSMKKYS 120
LSIGSGWSVS?LKGNNLIWTL?KDSAGEVRQI?TFRDLSDKFN?AYLANKWVFI?TITNDRLSSA 180
NLYINGVLMG?SAEITGLGAI?REDNNITLKL?DRCNNNNQYV?SIDKFRIFCK?ALNPKEIEKL 240
YTSYLSITFL?RDFWGNPLRY?DTEYYLIPVA?YSSKDVQLKN?ITDYMYLTNA?PSYTNGKLNI 300
YYRRLYSGLK?FIIKRYTPNN?EIDSFVRSGD?FIKLYVSYNN?NEHIVGYPKD?GNAFNNLDRI 360
LRVGYNAPGI?PLYKKMEAVK?LRDLKTYSVQ?LKLYDDKDAS?LGLVGTHNGQ?IGNDPNRDIL 420
IASNWYFNHL?KDKTLTCDWY?FVPTDEGWTN?D 451
Claims (5)
1. the tetanus toxin receptor combination region Hc is the dna sequence dna of SEQ ID No.1 in the sequence table.
2. contain the said expression carrier of claim 1.
3. the transgenic cell line that contains the said gene of claim 1.
4. method of expressing the said tetanus toxin receptor combination region Hc of claim 1 proteins encoded; Be that the recombinant expression vector that will contain the said tetanus toxin receptor combination region Hc of claim 1 imports host cell, express obtaining tetanus toxin receptor combination region Hc coding soluble proteins.
5. the application of the described tetanus toxin receptor combination region Hc of claim 1 in preparation tetanus toxin subunit vaccine.
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| WO2011143557A2 (en) * | 2010-05-14 | 2011-11-17 | The Children's Hospital Of Philadelphia | Humanized ttc and methods of use thereof |
| CN105542004B (en) * | 2016-01-12 | 2019-02-19 | 中国人民解放军军事医学科学院生物工程研究所 | A kind of neutralizing monoclonal antibody of anti-tetanus toxin and its application |
| CN106520870B (en) * | 2016-12-29 | 2019-12-13 | 四川自豪时代药业有限公司 | High-density fermentation method for exogenously expressing tetanus toxin receptor binding region Hc |
| CN110025779A (en) * | 2019-04-29 | 2019-07-19 | 中国兽医药品监察所 | A kind of nontoxicity clostridium tetani genetic engineering subunit vaccine and its production method |
| CN109942718B (en) * | 2019-04-29 | 2020-12-25 | 中国兽医药品监察所 | Non-toxic tetanus toxin and clostridium perfringens beta toxin recombinant fusion protein |
| CN110041437B (en) * | 2019-04-29 | 2020-12-25 | 中国兽医药品监察所 | Non-toxic tetanus toxin and novyi alpha toxin recombinant fusion protein |
| CN111909265B (en) * | 2020-08-25 | 2022-02-11 | 中国人民解放军军事科学院军事医学研究院 | Humanized antibody combined with tetanus toxin heavy chain C-terminal domain and application |
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| Adriana V Ribas等.High-level expression of tetanus toxin fragment C-thioredoxin fusion protein in Escherichia coli.《Biotechnology and applied biochemistry》.2000,第31卷(第2期),91-94. * |
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