CN102199611B - Clostridium difficile exotoxin A carboxy-terminal gene sequence with optimized codon and nucleic acid vaccine thereof - Google Patents

Abstract

The invention belongs to the technical field of biomedicine, and relates to a Clostridium difficile exotoxin A carboxy-terminal gene fragment with an optimized codon and a nucleic acid vaccine thereof. In the gene sequence with the optimized codon, the codon usage biases of mammalian cells and Escherichia coli are considered; and the Clostridium difficile vaccine consists of the exotoxin A carboxy-terminal gene sequence with the optimized codon and a eukaryotic expression vector pJW4303. The Clostridium difficile exotoxin A carboxy-terminal gene fragment with the optimized codon can be used for constructing the nucleic acid vaccine, effectively stimulates an immune system of a host after a mammal is immunized so as to produce better humoral immune response, is also applicable to the prokaryotic expression of a protein in the Escherichia coli, and lays a foundation for the batch acquisition of the protein.

Description

Codon optimized clostridium difficile exotoxin A carboxy-terminal gene sequence and nucleic acid vaccine thereof

Technical field

The invention belongs to the biological medicine technology field, relate to codon optimized clostridium difficile exotoxin A carboxy-terminal gene fragment and nucleic acid vaccine thereof.

Background technology

Clostridium difficile is a kind of Gram-positive, the bacillus fusiformis of brood cell, obligate anaerobic is arranged.Current, the prevention of C. difficile infection, diagnosis, treatment are faced with numerous difficulties.On the one hand, clostridium difficile extensively is present in the institute in the environment, and can tolerate sterilizing agent commonly used in the multiple institute as the brood cell of its dormancy form, and it has become the main pathogens of the ward infection diarrhoea that is only second to Campylobacter; Metronidazole, vancomycin, as two kinds of main medicines for the treatment of C. difficile infection, their resistance is also constantly being accumulated, is being propagated, and curative effect reduces increasingly, and for the treatment of recurrent cases, these two kinds of medicines present incompetent state.In addition on the one hand, up to the present, there is no effective, commercial vaccine and come out; ELISA detection kit based on toxin is expensive, is difficult to clostridium difficile is carried out routine clinical detection, also is not suitable for large-scale epidemiology survey and long term monitoring; Therapeutic antibodies is as a kind of safe, effective, cheap Substitutes For Antibiotic, and having a bright future but still is in probe phase.So, be badly in need of exploitation safety, effectively, convenient, cheap clostridium difficile vaccine protects the high risk population; Simultaneously, strive in the corresponding monoclonal antibody of the basis of vaccine exploitation, for diagnostic reagent and therapeutic antibodies are laid solid foundation.

Clostridium difficile enters digestive tube by fecal-oral route, then secrete extracellular toxin and cause diarrhoea.Wherein, toxin A is a kind of enterotoxin, is again a kind of cytotoxin, is the Major Virulence Factors that clostridium difficile causes a disease.Research finds, TcdA only has when its carboxyl terminal can bring into play toxic action after the target cell surface receptor is combined; The molecular surface structure of TcdA is most of to be covered by its carboxyl terminal; For the antibody of clostridium difficile exotoxin A carboxy-terminal, energy establishment its intestines toxicity and cytotoxicity have protective effect.So clostridium difficile exotoxin A carboxy-terminal is the ideal structure territory of vaccine research and antibody exploitation.

Nucleic acid vaccine (nucleic vaccine), have another name called gene vaccine (gene vaccine) or dna vaccination (DNA vaccine), its essence is that the carrier for expression of eukaryon that contains antigen gene is imported into and is absorbed by zooblast behind the animal body and express corresponding antigen protein, thereby induces body to the immune response of this albumen.As emerging in recent years a kind of vaccine, it is attracting people with its unique advantage: (1) antigen is synthetic similar to the natural infection of pathogenic agent with the submission process, by MHC I class and the direct submission immunity system of II quasi-molecule.The particularly immune response of inducing producing specificity CD8+ lymphocyte (CTL), this is that inactivated vaccine and subunit vaccine can not be compared; (2) immunogenic unicity.Only have the antigen gene on the carrier to obtain expressing in cell, itself does not have antigenicity carrier.And the virus live vector vaccine of restructuring also has the many albumen of himself to obtain expressing except destination gene expression; (3) be easy to make up and prepare, good stability, with low cost, be suitable for scale operation.But the goal gene overwhelming majority who is used at present making up nucleic acid vaccine comes from the prokaryotic organism such as virus or bacterium, and the research of vaccine and application are mainly eukaryote, such as mouse, and macaque or the senior Mammals such as human.Because prokaryotic organism and eukaryote there are differences at codon usage bias; the foreign gene that causes making up nucleic acid vaccine can not effective expression in the mammalian hosts body, therefore just effectively the immunity system of stimulation of host produce preferably immanoprotection action.This is to cause the lower major cause of present nucleic acid vaccine immunity originality.

Summary of the invention

The object of the invention is to overcome defects, a kind of codon optimized clostridium difficile exotoxin A carboxy-terminal gene sequence is provided.

Another object of the present invention provides a kind of clostridium difficile extracellular toxin nucleic acid vaccine that is made up by said gene.

Purpose of the present invention is achieved through the following technical solutions:

A kind of codon optimized clostridium difficile exotoxin A carboxy-terminal gene sequence, sequence are SEQ ID NO.1

A kind of clostridium difficile extracellular toxin nucleic acid vaccine is that clostridium difficile exotoxin A carboxy-terminal gene sequence and the carrier for expression of eukaryon of SEQ ID NO.1 forms by sequence.

Described carrier for expression of eukaryon is pJW4303.

The construction step of codon optimized clostridium difficile exotoxin A carboxy-terminal gene sequence provided by the invention and nucleic acid vaccine thereof is as follows:

(1) design of codon optimized clostridium difficile exotoxin A carboxy-terminal gene sequence and synthetic

At first choose 978 base sequences in TcdA gene carboxyl terminal end, then use the gene order of software MacVector7.2 analysis of encoding, find out its codon usage bias and find out simultaneously from the Mammals codon usage bias, use the different codon site of preference with e. coli codon.Use the identical codon of preference for Mammals with intestinal bacteria, with Mammals and intestinal bacteria all the codon of preference substitute and use the different codon of preference in the TcdA gene, then design codon optimized clostridium difficile exotoxin A carboxy-terminal sequence, and obtained codon optimized clostridium difficile exotoxin A carboxy-terminal gene sequence through the chemosynthesis of genome company.The protein amino acid sequence that codon optimized sequence is coded and original aminoacid sequence are consistent.For example: the triplet codon of coding Isoleucine Ile mainly is ATT in the TcdA gene, and in the senior mammalian genes groups such as the mankind, mouse, mainly be ATC, in intestinal bacteria, mainly be ATC also, when codon optimized, can select all codon ATC of preference of mammalian cell and intestinal bacteria.Clostridium difficile exotoxin A carboxy-terminal gene sequence after the optimization is SEQ ID NO.1, and the sequence before optimizing is SEQ ID NO.2.

(2) the recombinant vectors pMK-RQ-TcdA-C that contains target sequence that genome company is provided carries out enzyme and cuts, reclaim test kit (TaKaRa Agarose Gel DNA Purification Kit Ver.2.0 with dna gel, the precious biotech firm in Dalian) the purpose fragment of recovery purifying 995bp, this fragment is for be connected with respectively the gene order of Pst I and BamH I restriction enzyme site at codon optimized clostridium difficile exotoxin A carboxy-terminal gene sequence (TcdA-C) two ends, so that the structure of nucleic acid vaccine.

(3) gene fragment clone that step (2) is obtained obtains recombinant plasmid pJW4303-TcdA-C in carrier for expression of eukaryon pJW4303.After restructuring plasmid extraction, enzyme are cut, checked order, determine to have obtained plasmid in line.

Beneficial effect of the present invention:

Compare with wild type gene, the codon frequency of occurrences of the codon of mammalian cell preference and intestinal bacteria preference increases in the codon optimized gene, but the clostridium difficile exotoxin A carboxy-terminal aminoacid sequence of its coding is constant, thereby makes it be more suitable for protein expression in mammalian cell and intestinal bacteria.

Because there is the Preference of codon in the nature biotechnology body; in the heterologous host body, be difficult to effective expression from the clostridium difficile exotoxin A carboxy-terminal gene clone in pathogenic agent source; therefore the effective immunity system of stimulation of host just makes it to produce preferably immanoprotection action.In order to improve the expression efficiency of heterologous gene in Mammals and intestinal bacteria, often need nucleotide coding sequence is optimized.Owing at present the optimization of nucleotide sequence still do not had unified standard or principle.Therefore for aminoacid sequence, different researchists can design expression and the manufacturing that different nucleotide sequences is used for target polypeptides fully, and correspondingly expression efficiency also may there are differences.Nucleotide coding sequence according to we optimize has overcome defects, has improved the clostridium difficile exotoxin A carboxy-terminal protein expression level.And the gene after will optimizing is used for making up nucleic acid vaccine, and the immunity system that can effectively stimulate the host behind immune Mammals has produced preferably humoral immune reaction.

The contriver enters carrier for expression of eukaryon with the clostridium difficile exotoxin A carboxy-terminal gene Direct Cloning of codon optimization; made up clostridium difficile nucleic acid vaccine pJW4303-TcdA-C; this vaccine can be in eukaryotic cell 293T cell effective expression; immune animal can stimulate the generation specific antibody, and passive protection test shows that the immunize rabbit serum of this dna vaccination can obviously reduce the mortality ratio of BALB/c mouse.This shows that the pJW4303-TcdA-C dna vaccination has good immunogenicity stimulates the generation specific antibody, and these polyclonal antibodies has good neutralization activity.

Description of drawings

The codon preference comparative result of Fig. 1 wild-type and codon optimized clostridium difficile exotoxin A carboxy-terminal gene sequence

A be wild-type and codon optimized clostridium difficile exotoxin A carboxy-terminal gene sequence in the comparison (index＞1 is mammalian cell institute preference) of mammalian cell expression preference, ordinate zou represents preference function, X-coordinate represents the nucleotides sequence column position.Left figure is the wild-type clostridium difficile exotoxin A carboxy-terminal gene sequence, and right figure is codon optimized clostridium difficile exotoxin A carboxy-terminal gene sequence.

B be wild-type and codon optimized clostridium difficile exotoxin A carboxy-terminal gene sequence in the comparison (index＞1 is intestinal bacteria institute preference) of escherichia coli expression preference, ordinate zou represents preference function, X-coordinate represents the nucleotides sequence column position.Left figure is the wild-type clostridium difficile exotoxin A carboxy-terminal gene sequence, and right figure is codon optimized clostridium difficile exotoxin A carboxy-terminal gene sequence.

The enzyme of Fig. 2 plasmid pJW4303-TcdA-C is cut the result

M, marker; 1～3 swimming lane is respectively first clone who selects and cuts without enzyme, through BamH I single endonuclease digestion, through the electrophorogram of Pst I and BamH I double digestion; All the other the like be respectively the 2nd, 3,4,5 clone and cut without enzyme, through BamH I single endonuclease digestion, through the electrophorogram of Pst I and BamH I double digestion.

The Western blot analytical results of the 293T cell target protein expression of Fig. 3 transfection pJW4303-TcdA-C

1:pJW4303-TcdA-C transfection lysate; 2:pJW4303-TcdA-C transfection supernatant; 3:pJW4303 transfection lysate; 4:pJW4303 transfection supernatant.Antigen is culture supernatant or the lysate of pJW4303 empty carrier, pJW4303-TcdA-C transfection 293T cell, and primary antibodie is the immunize rabbit serum of dilution in 1: 500.

IgG antibody response time curve after Fig. 4 pJW4303, the pJW4303-TcdA-C immunity BALB/c mouse

A is the IgG antibody response time curve after the pJW4303 immunity BALB/c mouse, totally 6 mouse (M1-M6);

B is the IgG antibody response time curve after the pJW4303-TcdA-C immunity BALB/c mouse, totally 7 mouse (M7-M13).Arrow represents immune time point.

Western blot analytical results after Fig. 5 pJW4303-TcdA-C immunity New Zealand white rabbit

1:pJW4303-TcdA-C transfection lysate; 2:pJW4303-TcdA-C transfection supernatant; 3:pJW4303 transfection lysate; 4:pJW4303 transfection supernatant.Used antigen is culture supernatant or the lysate of pJW4303 empty carrier, pJW4303-TcdA-C transfection 293T cell, and primary antibodie is the immunize rabbit serum of dilution in 1: 500.

The highest titre of specific IgG antibodies after Fig. 6 pJW4303, the pJW4303-TcdA-C immunity New Zealand white rabbit

PJW4303 represents the highest titre of specific IgG antibodies after the empty carrier immunity New Zealand white rabbit, totally 5 New Zealand white rabbit;

PJW4303-TcdA-C represents the highest titre of specific IgG antibodies after the pJW4303-TcdA-C dna vaccination immunity New Zealand white rabbit, totally 5 New Zealand white rabbit.

The passive protection test of Fig. 7 pJW4303-TcdA-C immunize rabbit serum on HT-29 clone

A figure: cellular form figure, 1, do not add 4CTU clostridium difficile VPI 10463 nutrient solution ultrafiltration things (normal cell contrast) in the cell hole; 2, only add 4CTU clostridium difficile VPI 10463 nutrient solution ultrafiltration things (toxin contrast) in the cell hole; 3, add in the cell hole to attack malicious content be 4CTU clostridium difficile VPI 10463 nutrient solution ultrafiltration things and the pJW4303-TcdA-C immunize rabbit serum pre-composition that dilutes at 1: 200; 4, add in the cell hole to attack malicious content be 4CTU clostridium difficile VPI 10463 nutrient solution ultrafiltration things and the pJW4303 immunize rabbit serum pre-composition that dilutes at 1: 100.

B figure: the NAT of pJW4303-TcdA-C immunize rabbit serum on HT-29 clone.

The passive protection test of Fig. 8 pJW4303-TcdA-C immunize rabbit serum on BALB/c mouse

" None " is commercial pure clostridium difficile Toxin A 100ng; " TcdA-C " is rabbit anteserum 11 μ l after the commercial pure clostridium difficile ToxinA 100ng+pJW4303-TcdA-C immunity; " Pre-bleed " is rabbit anteserum 11 μ l before the commercial pure clostridium difficile ToxinA 100ng+pJW4303-TcdA-C immunity.

Embodiment

The design of the clostridium difficile exotoxin A carboxy-terminal gene sequence that embodiment 1. is codon optimized is with synthetic

At first use software MacVector 7.2 analysis of encoding clostridium difficile exotoxin A carboxy-terminal gene sequences, find out its codon usage bias and use the different site of preference from Mammals, e. coli codon.For using the different codon site of preference, usefulness mammalian cell and the intestinal bacteria all codon of preference substitute, and design codon optimized clostridium difficile exotoxin A carboxy-terminal gene sequence.The above-mentioned codon optimized coded protein amino acid sequence consensus amino acid sequence original with it of gene order.Hand over U.S. GENEART company synthetic the sequence that designs, the carrier pMK-RQ that packs into is built into recombinant plasmid pMK-RQ-TcdA-C.Confirm that through order-checking synthetic sequence is correct.

To carry out codon optimized site in order clearly showing, now the TcdA-C nucleotide sequence after codon optimized and codon optimized front TcdA-C nucleotide sequence to be compared.Comparative result following (* is terminator codon):

ATG ATT AAA TTA AAA TTT GGT GTT TTT TTT ACA GTT TTA CTA TCT TCA GCA TAT before optimizing

ATG AT after optimizing CAA G CT GAAA TT CGG CGT GTT CTT CAC CGT G CT GCT G AGCTC CGCC TA C

Amino acid M I K L K F G V F F T V L L S S A Y

GCA CAT GGA ACA CCT CAA AAT ATT ACT GAT TTG TGT GCA GAA TAC CAC AAC ACA before optimizing

GC after optimizing CCA CGG CAC CCC GCA GAA CAT CAC CGA C CTG TG CGC CGA GTAC CAC AAC AC C

Amino acid A H G T P Q N I T D L C A E Y H N T

CAA ATA CAT ACG CTA AAT GAT AAG ATA TTT TCG TAT ACA GAA TCT CTA GCT GGA before optimizing

CA after optimizing GAT CCA CAC CCT GAA CGA CAA AAT CTT CTC CTA CAC CGA GTC CCT GGC GGG C

Amino acid Q I H T L N D K I F S Y T E S L A G

AAA AGA GAG ATG GCT ATC ATT ACT TTT AAG AAT GGT GCA ACT TTT CAA GTA GAA before optimizing

AA after optimizing G CG CGAG ATG GC CATC AT CAC CTT CAAG AA CGG CGC GAC CTT CCA GGT GGA G

Amino acid K R E M A I I T F K N G A T F Q V E

GTA CCA GGT AGT CAA CAT ATA GAT TCA CAA AAA AAA GCG ATT GAA AGG ATG AAG before optimizing

GT after optimizing GCC GGG CAG CCA GCA CAT CGA CTC CCA GAA GAAA GC CAT CGA G CG CATG AAG

Amino acid V P G S Q H I D S Q K K A I E R M K

GAT ACC CTG AGG ATT GCA TAT CTT ACT GAA GCT AAA GTC GAA AAG TTA TGT GTA before optimizing

GA after optimizing CACC CTG CG CAT CGC CTA CCT GAC CGA GGC CAA GGT GGA GAAG CT GTG CGT G

Amino acid D T L R I A Y L T E A K V E K L C V

TGG AAT AAT AAA ACG CCT CAT GCG ATT GCC GCA ATT AGT ATG GCA AAT TAA before optimizing

TGG AA after optimizing CAA CAA GAC CCC GCA CGC CAT CGC GGC CAT CAG CATG GC GAA CTAA

Amino acid W N N K T P H A I A A I S M A N *

The Preference change has relatively occured in above-mentioned codon optimized clostridium difficile exotoxin A carboxy-terminal gene sequence and wild-type.Software MacVector 7.2 analog results of Fig. 1 can be found out, compare with wild type gene, the codon frequency of occurrences of the codon of mammalian cell preference and intestinal bacteria preference increases in the codon optimized gene, but their coded aminoacid sequences are constant, thereby make it be more suitable for protein expression in mammalian cell and intestinal bacteria.

The structure of embodiment 2. carrier for expression of eukaryon pJW4303-TcdA-C

(1) acquisition of TcdA-C fragment, the linear large fragment of plasmid pJW4303: use respectively Pst I and BamH I double digestion plasmid pJW4303 and pMK-RQ-TcdA-C (synthetic by U.S. GENEART company).The endonuclease reaction system is: 10 * BufferTango ^TM4 μ l, plasmid (pJW4303, pMK-RQ-TcdA-C) 10 μ l, Pst I 1.5 μ l, BamHI 1.5 μ l, moisturizing to 40 μ l, 37 ℃, 2h.

(2) enzyme is cut product purification: above-mentioned enzyme is cut product behind the agarose gel electrophoresis of 10g/L, place under the Ultraviolet Detector, reclaim test kit (Agarose Gel DNA Purification Kit Ver.2.0 according to gel, the precious biotech firm in Dalian) specification sheets, cutting-out contains the gel of purpose fragment, analytical balance claims the quality of blob of viscose, presses the volume that 1mg=1 μ l calculates blob of viscose.Add the DR-I Buffer of 4 times of volumes, place 75 ℃ of water-bath heating and melting blob of viscoses, be interrupted vibration and mix, until blob of viscose melts fully, add the DR-II Buffer of DR-I Buffer 1/2 volume, fully mixing solution.Centrifugal adsorbing column is placed on the collection tube, shifts mixing solutions to adsorption column, 12000rpm, centrifugal 1min.Abandon filtrate, add 500 μ l Rinse A liquid, 12000rpm, centrifugal 30s.Abandon filtrate, add 700 μ l Rinse B liquid, 12000rpm, centrifugal 30s, repeated washing are once.Adsorption column is placed on the new Ep pipe, and the film central authorities dropping 25 μ l sterile purified waters at adsorption column leave standstill 60s.12000rpm, centrifugal 1min, the elutriant in the Ep pipe is target DNA solution at this moment.Measure the concentration of dna solution, with 10g/L agarose gel electrophoresis analysis rubber tapping purification effect, elutriant is kept in-20 ℃ of refrigerators for subsequent use.

(3) ligation: with the T4DNA ligase enzyme purpose fragment is connected with the linear large fragment of plasmid pJW4303, obtains the pJW4303-TcdA-C recombinant expression plasmid, be the nucleic acid vaccine of TcdA provided by the present invention.The ligation system is: 10 * T4DNA Ligase Buffer, 1 μ l, and linearizing pJW43031 μ l, the purpose fragment 7 μ l of purifying, T4DNA Ligase 1 μ l, mixing is placed 16h for 16 ℃.Connector transforms the HB101 competent cell.

The evaluation of embodiment 3. recombinant plasmid pJW4303-TcdA-C

3.1pJW4303-TcdA-C transform respectively the HB101 competent cell

1) 10 μ l connectors is joined in the Ep pipe that 100 μ l HB101 competent cells are housed the tube wall several of flapping gently, abundant mixing, ice bath 30min.

2) the Ep pipe is placed 42 ℃ of water-bath 90s.

3) in the Ep pipe, slowly add LB substratum 0.5mL, 37 ℃, 80rpm, jolting 45min.

4) bacterium liquid is coated on the LB flat board that contains penbritin (0.1g/L), 37 ℃, overnight incubation.

3.2 screening positive clone

5 single bacterium colonies of picking are inoculated in respectively in 5 culture test tubes (the LB substratum that contains the 0.1g/L penbritin) at random, and 37 ℃, 200rpm jolting, overnight incubation.

3.3 extract in a small amount recombinant plasmid pJW4303-TcdA-C (plasmid extracts test kit in a small amount: TaKaRa MiniBEST PlasmidPurification Kit Ver.2.0, TaKaRa company)

1) slowly be drawn onto in aseptic super clean bench in the 1.5ml centrifuge tube shaking the bacterium that spends the night in 3.2, remaining a small amount of bacterium liquid is stored in 4 ℃ in the culture test tube.

2) bacterium liquid in the centrifuge tube, the centrifugal 2min of 12000rpm abandons supernatant under the normal temperature.

3) add the 250 μ l Solution I bacterial precipitation that fully suspends.

4) add 250 μ l Solution II, gentleness is put upside down 5-6 time, forms clear solution.

5) the Solution III of 4 ℃ of precoolings of adding 400 μ l, gentleness is put upside down 5-6 time, and then room temperature leaves standstill 2min.

6) room temperature 12000rpm, centrifugal 10min.

7) Spin Column is placed on the Collection Tube, with 6) in supernatant liquor join among the Spin Column, the centrifugal 1min of 12000rpm abandons filtrate.

8) 500 μ l Rinse A are added among the Spin Column, the centrifugal 30s of 12000rpm abandons filtrate.

9) 700 μ l Rinse B are added among the Spin Column, the centrifugal 30s of 12000rpm abandons filtrate.

10) repeating step 9.

11) Spin Column is placed on the 1.5mlEp, at the sterile purified water of the dropping 60 μ l of film central authorities, room temperature leaves standstill 1min

12) the centrifugal 1min of 12000rpm, elutriant is the solution that contains plasmid.

3.4 cutting, enzyme identifies plasmid pJW4303-TcdA-C

Plasmid pJW4303-TcdA-C BamH I single endonuclease digestion, Pst I and BamH I double digestion.Single endonuclease digestion reaction system (10 μ l): 10 * Buffer Tango ^TM1 μ l, plasmid (0.22mg/mL) 2 μ l, BamH I 0.5 μ l, moisturizing to 10 μ l.Double digestion reaction system (10 μ l): 10 * Buffer Tango ^TM1 μ l, plasmid (0.22mg/mL) 2 μ l, Pst I 0.25 μ l, BamH I 0.25 μ l, moisturizing to 10 μ l.Hatch 2h for 37 ℃.Add 1 μ l, 10 * Loading buffer and stop endonuclease reaction.10g/L agarose gel electrophoresis observations, enzyme are cut the rear electrophoresis collection of illustrative plates and are seen Fig. 2, and Fig. 2 shows that 5 clones all make up correctly.Enzyme is cut the correct bacterium of evaluation serve the order-checking of Hai Yingjun company, the correct bacterium of checking order is drawn flat board three times, and optional wherein two mono-clonal bacteriums preserve.

A large amount of preparations of embodiment 4. plasmid pJW4303-TcdA-C (the large extraction reagent kit of plasmid is QIAGEN Plasmid MegaKit (5), Qiagen company)

1) draw to identify that correct bacterium preserves liquid 5 μ l and is inoculated in the LB nutrient solution that 5ml contains penbritin, 37 ℃, 200rpm, overnight growth.

2) by 1: 500 with 1) in cultivate bacterium liquid and be inoculated in the LB nutrient solution that 1000ml contains penbritin, 37 ℃, 200rpm, overnight growth.

3) second day moves on to bacterium in the 250ml centrifugal bottle, and 4 ℃, the centrifugal 15min of 6000g abandon supernatant, collects bacterium.

4) add 50ml damping fluid P1, repeatedly jolting all is resuspended in the solution bacterium.

5) add 50ml damping fluid P2, gentleness is put upside down 5-6 time, and solution is even blueness, leaves standstill 5min.

6) add 50ml damping fluid P3, gentleness is put upside down 5-6 time, and blue solution disappears, the solution layering, and the upper strata is solid oyster white agglomerate, lower floor is limpid liquid, places 30min on ice.

7) 4 ℃, 21000g, centrifugal 30min,

8) supernatant is transferred to another centrifugal bottle, 4 ℃, 21000g, centrifugal 15min.

9) in adsorption column, add QBT damping fluid 35ml equilibrium adsorption post.

10) supernatant that obtains in the step 8 is added in the adsorption column, filters, abandon filtrate.

11) add lavation buffer solution QC 200ml, cross post, abandon filtrate.

12) add elution buffer QF 35ml, cross post, collect filtrate.

13) add the 24.5ml Virahol in the liquid to collecting, 4 ℃, 16000g, centrifugal 30min abandons supernatant.

14) add 7ml 70% ethanol, the centrifugal 10min of normal temperature 16000g abandons supernatant.

14) will there be the centrifuge tube of precipitation naturally to dry in super clean bench, then use 1ml physiological saline solution plasmid agglomerate.

15) (wavelength 260nm～280nm) measures plasmid concentration and 260/280 ratio in the extracting gained solution to ultraviolet spectrophotometry.

Embodiment 5. cell transfectings

The 293T cell uses the DMEM high glucose medium that contains 100U/ml penicillin, 0.1mg/ml Streptomycin sulphate and 10% foetal calf serum at 37 ℃, 5%CO ₂Be cultured to logarithmic phase in the saturated humidity incubator, after the 2.5g/L trysinization, with 5.0 * 10 ⁶Individual cell (6mL) is inoculated in the 10cm culture dish, when waiting to grow to 80% fusion, carries out cell transfecting according to the PEI infection protocol.Get PEI 75 μ l, pJW4303-TcdA-C 15 μ g, add DMEM high glucose medium to the 825 μ l that contains 100U/ml penicillin, 0.1mg/ml Streptomycin sulphate, abundant mixing, incubated at room 15min, then above-mentioned mixed solution is added in the culture dish and shakes gently to make and mix, simultaneously with pJW4303 empty plasmid transfectional cell as negative control.Change behind the 8h and do not contain the DMEM nutrient solution that serum contains 100U/ml penicillin, 0.1mg/ml Streptomycin sulphate.Gather in the crops culture supernatant and cell pyrolysis liquid after continuing to cultivate 48h, carry out Western blot and analyze.Analytical results as shown in Figure 3.Can detect the expression of specific proteins among Fig. 3 behind the demonstration pJW4303-TcdA-C transfection 293T cell in supernatant and cell pyrolysis liquid, its apparent molecular weight is about 36kDa.The existence that the supernatant of negative control empty carrier pJW4303 transfection 293T cell and lysate do not detect specific proteins.Western blot the analysis showed that the pJW4303-TcdA-C nucleic acid vaccine can be at the corresponding albumen of 293T cell inner expression.The nucleic acid vaccine of explanation after codon optimized can be in host cell expressing protein.

Wherein, transfectional cell results step is: draw cell conditioned medium liquid, and 2500rpm, room temperature, centrifugal 10min, it is ℃ frozen to draw supernatant-20.With PBS (concentration 10mM, pH7.2) with cell wash-out from culture dish, collecting cell suspension, 2500rpm, room temperature, centrifugal 10min, abandon supernatant, add lysate (50mM Tris-HCl PH7.6,150mM NaCl, 1%Triton adds 2%100mM PMSF (phenylmethylsulfonyl fluoride) before use), hatch 15min on ice, 12000rpm, 4 ℃, centrifugal 60min, collect supernatant liquor ,-20 ℃ frozen.

The research of embodiment 6. codon optimized pJW4303-TcdA-C nucleic acid vaccine immunity originality

After nucleic acid vaccine makes up and expresses successfully, laboratory animal is carried out immunity, detect the immunogenicity and the specificity of inducing the antibody of generation of codon optimized clostridium difficile exotoxin A carboxy-terminal nucleic acid vaccine by ELISA and Western blot method.

6.1pJW4303-TcdA-C immune BALB/c mouse and New Zealand white rabbit, experimental design is as follows:

Table 1pJW4303-TcdA-C immunity BALB/c mouse

Group	Quantity (only)	Nucleic acid vaccine	Dosage
				A
	6	Empty carrier pJW4303	100μg
				B
	7	pJW4303-Tcd A-C	100μg

Such as table 1, with empty carrier pJW4303 and each immune one group of BALB/c mouse of pJW4303-TcdA-C.The corresponding plasmid of intramuscular injection 100 μ g, (the syringe needle depth of penetration is 2mm at least to carry out electrotransfection in the body in the injection site with the WJ-2002 living gene importing equipment immediately after the injection, electrotransfection parameter: voltage 50V, positive and negative each 3 times of pulse number, the wide 30ms of ripple, frequency 30Hz), it is effective to be considered as electrotransfection with mouse leg muscle generation shake.Carry out dna immunization in the 0th, 2,4,8 weeks, two weeks blood sampling before each immunity, after the 6th week, last immunity, detect special IgG antibody response in the serum with the ELISA method, estimate the pJW4303-TcdA-C nucleic acid vaccine and in the BALB/c mouse model, induce the ability that produces humoral immunization.

Table 2pJW4303-TcdA-C immunity New Zealand white rabbit

Group	Quantity (only)	Nucleic acid vaccine	Dosage
				A
	5	Empty carrier pJW4303	200μg
				B
	5	pJW4303-Tcd A-C	200μg

Such as table 2, with empty carrier pJW4303 and each immune one group of New Zealand white rabbit of pJW4303-TcdA-C.The corresponding plasmid of intramuscular injection 200 μ g, (the syringe needle depth of penetration is 2mm at least to carry out electrotransfection in the body in the injection site with the WJ-2002 living gene importing equipment immediately after the injection, electrotransfection parameter: voltage 100V, positive and negative each 6 times of pulse number, the wide 60ms of ripple, frequency 60Hz), it is effective to be considered as electrotransfection with rabbit leg muscle generation shake.Carry out dna immunization in the 0th, 2,4,8 weeks, two weeks blood sampling before each immunity, after the 6th week, last immunity.Detect specific IgG antibody response in the serum with Western blot, estimate the pJW4303-TcdA-C nucleic acid vaccine and in the New Zealand white rabbit model, induce the ability that produces humoral immunization.

6.2ELISA TcdA-C specific IgG in the detection serum

Detect special IgG antibody response in the serum with the ELISA method, estimate the pJW4303-TcdA-C nucleic acid vaccine and in BALB/c mouse, New Zealand white rabbit model, induce the ability that produces humoral immunization.

1) pJW4303-TcdA-C transfection product is as antigen coated elisa plate (using PBS pH7.2-7.4 as coating buffer, the dilution in 1: 5 of transfection supernatant, transfection cracking dilution in 1: 10), and every hole 100 μ l, spend the night by 4 ℃.

2) abandon coating buffer, wash plate 5 times (PBST constitutes 10mMPBS and 0.05%Tween-20) with 1XPBST.

3) 5% skim-milk (PBS, 0.05%Tween-20,5% skim-milk) is 37 ℃, sealing 1h, every hole 200 μ l.

4) abandon confining liquid, wash plate 5 times with 1XPBST.

5) primary antibodie is serum to be detected, and extent of dilution is 1: 500.Every hole adds 100 μ l, 37 ℃, hatches 1h (primary antibodie diluent: 4% whey-protein, 0.5%Tween-20, PBS).

6) abandon primary antibodie, wash plate 5 times with 1XPBST.

7) two anti-are sheep anti mouse or the goat anti-rabbit igg of HRP mark, and extent of dilution is 1: 5000, and every hole adds 100 μ l, 37 ℃, hatches 1h.(two anti-diluents: 4% whey-protein, 0.5%Tween-20, PBS).

8) abandon two and resist, wash plate 5 times with 1XPBST.

9) TMB colour developing (TMB solution formula: 1 in TMB tablet, 0.1M phosphoric acid citrate buffer (the pH value is 5.0) 5ml, distilled water 5ml, 30% hydrogen peroxide, 2 μ l), every hole adds 100 μ l, room temperature, 3.5min, every hole adds the H of 50 μ l 1M ₂SO ₄Color development stopping.

10) each hole A450 value is measured and recorded to microplate reader, calculates multiple hole mean value, and as the cut-off value, and immune metapore OD value is less than 0.05 also removal with 2.1 times of preimmune serum OD value.

BALB/c mouse serum T cdA-C specific IgG antibodies answering time curve as shown in Figure 4.Show among the figure in the rear 2 all pJW4303-TcdA-C dna vaccination group serum of for the second time immunity to detect the TcdA-C specific IgG, and along with the increase immune response strength of immune time improves gradually.But the serum of pJW4303 empty carrier immune mouse does not detect antibody response.

The highest titre of New Zealand white rabbit serum T cdA-C specific IgG antibodies as shown in Figure 6.Show among the figure in the rabbit anteserum in 2 weeks after the 4th immunity, the TcdA-C immune group has the specific antibody of higher titre, pJW4303 empty carrier immune group does not then detect antibody response, has significant difference (p＜0.05) between two groups the highest titre of IgG antibody.

6.3Western blot detects specific IgG in the serum

1) each 20 μ l of pJW4303-TcdA-C transfection 293T lysis and supernatant, pJW4303 transfection 293T lysis and supernatant add 5x sample-loading buffer 5 μ l, 100 ℃, boil 10min.

2) separation gel (7.5ml 30% acrylamide soln, 3.7ml 1MTris/Cl pH8.8,150 μ l 10%SDS, 150 μ l, 10% ammonium persulfate, 6 μ l TEMED) of preparation 15%, encapsulating, liquid level top Jia Shui, polymerization 20～30min under the room temperature.

3) abandon water, prepare again 5% spacer gel (960 μ l, 30% acrylamide soln, 740 μ l 1MTris/Cl PH6.8,60 μ l10%SDS, 60 μ l, 10% ammonium persulfate, 5 μ l TEMED), insert broach at spacer gel, after glue condenses fully, pull up broach.

4) sample of will be above-mentioned handling well adds in the glue hole and carries out electrophoresis, first 20mA, 1h, 40mA then, 2h.

5) 100v, 1h forwards the albumen on the glue on the pvdf membrane to.

6) film that takes a turn for the better seals with 5% skim-milk, and 37 ℃, 1h.

7) wash film twice with 1xPBST.

8) film is immersed in the serum of dilution in 1: 500,4 ℃, spends the night.

9) discard serum, wash film 6 times with 1xPBST, every minor tick 10min.

10) discard washing lotion, add the goat anti-rabbit igg of 1: 5000 dilution HPR mark, 37 ℃, 1h.

11) discard two and resist, wash film 6 times with 1xPBST, every minor tick 10min.

12) luminous agent is added on the film, scotography.

The results are shown in Figure 5: detect specific antibody in the serum that can obtain after pJW4303-TcdA-C dna vaccination immunity New Zealand white rabbit, it is all reactionless that its apparent molecular weight is about supernatant, the cracking of 36kDa and these antibody and pJW4303 transfection.

Embodiment 7. cell toxicity tests

7.1 from culture supernatant partial purification toxin

1) with clostridium difficile VPI 10463 (the biological product of USS collecting center,

Numbering: 43255 ^TM, lower with) be inoculated in bioM é rieux company

In 259790 culturing bottles (pancreas peptone soybean broth substratum (TSB), 40ml), 37 ℃, incubated overnight.

2) get 1) in bacterium liquid 1ml be inoculated in

259790 culturing bottles, 37 ℃, 72h.

3) results bacterium liquid, 4 ℃, 6000g, centrifugal 15min.

4) get supernatant, the 0.45um filter filters.

5) with Amicon Mltra-15 Centrifugal Filter Unit with Mltracel-100 membrane above-mentioned filtered liquid is carried out ultrafiltration and concentration, centrifugal condition is: 4 ℃, and 5000g, 30min.Concentrated solution places-70 ℃ of preservations.

7.2 cell toxicity test

1) HT-29 cell (typical case's culture collection council of Chinese Academy of Sciences cell bank/Shanghai Inst. of Life Science, CAS cell resource center, Chinese) use the DMEM high glucose medium that contains 100U/ml penicillin, 0.1mg/ml Streptomycin sulphate, 10% heat-inactivated fetal bovine serum at 37 ℃, 5%CO ₂, be cultured to logarithmic phase in the saturated humidity incubator.

2) with after the 2.5g/L trysinization, with 1 * 10 ⁴/ hole is inoculated in 96 well culture plates, and 37 ℃, 5%CO ₂, cultivate 24h in the saturated humidity incubator.

3) discard substratum, the PBS that adds 37 ℃ of preheatings of 200 μ l washs one time.

4) with the clostridium difficile nutrient solution ultrafiltration concentrate that obtained in the continuous 2 times of dilutions 7.1 of the DMEM high glucose medium that contains 100U/ml penicillin, 0.1mg/ml Streptomycin sulphate, 10% heat-inactivated fetal bovine serum, each concentration dilution liquid joins in the 96 porocyte culture dish according to the rule in 100 μ l/ holes, 3 multiple holes; Control wells adds the substratum that does not contain toxin.37 ℃, cultivate 24h in the 5%CO2, saturated humidity incubator.

5) behind the 24h in Nikon EC LJPSE TE2000-S microscopically observation of cell form.The toxin lowest dose level that will cause the hole inner cell all to justify contractingization is defined as 1CTU (cytotoxic unit).

7.3 toxin neutralization test

1) prepare the HT-29 cell, step with 7.2 1), 2), 3).

2) be initial extent of dilution at 1: 100, rabbit anteserum with the continuous twice dilution of the DMEM high glucose medium that contains 100U/ml penicillin, 0.1mg/ml Streptomycin sulphate, 10% heat-inactivated fetal bovine serum pJW4303-TcdA-C immunity, each dilution serum 100 μ l and the volume that contains 4CTU toxin amount are that the clostridium difficile nutrient solution ultrafiltration concentrate diluent of 100 μ l mixes, and hatch 1h for 37 ℃.

3) said mixture is added in 3 multiple holes, 37 ℃, 5%CO ₂, cultivate 24h in the saturated humidity incubator.

4) behind the 24h in Nikon ECLIPSE TE2000-S microscopically observation of cell form.Answer the hole inner cells with 3 and all be defined as NAT without the inverse of the serum greatest dilution of justifying contractingization.

The results are shown in Figure 7.Fig. 7 A shows that under the toxin dose of 4CTU, the rabbit anteserum of pJW4303-TcdA-C immunity can be protected the HT-29 cell and not occur to change such as the cellular form of circle contractingization; Fig. 7 B shows, the rabbit anteserum of pJW4303-TcdA-C immunity when 3200 times of dilutions still can in and the toxin of 4CTU dosage and make the HT-29 cell that the circle contractingization not occur.

Embodiment 8. commercial pure clostridium difficile Toxin A attack the passive protection test of malicious BALB/c mouse

Get 36 of BALB/c mouse, be divided at random 3 groups, by table 3 intraperitoneal injection content (all " administration contents " all hatch 1h at 37 ℃ after mixing).Every day, the interval was observed once in 12 hours, and continuous 14 days, the death condition of record mouse.

Table 3

The results are shown in Figure 8, as shown in the figure: " None " and " Pre-bleed " two groups of mouse are all dead in 36 hours, then still 100% survival when observing terminal point of " TcdA-C " group mouse.This shows that the pJW4303-TcdA-C dna vaccination has good immunogenicity stimulates the generation specific antibody, and these polyclonal antibodies has good neutralization activity.

The part that the present invention does not relate to all prior art that maybe can adopt same as the prior art is realized.

Related reagent information such as following table among the embodiment:

Claims (2)

1. codon optimized clostridium difficile exotoxin A carboxy-terminal gene sequence, sequence is SEQ ID NO.1.

2. a clostridium difficile extracellular toxin nucleic acid vaccine is characterized in that this vaccine is comprised of clostridium difficile exotoxin A carboxy-terminal gene sequence claimed in claim 1 and carrier for expression of eukaryon pJW4303.

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