CN101538574B - Codon-optimized cholera enterotoxin CTB gene and cholera nucleic acid vaccine thereof - Google Patents

Abstract

The invention belongs to the technical field of biological medicine and relates to a codon-optimized cholera enterotoxin CTB gene and a cholera nucleic acid vaccine thereof. The CTB gene takes preferences during the use of mammalian cells and colon bacillus codons, and the related cholera nucleic acid vaccine comprises the CTB gene and a eukaryotic expression vector pJW4303. The CTB gene optimized by the codon can be used for constituting a nucleic acid vaccine and remarkably improve the protein expression amount in a mammalian parasitifer body, and the constituted nucleic acid vaccine effectively stimulates the immunity system of the parasitifer so that the parasitifer generates better immunological reaction of body fluid and better cellular immune response. In addition, the CTB gene can be used for expressing CTB protein in colon bacillus so as to lay a foundation for future protein extraction or purification in the colon bacillus of prokaryote.

Description

A kind of codon optimized cholera enterotoxin CTB gene and cholera nucleic acid vaccine thereof

Technical field

The invention belongs to the biological medicine technology field, relate to a kind of codon optimized cholera enterotoxin CTB gene and cholera nucleic acid vaccine thereof.

Background technology

Nucleic acid vaccine (nucleic acid vaccine) has another name called gene vaccine (gene vaccine) or dna vaccination (DNAvaccine); Its essence is the carrier for expression of eukaryon that contains pathogenic agent protection antigen gene; After it is imported into animal body; The antigen protein that can be absorbed and express pathogenic agent by the animal cell, thus induce body to this proteic immunoreation.As emerging in recent years a kind of vaccine, it is attracting people with its unique advantage: (1) antigen is synthetic similar with the natural infection of cause of disease with the submission process, through MHC I class and the direct submission immunity system of II quasi-molecule.The particularly immunoreation of specific C D8+ lymphocyte (CTL), this is that inactivated vaccine and subunit vaccine can not be compared; (2) immunogenic unicity has only the required antigen gene transfered cell of coding to obtain expressing, and itself does not have antigenicity carrier.And the vector-viral vaccine of reorganization also has huge and complicated carrier proteins except destination gene expression; (3) be easy to make up and prepare, good stability, with low cost, be suitable for large-scale production.But the goal gene overwhelming majority who is used for making up nucleic acid vaccine at present comes from prokaryotic organism such as virus or bacterium, and the research of vaccine and application are mainly eukaryote, for example: mouse, senior Mammalss such as the macaque or the mankind.Because prokaryotic organism and eukaryote are used on the preference at codon and there are differences; The pathogen gene that causes being used for making up nucleic acid vaccine can not effective expression in the mammalian hosts body; Therefore the effective immunity system of stimulation of host just; Make it to produce immanoprotection action preferably, this is to cause one of lower reason of present nucleic acid vaccine immunity originality.

Summary of the invention

The objective of the invention is to overcome above-mentioned defective, design a kind of codon optimized cholera enterotoxin CTB gene and cholera nucleic acid vaccine thereof.

Technical scheme of the present invention is:

A kind of codon optimized cholera enterotoxin CTB gene, sequence is SEQ ID NO.1.

A kind of cholera nucleic acid vaccine is characterized in that this vaccine is that cholera enterotoxin CTB gene and the carrier for expression of eukaryon of SEQ ID NO.1 formed by sequence.

Described carrier for expression of eukaryon can be any dna vaccine vector, and the present invention uses carrier pJW4303, but the present invention is not limited thereto carrier.

The structure of codon optimized cholera nucleic acid vaccine provided by the invention comprises the steps:

(1) design of codon optimized CTB gene and synthetic

At first use the gene order of gene software MacVector 7.2 analysis of encoding cholera enterotoxins, find out its codon and use preference and use the sub-site of preference different ciphers with Mammals codon use 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 sub-site of preference different ciphers in the vibrio cholerae CTB gene; Artificial then according to concrete carrier cloning needs; Further the adjustment gene order is designed codon optimized CTB gene order, and has obtained codon optimized CTB gene through the chemosynthesis of genome company.The coded protein amino acid sequence of this codon optimized sequence will be consistent with original CTB aminoacid sequence.For example: the triplet codon of coding Isoleucine Ile mainly is ATT in the vibrio cholerae CTB genome; And in 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 for use.Target gene sequences after the optimization is SEQ ID NO.1, and the sequence before optimizing is SEQ ID NO.2, and amino acid sequence coded is SEQID NO.3.

(2) the recombinant vectors pUC18/CTB that contains target sequence that genome company is provided carries out enzyme and cuts; Reclaim the purpose fragment that test kit (TaKaRa Agarose Gel DNA Purification Kit Ver.2.0, the precious biotech firm in Dalian) reclaims the about 375bp of purifying with dna gel.

(3) the CTB gene fragment clone that step (2) is obtained obtains recombinant plasmid pJW4303/CTB in carrier for expression of eukaryon pJW4303.Through purifying, the enzyme of recombinant plasmid pJW4303/CTB are cut and check order identify after, confirmed to obtain the CTB nucleic acid vaccine after codon optimized.

Beneficial effect of the present invention:

Advantage and the effect of invention is the cholera enterotoxin B subunit gene after codon optimized; Not only can be used to make up nucleic acid vaccine but also significantly improve this gene at the intravital expressing quantity of mammalian hosts; Constructed nucleic acid vaccine has effectively stimulated host's immunity system, makes it to produce humoral immune reaction and cell immune response preferably; And owing to when sequences Design, correspondingly taken into account colibacillary codon use preference; Simulation through computer software MacVector 7.2; Find that this gene can be used for the albumen at expression in escherichia coli CTB; Can significantly improve the protein expression level of this gene in intestinal bacteria, this lays the foundation in the prokaryotic organism intestinal bacteria, carrying out protein expression later on.

Description of drawings

The codon preference of Fig. 1 wild-type and codon optimized CTB gene relatively

Figure 1A wild-type and codon optimized CTB gene are in the comparison (index＞1 is a mammalian cell institute preference) of the preference of mammalian cell expression, and ordinate zou is represented the preference index, and X-coordinate is represented the nucleotides sequence column position.Left side figure is a wild-type CTB gene, and right figure is codon optimized CTB gene.

Figure 1B wild-type and codon optimized CTB gene are in the comparison (index＞1 is an intestinal bacteria institute preference) of escherichia coli expression preference, and ordinate zou is represented the preference index, and X-coordinate is represented the nucleotides sequence column position.Left side figure is a wild-type CTB gene, and right figure is codon optimized CTB gene.

The proteic Western blot of Fig. 2 CTB analyzes

Fig. 2 A replys with CTB specific antibody in the Western blot analysis immunize rabbit serum

Used antigen is CTB albumen, and used antiserum(antisera) is an immunize rabbit serum, and extent of dilution is 1: 200.

Fig. 2 B Western blot analyzes the expression of CTB albumen in the 293T cell

The CTB:CTB dna vaccination; Vector:pJW4303, antigen are above-mentioned two kinds of plasmid transfection 293T cell pyrolysis liquids, and used antiserum(antisera) is an immunize rabbit serum, and extent of dilution is 1: 200.

Fig. 3 Balb/c mouse CTB specific IgG antibodies answering time curve

Arrow is represented immune time point; Left figure CTB dna vaccination group, totally six curves, every curve is represented an immune Response of Mice time curve; Right figure empty carrier control group, totally five curves, every curve is represented an immune Response of Mice time curve.

Fig. 4 NZw change of serum C TB specific IgG antibodies answering time curve

Arrow is represented immune time point, and every curve is represented the immune response time curve of a NZw.◆, ■, ▲ represent 3 NZws of CTB dna vaccination immunity, ◇,, △ represents 3 immune NZws of empty carrier respectively.

The protection test of Fig. 5 suckling mouse

The suckling mouse survival rate and the time relation curve of the suckling mouse protection test that rabbit anteserum dilution in 1: 1 vibrio cholerae carries out before the curve representation immunity of zero mark; ● suckling mouse survival rate and the time relation curve of the suckling mouse protection test that expression immunize rabbit serum dilution in 1: 1 vibrio cholerae carries out, the ★ ★ by the curve represent this group to compare survival rate with the empty carrier group there were significant differences P＜0.01; Suckling mouse survival rate and the time relation curve of the suckling mouse protection test that ▲ expression immunize rabbit serum dilution in 1: 10 vibrio cholerae carries out, the ★ by the curve represent this group to compare survival rate with the empty carrier group there were significant differences P＜0.05; representes the suckling mouse survival rate and the time relation curve of the suckling mouse protection test that immunize rabbit serum dilution in 1: 50 vibrio cholerae carries out.

Embodiment

Below in conjunction with accompanying drawing and embodiment the present invention is further described.

The design of the CTB gene that embodiment 1. is codon optimized and synthetic

At first use the gene order of gene software MacVector 7.2 analysis of encoding cholera enterotoxin B subunits (CTB), find out its codon and use preference and use preference and the sub-site of intestinal bacteria different ciphers with the Mammals codon.Use the identical codon of preference for Mammals with intestinal bacteria; With mammalian cell and intestinal bacteria all the codon of preference substitute and use the sub-site of preference different ciphers in the vibrio cholerae CTB gene; Artificial then according to concrete carrier cloning needs; Further the adjustment gene order is designed codon optimized CTB gene order.The coded protein amino acid sequence of this codon optimized gene order will be consistent with original CTB aminoacid sequence.Codon optimized CTB gene is synthetic by the precious biotech firm in Dalian, and the carrier pUC18 that packs into is built into recombinant plasmid pUC18/CTB.Confirm that through order-checking the synthetic sequence is correct.

Carry out codon optimized site in order clearly to show, existing nucleic acid vaccine after codon optimized compares with codon optimized preceding nucleic acid vaccine target gene sequences with CTB.Nucleic acid vaccine after codon optimized and codon optimized preceding nucleic acid vaccine gene order be (* is a terminator codon) relatively as follows:

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

Optimize back ATG AT 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

Optimize back GC 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

Optimize back CA 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

Optimize back AA 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

Optimize back GT 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

Optimize back GA 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

Optimize back TGG AA 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 *

Preference has relatively taken place and has sexually revised in above-mentioned codon optimized CTB gene and wild-type.As can be seen from Figure 1; 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 CTB aminoacid sequence of its coding is constant, thereby makes it be more suitable for the protein expression in mammalian cell and intestinal bacteria.

Embodiment 2.CTB dna vaccination makes up

Plasmid pJW4303 and pUC18/CTB (precious biotech firm is synthetic by Dalian) are carried out double digestion respectively with Pst I and BamH I.The endonuclease reaction system is: 10 * Buffer Tango ^TM4 μ L, 10 * BSA, 4 μ L, plasmid (pJW4303 or pUC18/CTB) 20 μ L, PstI 1 μ L, BamHI 1 μ L, moisturizing to 40 μ L.Enzyme is cut product behind the agarose gel electrophoresis of 7g/L, reclaims purpose fragment and the linearizing pJW4303 that test kit (Agarose Gel DNA Purification Kit Ver.2.0, the precious biotech firm in Dalian) reclaims the about 375bp of purifying respectively with gel.

Sepharose behind the electrophoresis is placed under the Ultraviolet Detector, downcut the gel that contains target DNA, analytical balance is claimed the quality of blob of viscose, presses the volume that 1mg=1 μ L calculates blob of viscose.Add the DR-I Buffer of 3 times of volumes, place 75 ℃ of water-bath heating and melting blob of viscoses, every separated 2min mixing once melts until blob of viscose fully, adds the DR-II Buffer of 1/2DR-I Buffer volume, fully mixing solution.Centrifugal adsorption column is placed on the collection tube, shifts mixing solutions to adsorption column, leave standstill 2min, 3600rpm, centrifugal 1min.Abandon filtrating, add 500 μ L Rinse A liquid, 3600rpm, centrifugal 30s.Abandon filtrating, add 700 μ LRinse B liquid, 3600rpm, centrifugal 30s, repeated washing are once.Void column 12000rpm, centrifugal 1min is placed in adsorption column on the Ep pipe, lets ethanol volatilize fully totally.In the central authorities of adsorption film, add 25 μ L elution buffers, 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 subsequent use.

Purpose fragment that will about 375bp with the T4DNA ligase enzyme is connected with linearizing pJW4303, makes up the pJW4303/CTB recombinant expression plasmid.Be the CTB dna vaccination of mentioning among this paper result.The ligation system is: 10 * T4 DNA Ligase Buffer, 1 μ L, and linearizing pJW4303 1 μ L, purifying purpose fragment 7 μ L, T4 DNA Ligase1 μ L, mixing is placed 16h for 16 ℃.Connector transforms the HB101 competent cell.

Embodiment 3. recombinant plasmid pJW4303/CTB identify

3.1pJW4303/CTB transform 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, go to ice bath immediately, leave standstill 2min.

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

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

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 ℃, 250rpm jolting, overnight cultures.

3.3 extract recombinant plasmid pJW4303/CTB (plasmid extracts test kit in a small amount: QIAGEN Plasmid Mini Kit (50), Qiagen company) in a small amount

1) in aseptic super clean bench, slowly be drawn onto 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, 6000g under the normal temperature, centrifugal 10min abandons supernatant, collects bacterium.

3) add the resuspended damping fluid of 250 μ l in bacterial precipitation, jolting repeatedly, until can't see bacterial aggregate, bacterium all is resuspended in the solution.

4) add 250 μ l lysis buffers in the resuspended liquid of above-mentioned thalline, gentleness is put upside down 5-6 time, and solution is even blueness, leaves standstill 5min.

5) in the liquid that adding 350 μ l level pads obtain in (4), gentleness is put upside down 5-6 time, and blue solution disappears, the solution layering, and the upper strata is blocky oyster white agglomerate, and lower floor is limpid liquid, and room temperature leaves standstill 2min.

6) 12000g, centrifugal 10min draws supernatant, is transferred to another centrifuge tube.Centrifugal 10min under this condition.

7) supernatant is joined in the centrifugal adsorption column, 12000g, centrifugal 1min abandons filtrating.

8) adsorption column 12000g again, centrifugal 1min discards filtered solution.

9) in centrifugal adsorption column, add lavation buffer solution 25 μ l, 12000g, centrifugal 1min collects filtrating.

10) again with step 9 repetitive operation 1 time.

11) promptly contain plasmid in the liquid of twice collection.

3.4 extracting in a small amount plasmid pJW4303/CTB carries out enzyme and cuts evaluation

Plasmid pJW4303/CTB is with BamH I single endonuclease digestion, and Pst I and BamH I carry out double digestion, Sac I single endonuclease digestion.Double digestion total reaction system (10 μ L): 10 * Buffer Tango ^TM1 μ L, 10 * BSA, 1 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.Single endonuclease digestion system (10 μ L): 10 * Buffer Tango ^TM1 μ L, plasmid (0.22mg/mL) 2 μ L, BamH I or Sac I 0.5 μ L, moisturizing to 10 μ L.Hatch 1h for 37 ℃, add 1 μ L10 * Loading buffer and stop endonuclease reaction, 10g/L agarose gel electrophoresis observations.The correct bacterium of evaluation is served the Hai Shenggong order-checking, and the correct bacterium of checking order is drawn flat board three times, preserves two mono-clonal bacteriums.

A large amount of preparations of embodiment 4. nucleic acid vaccines (the big extraction reagent kit of plasmid is QIAGEN Plasmid Mega Kit (5), Qiagen company)

1) absorption identifies that correct bacterium preservation liquid 5 μ L are inoculated in 5ml and contain in the LB nutrient solution of penbritin, 37 ℃, and 200rpm, overnight growth.

2) by 1: 500 with 1) in culture bacteria liquid be inoculated in 1000ml and contain in the LB nutrient solution of penbritin, 37 ℃, 200rpm, overnight growth.

3) bacterium moved on in the 250ml centrifugal bottle in second day, 4 ℃, the centrifugal 15min of 6000g abandon supernatant, collect bacterium.

4) the resuspended bacterial precipitation of 50ml damping fluid P1, jolting repeatedly, until can't see bacterial aggregate, bacterium all is resuspended in the solution.

5) 50ml damping fluid P2 adds resuspended liquid in centrifugal bottle, and gentleness is put upside down 5-6 time, and solution is even blueness, leaves standstill 5min.

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

7) 4 ℃, 20000g, centrifugal 30min keeps supernatant, is transferred to another centrifugal bottle.Under this condition again with the centrifugal 10min of supernatant.

8) level pad QBT35ml wet balanced extraction column.

9) supernatant that obtains in (7) is added extraction column, cross post naturally, discard filtered solution.

10) add lavation buffer solution QC200ml, cross post naturally, discard filtered solution.

11) add elution buffer QF35ml, cross post naturally, collect filtered solution.

12) in collecting liquid, add the 24.5ml Virahol, 4 ℃, 15000g, centrifugal 30min abandons supernatant.

13) with precipitating the centrifugal 10min of normal temperature 15000g, supernatant discarded in the resuspended centrifuge tube of 7ml 70% ethanol.

14) will there be sedimentary centrifuge tube to dry naturally, the 1ml physiological saline solution in super clean bench.

15) (wavelength 260nm～280nm) is plasmid content quantitatively for ultraviolet spectrophotometry.

16) add elution buffer QF35ml, again with pillar wash-out 1 time.

17) for improving the utilization ratio of adsorption column, the second day solution with autogamy carries out 1 plasmid again by the step of 1-15 and extracts in a large number.

18) plasmid with extracted twice mixes, and carries out digestion with restriction enzyme and identifies.

Embodiment 5. cell transfectings

The 293T cell is with containing containing of 10% foetal calf serum of 37 ℃ of two anti-DMEM high glucose mediums, 5%CO ₂Be cultured to logarithmic phase in the saturated humidity incubator, after the 2.5g/L trysinization, with 1.0 * 10 ⁶Individual cell (6mL) is inoculated in the 60mm petridish, begins transfection when waiting to grow to 80% fusion.Carry out cell transfecting according to the PEI transfection method, get PEI 50 μ L, join 930 μ L and contain in two anti-DMEM high glucose mediums; Other gets 8.0 μ g pJW4303/CTB recombinant plasmids and joins in the above-mentioned nutrient solution, mixing gently, room temperature; Hatch 15min; Then 1ml PEI/DNA mixture is added in the culturing bottle and shakes gently to make and mix, simultaneously with pJW4303 empty plasmid transfectional cell as negative control, the replaceable serum that do not contain contains two anti-DMEM nutrient solutions behind the 10h.Carry out Western blot analysis after continuing to cultivate 72h.Analytical results is shown in Fig. 2 B.Can in the lysate extracting solution, detect the expression of specific proteins among the figure behind the demonstration CTB dna vaccination transfection 293T cell.The apparent molecular weight of monomer whose is about 13kDa, and the lysate of negative control empty carrier pJW4303 transfection 293T cell does not have specific proteins to exist.Engram analysis shows that the CTB dna vaccination can be at the 293T cell inner expression.

Transfectional cell results: draw cell conditioned medium liquid, 2500rpm, room temperature, centrifugal 10min, it is ℃ frozen to draw supernatant-20.(concentration 10mM is pH7.2) with cell wash-out from petridish, collecting cell suspension, 2500rpm, room temperature with PBS; Centrifugal 10min abandons supernatant, adds lysate (50mM Tris-HCl Ph7.6,150mM NaCl; 1%Triton is with preceding adding 2%100mM PMSF (PMSF)), hatch 20min on ice, 12000rpm; 4 ℃, centrifugal 60min collects supernatant, and-20 ℃ frozen.

The Studies on Immunogenicity of the codon optimized cholera enterotoxin B of embodiment 6. subunit nucleic acid vaccine

After codon optimized CTB nucleic acid vaccine makes up and expresses successfully,, detect the immunogenicity of codon optimized cholera nucleic acid vaccine through experimental animal (like mouse, rabbit etc.) is carried out nucleic acid vaccine immunity.Detect the antibody mediated immunity of CTB antigen-specific replys with ELISA and Westernblot.

This institute comprises two types with animal, Balb/c mouse and new zealand rabbit.Because immunizing rabbit can produce a large amount of immune serums, can enough carry out the research of antibody test and the effect of antibody passive protection, and rabbit intestinal segment ligation test can be carried out the initiatively research of provide protection of vaccine immunity.Therefore, in the research that the present invention relates to immunoprotection, will use the rabbit immunity as animal model.And mouse is raised conveniently, single easy operation during immunity, and blood sampling is simple, and therefore selecting mouse for use is that this tests another kind of animal model.Induce generation antibody to have ubiquity with two kinds of animal checking CTB dna vaccinations (pJW4303/CTB), the effect of inducing antibody to produce is all arranged at multiple animal immune.

6.1 the Balb/c mouse immune, table 1 is seen in experimental design:

Table 1CTB dna vaccination and empty carrier immunity Balb/c mouse and grouping

Group	Quantity	Dna vaccination	Dosage
				A
	6	The CTB dna vaccination	100μg
				B
	5	The empty carrier contrast	100μg

Contrast each immune one group of Balb/c mouse with CTB dna vaccination and empty carrier (pJW4303).Intramuscular injection, the immunity of live body gene lead-in mode guarantee syringe needle depth of penetration 2mm, inject vaccine; Observation injection site protuberance; Carry out (the technical parameter: voltage 50V, positive and negative each 3 times of pulse number, the wide 30ms of ripple of electrotransfection in the body in the injection site with WJ-2002 live body gene introducing apparatus immediately after the injection; Frequency 30Hz), it is effective to be regarded as electrotransfection with mouse leg muscle generation shake.Carry out dna immunization in the 0th and 2 weeks, before each immunity with after the last immunity, took a blood sample in two weeks and collection serum.Detect the special IgG antibody response of CTB in the serum with the ELISA method, evaluate CT B dna vaccination is induced the ability that produces humoral immunization in mouse model.Balb/c mouse CTB specific IgG antibodies answering time curve is as shown in Figure 3.The result shows CTB group mouse two weeks of back of immunity for the first time among the figure, just can detect the CTB specific IgG antibodies in the peripheral blood, and immunity back two all antibody horizontals significantly raise for the second time, and the highest antibody titers reaches 1: 364500.The serum of empty carrier pJW4303 immune mouse does not detect the CTB specific antibody, and the result confirms in the mouse body, to produce after the CTB immunity antibody response of antigen-specific.Arrow is represented immune time point, and every curve is represented a mouse, left figure CTB dna vaccination group, and right figure empty carrier control group, five curves of empty carrier group are almost overlapping.

6.2 new zealand rabbit is immune, table 2 is seen in experimental design:

Table 2CTB dna vaccination and empty carrier immunizing rabbit and grouping

Group	Quantity (only)	Dna vaccination	Dosage
				A
	3	The CTB dna vaccination	200μg
				B
	3	The empty carrier contrast	200μg

Contrast each immune one group of new zealand rabbit with CTB dna vaccination and empty carrier (pJW4303).Intramuscular injection, the immunity of live body gene lead-in mode; Guarantee syringe needle depth of penetration 2mm; Carry out (the technical parameter: voltage 100V, positive and negative each 6 times of pulse number, the wide 60ms of ripple of electrotransfection in the body in the injection site with WJ-2002 live body gene introducing apparatus immediately after the injection; Frequency 60Hz), it is effective to be regarded as electrotransfection with rabbit leg muscle generation shake.Carry out dna immunization in the 0th and 2 weeks, before each immunity with after the last immunity, took a blood sample and collection serum and ight soil in two weeks.Detect the special IgG antibody response of CTB in serum and the ight soil with the ELISA method, evaluate CT B dna vaccination is induced the ability that produces body fluid and mucosal immunity in the rabbit animal model.NZw change of serum C TB specific IgG antibodies answering time curve is as shown in Figure 4.Show CTB dna vaccination and empty carrier pJW4303 3 new zealand rabbits of immunity respectively among the figure, in 0,2, the immunity of 4,8 weeks is collected serum with 2 weeks of immunity back before the immunity.All can detect the CTB specific IgG in 3 rabbit serum of immunity back 2 all CTB dna vaccination groups for the first time; And increase along with immune time; CTB dna vaccination group rabbit anteserum CTB specific antibody level increases gradually; The 4th immunity back during 2 weeks antibody on average the highest titre be 1: 1093500, but the serum of 3 empty carrier immune rabbits does not detect antibody response.Arrow is represented immune time point.

Embodiment 7. immunne responses detect

7.1 ELISA detects in the serum and CTB specific IgG in the ight soil:

1) the antigen coated elisa plate of CTB (use PBS pH7.2-7.4 as coating buffer, the CTB protein concentration is 1 μ g/ml), 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) one anti-is serum to be detected, and initial extent of dilution is 1: 500, carries out 1: 3 doubling dilution again.Every hole adds 100 μ l, 37 ℃, hatches 1h.(an anti-diluent: 4% whey-protein, 0.5%Tween-20, PBS).If one anti-is the ight soil supernatant, carry out dilution in 1: 4, hatch 1h for 37 ℃, every hole adds 100 μ l.

6) abandon one and resist, wash plate 5 times with 1XPBST.

7) biotin labeled sheep anti-mouse igg (concentration is 1mg/ml, 1: 1000), 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) HRP mark streptavidin (concentration is 1mg/ml, 1: 2000), every hole adds 100 μ l, 37 ℃, hatches 1h.(diluent: 4% whey-protein, 0.5%Tween-20, PBS).

10) abandon HRP mark streptavidin, wash plate 5 times with 1XPBST.

11) 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 % ydrogen peroxide 50,2 μ l) every hole adds 100 μ l, room temperature, and 3.5min, every hole adds the H of 50 μ l1M ₂SO ₄Color development stopping.

12) each hole A450 value is measured and write down to ELIASA, calculates multiple hole MV, 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.

7.2Western blot detects specific IgG in the serum:

1) CTB dna vaccination transfection 293T cell pyrolysis liquid supernatant, pJW4303 transfection 293T cell pyrolysis liquid supernatant 20 μ l (if antigen is CTB albumen, get 5 μ g, be diluted to 20 μ l) add 5x sample-loading buffer 5 μ l, 100 ℃, boil 10min.

2) at first prepare 15% separation gel (7.5ml 30% acrylamide soln, 3.7ml 1MTris/Cl pH8.8,150 μ l10%SDS, 150 μ l, 10% ammonium persulfate, 6 μ l TEMED), encapsulating, the liquid level top adds entry, polymerization 20～30min under the room temperature.

3) abandon water, the spacer gel of refabrication 5% (960 μ l, 30% propylene phthalein amine aqueous solution, 740 μ l 1MTris/Cl PH6.8; 60 μ l 10%SDS, 60 μ l, 10% ammonium persulfate, 5 μ l TEMED); On spacer gel, insert broach, treat that glue condenses fully after, pull up broach.

4) the above-mentioned sample of handling well is added carry out electrophoresis, 20mA, 1h, 40mA, 2h in 15% glue.

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

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, each 10min with 1xPBST.

10) discard washing lotion, add the goat anti-mouse igg of 1: 10000 dilution HPR mark, 37 ℃, 1h.

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

12) luminous agent is added on the film, scotography, and the result sees Fig. 2.

Embodiment 8. suckling mouses protection experimental study

8.1 strains separation

Vibrio cholerae is provided by Jiangsu Province CDC, selects 10 strain bacteriums from the cholera epidemic strain lyophilized powder of its reservation, aseptic super clean bench with bacterolysis in saline water; Bacterial suspension is inoculated on the nutrient agar plate, 37 ℃, incubated overnight; The picking mono-clonal is drawn flat board three times again, gets an amount of bacterium and does slide agglutination test, isolates 4 strains of O1 crowd Eltor vibrio cholerae; Two strain rice leaves are respectively E.V.T 220 and 391.Two strain coulees are respectively V _48-65And V ₅₆

8.2 vibrio cholera toxin gene identification

8.2.1 the preparation of template

With bacterial strain to be checked on the ordinary nutrient agar flat board 37 ℃ cultivated 18-24 hour, scrape and get 2-3 bacterium colony mixing in 200 μ l sterilization deionized water, boil 15-30min, cooling is 10000rpm afterwards, centrifugal 2min gets supernatant and is used for pcr amplification.

8.2.2O1 crowd's toxin gene detects

Purpose: detect the virulence gene that our isolating vibrio cholerae contains, identify virulent strain.Because most important two virulence genes of vibrio cholerae are cholera enterotoxin A subunit and toxin-coregulated pili A subunit; The former can cause watery diarrhea; The latter is mainly sticked relevant with bacterium at enteron aisle; So also mainly detect these two virulence genes of cholera enterotoxin A subunit and toxin-coregulated pili A subunit in this research, identify the vibrio cholerae that contains these two kinds of virulence genes and come further to do challenge test.

(1). primer sequence

The upstream primer sequence of cholera enterotoxin A subunit gene (564bp) is SEQ ID NO.4; The downstream primer sequence is SEQ ID NO.5;

The upstream primer sequence of toxin-coregulated pili A subunit gene (1kb) is SEQ ID NO.6, and the downstream primer sequence is SEQID NO.7.

(2). reaction system: upstream primer 2 μ l

Downstream primer 2 μ l

10×buffer 5μl

15mmolMgcl ₂ 4μl

10mmol dNTPs 4μl

Taq enzyme (3U/ μ l) 0.5 μ l

Template 1 μ l

Deionized water 15 μ l

Total system 20 μ l

Experimental system is with the negative contrast of E.coli HB101.

(3). reaction conditions:

94℃ 5min

72℃ 10min

Through finding that O1 crowd Eltor type vibrio cholerae two strain rice leaves (E.V.T 220 and 391) and two strain coulees that we choose (are respectively V behind the above PCR method amplification virulence gene _48-65And V ₅₆) all contain CTB and two kinds of virulence genes of TcpA.

8.2.3 the concentration of needed vibrio cholerae when confirming 50% lethal quantity

1) birth 3～5d CD-1 suckling mouse moves apart female mouse 3h;

2) (bacterium is measured from 2x10 to inject 50 μ l Ai Ertuo type vibrio cholerae through the filling stomach ⁵Cfu/ml～2x10 ⁸Cfu/ml), suckling mouse is placed 30 ℃ barrier;

3) per 3～6h observes once, up to 48h;

4) confirm the vibrio cholerae concentration of 50% lethal dose.

8.3 suckling mouse protection test

1) V _48-65Type vibrio cholerae (Jiangsu Province CDC preserves cholera O1 crowd bacterial strain) is drawn dull and stereotyped; On the ordinary nutrient agar flat board, cultivate 18-24h for 37 ℃, scrape and get bacterium colony, bacterial suspension is inoculated in the LB substratum; Cultivate 16h for 37 ℃, it is 1x10 that the Maxwell turbidimeter is regulated bacterial concentration ⁸Cfu/ml;

2) vibrio cholerae mixes (preceding and immune serum is 56 ℃ with immunity, hatches 30min deactivation complement) 37 ℃ respectively with before the immunity with immune serum, hatches 1h;

3) in the suckling mouse stomach, pour into vibrio cholerae+hot deactivation preimmune serum liquid of 5 times of LD50 respectively; The vibrio cholerae of 5 times of LD50+hot deactivation immune serum liquid; The suckling mouse amount of death in every group writes down the quantity of each time point suckling mouse in the observation 48h, and the result is as shown in Figure 5.Can know that by Fig. 5 the rabbit anteserum of CTB nucleic acid vaccine immunity can block the pathogenic effects of vibrio cholerae, suckling mouse is produced effective immunoprotection.

The present invention does not relate to all identical with the prior art prior art that maybe can adopt of part and realizes.

Related reagent information such as following table among the embodiment:

Taq enzyme Promega company

T4DNA ligase enzyme Promega company

The precious biotech firm in DNA marker (DL 3000) Dalian

Restriction enzyme BamHI, Pst I and Sac I Fermentas company

Dna gel reclaims the precious biotech firm in test kit Dalian

DMEM high glucose medium Hyclone company

RPM1640 cell culture fluid Hyclone company

Foetal calf serum Gibico company

Plasmid extracts test kit Qiagen company in a small amount

The a large amount of test kit Qiagen companies that extract of grain

PEI Invitrogen company

The sheep anti-mouse igg BD company of HRP mark

The streptavidin SouthernBiotech company of HRP mark

The goat anti-rabbit igg SouthernBiotech company of HRP mark

Biotin labeled sheep anti-mouse igg SoutherbBiotech company

Biotin labeled goat anti-rabbit igg SouthernBiotech company

CTB protein Sigma company

TMB tablet Sigma company

Sequence table

< 110>Huang Zuhu, Xu Guifang

< 120>a kind of codon optimized cholera enterotoxin CTB gene and cholera nucleic acid vaccine thereof

<160>7

<210>1

<211>375

<212>DNA

< 213>artificial sequence

<220>

< 223>codon optimized CTB gene order

<400>1

atgatcaagc tgaaattcgg cgtgttcttc accgtgctgc tgagctccgc ctacgcccac 60

ggcaccccgc agaacatcac cgacctgtgc gccgagtacc acaacaccca gatccacacc 120

ctgaacgaca aaatcttctc ctacaccgag tccctggcgg gcaagcgcga gatggccatc 180

atcaccttca agaacggcgc gaccttccag gtggaggtgc cgggcagcca gcacatcgac 240

tcccagaaga aagccatcga gcgcatgaag gacaccctgc gcatcgccta cctgaccgag 300

gccaaggtgg agaagctgtg cgtgtggaac aacaagaccc cgcacgccat cgcggccatc 360

agcatggcga actaa 375

<210>2

<211>375

<212>DNA

< 213>native sequences derives from vibrio cholerae (Vibrio cholerae)

<220>

< 223>the CTB gene order of vibrio cholerae (Vibrio cholerae)

<400>2

atgattaaat taaaatttgg tgtttttttt acagttttac tatcttcagc atatgcacat 60

ggaacacctc aaaatattac tgatttgtgt gcagaatacc acaacacaca aatacatacg 120

ctaaatgata agatattttc gtatacagaa tctctagctg gaaaaagaga gatggctatc 180

attactttta agaatggtgc aacttttcaa gtagaagtac caggtagtca acatatagat 240

tcacaaaaaa aagcgattga aaggatgaag gataccctga ggattgcata tcttactgaa 300

gctaaagtcg aaaagttatg tgtatggaat aataaaacgc ctcatgcgat tgccgcaatt 360

agtatggcaa attaa 375

<210>3

<211>124

<212>PRT

< 213>native sequences derives from vibrio cholerae (Vibrio cholerae)

<220>

< 223>cholera enterotoxin B subunit gene amino acid sequence coded

<400>3

Met Ile Lys Leu Lys Phe Gly Val Phe Phe Thr Val Leu Leu Ser

1 5 10 15

Ser Ala Tyr Ala His Gly Thr Pro Gln Asn Ile Thr Asp Leu Cys

20 25 30

Ala Glu Tyr His Asn Thr Gln Ile His Thr Leu Asn Asp Lys Ile

35 40 45

Phe Ser Tyr Thr Glu Ser Leu Ala Gly Lys Arg Glu Met Ala Ile

50 55 60

Ile Thr Phe Lys Asn Gly Ala Thr Phe Gln Val Glu Val Pro Gly

65 70 75

Ser Gln His Ile Asp Ser Gln Lys Lys Ala Ile Glu Arg Met Lys

80 85 90

Asp Thr Leu Arg Ile Ala Tyr Leu Thr Glu Ala Lys Val Glu Lys

95 100 105

Leu Cys Val Trp Asn Asn Lys Thr Pro His Ala Ile Ala Ala Ile

110 115 120

Ser Met Ala Asn

<210>4

<211>22

<212>DNA

< 213>artificial sequence

<220>

< 223>cholera enterotoxin A subunit gene upstream primer

<400>4

cgggcagatt ctagacctcc tg 22

<210>5

<211>23

<212>DNA

< 213>artificial sequence

<220>

< 223>cholera enterotoxin A subunit gene downstream primer

<400>5

cgatgatctt ggagcattcc cac 23

<210>6

<211>21

<212>DNA

< 213>artificial sequence

<220>

< 223>toxin-coregulated pili A subunit gene upstream primer

<400>6

ccctctctat gtgaatgttg c 21

<210>7

<211>21

<212>DNA

< 213>artificial sequence

<220>

< 223>toxin-coregulated pili A subunit gene downstream primer

<400>7

ctctaactcc cagcagcgac c 21

Claims (4)

1. codon optimized cholera enterotoxin CTB gene, sequence is SEQ ID NO.1.

2. cholera nucleic acid vaccine is characterized in that this vaccine is that cholera enterotoxin CTB gene and the carrier for expression of eukaryon of SEQ ID NO.1 formed by sequence.

3. cholera nucleic acid vaccine according to claim 2 is characterized in that described carrier for expression of eukaryon can be any dna vaccine vector.

4. cholera nucleic acid vaccine according to claim 3 is characterized in that described carrier for expression of eukaryon is pJW4303.

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