CN1231586C - Pleiomorphic saccharomyces hansenii expression recombination cholere toxin B subunit gene and its application - Google Patents

Abstract

The present invention belongs to the field of protein expression gene engineering, more specifically an industrialization research field for expressing foreign genes by using a Hansen yeast as a cell factory. Because the relationship of comma bacillus is relatively far from the Hansen yeast on an evolutionary relationship, the comma bacillus belongs to a prokaryotic organism and a codon using method of cholera toxin B subunit genes (GTB) in the protein translation process makes a great difference from the Hansen yeast; therefore, the translation can be stopped anomaly. In order to increase the expression amount of the GTB in the Hansen yeast, coding genes of the GTB are redesigned by a multi-variation statistical analysis method according to the codon using method of high expression genes of the Hansen yeast in the field, and an expression product can be used for preventing diarrhea resulted from the comma bacillus and can be used as a mucous membrane immunologic adjuvant to increase the immune effect of a plurality of vaccines noculated by a mucous membrane path. The CTB genes designed in the field can be expressed in a fusion way in the Hansen yeast with other antigenic genes, and an expression product can be used as an oral vaccine.

Description

A kind of multiple-shaped nuohan inferior yeast express recombinant Cholera Toxin B Subunit Gene and application thereof

Technical field

The invention belongs to protein expression genetically engineered field, the multiple-shaped nuohan inferior yeast (Hansenulapolymorpha) that relates to specifically utilize a kind of recombinant cholera toxin CTB subunit gene and utilization to contain this gene efficiently expresses CTB as cell factory and utilizes CTB and the application of fusion rotein in preparation cholera vaccine and adjuvant.

Background technology

Cholera (Cholera) is one of the world's seven pandemic disease, China is decided to be category A infectious disease, it is a kind of infectious intestinal disease that is caused by vibrio cholerae (Vibrio cholerae), causes people and mammiferous acute diarrhea during morbidity, if untimely treatment will soon be dead.(Cholera toxin CT) is a kind of heat-labile toxin of vibrio cholerae excretory to Toxins,exo-, cholera.The toxicity of CT is very strong, and the people is especially responsive to CT, and 8 μ g CT just can cause strong diarrhoea.CT is made up of two kinds of subunits, and promptly an A subunit (CTA, toxicity part) and 5 B subunits (CTB, bound fraction, nontoxicity) are with the AB of non-covalent form composition ₅The polymer activated protein.Wherein B subunit (CTB) maturation protein is made up of 108 amino acid.CTB can combine with the Ganglioside GM1 that most of mammalian cell surfaces all have, in conjunction with after can activated G protein, activatory G albumen is activated adenyl cyclase thereupon, then cause cAMP concentration rising in the cell, cause chlorine ion concentration to increase, sodium ion absorbs and is suppressed, and causes serious watery diarrhea syndromes.

The mucomembranous surface that digestive tube, respiratory tract, urogenital tract etc. have a huge surface area is to include the main place that the microorganism of harm contacts with allogenic material; be the protection mucosal tissue; immunity system herein is very complicated and connect each other, is formed in actual and the relatively independent system of systemic immunity on anatomy and the function.Contain wherein in the body that lymphocytic enteron aisle is a best part in this immunity system 70% or more, and be secretory IgA (sIgA) the main immunoglobulin that enteron aisle and other mucomembranous surfaces provide specific immune to protect.CTB has very strong immunogenicity and adjuvanticity, is one of maximum mucosal adjuvant of current research.McKenzie and Halsey confirms that anti-HRP antibody (IgA) that CTB and HRP conjugate produce and mucosal immune response are than injecting strong several 10 times of HRP or CTB and HRP mixture (not being conjugate) separately.Guyon-Gruaz finds that the 30-50AA of independent oral CTB or 50-75AA also can produce the IgA of higher titre.1988, Bessenand Fischetti etc. were with after A group streptococcus M albumen and the CTB coupling, and the oral immunity mouse can significantly reduce mouse to streptococcic infection.1989, (A/PR/8/34 (PR-8), H1N1) vaccine nasal feeding simultaneously mouse still can protect mouse to avoid strong virus attack after 16 weeks to Tamura fully with CTB and influenza virus.Hirabayashi finds again that for CTB and influenza virus nasal feeding is better with parenteral route approach (subcutaneous injection etc.) than oral.Rudin shows also that to the research of Sweden's 15 routine male volunteers CTB is more excellent than per os approach in the nasal route inoculation.1992, Vajdy andLycke discovered KLH's, utilizes CTB can excite secular immunne response (at least 22 months) as mucosal adjuvant, thus the invasion and attack of more effective opposing pathogenic micro-organism.Therefore, the effect of CTB not only is to increase antibody horizontal, and can also increase the submission dynamics of antigen to mucous membrane, it is also that as immunogen it can and can excite lasting immunity to reply in conjunction with assembly of the Peyer ' s on the intestinal epithelial cells and mesenteric lymph nodes.1998, Wu and Russell utilizes streptococcus surface antigen, has studied the adjuvant effect of recombinant C TB (rCTB), and he finds that rCTB can significantly strengthen the IgG antibody horizontal of mucous membrane IgA and whole body, if add trace holotoxin CT (5mg), can make antibody horizontal reach maximum.Can improve adjuvant effect though add the CT of trace, consider that from the angle of health recombinant C TB is safer, reliable.Recent years, people are positioned at the forward position of mucosal immunity always to the research of CTB.Isaka for example, M utilizes recombinant C TB to improve the immune effect of Toxoid,tetanus, in the presence of rCTB, adopts the method for nasal feeding inoculation, cost is reduced greatly, and he uses the same method and has also improved the immune effect of diphtheria toxin and recombinant hepatitis B surface antigen; Shen and Olive and Dunn utilize CTB also to improve the immune effect of suis peptide vaccine in mouse.Mattos Areas has synthesized the 6XHis-tagged CTB of 359bp respectively according to intestinal bacteria/milk-acid bacteria/Salmonella typhimurium codon preference, and expresses in intestinal bacteria, and expression amount can reach 13mg/L.Sun can significantly reduce the schistosomicide mortality of mice with schistosome ovum antigen and CTB conjugate immune mouse.In recent years, the researchist is also clearer to the understanding of the immune effect of CTB, such as, George-Chandy is respectively by studying the promoter action of CTB to antigen presenting cell with chemical process and gene fusion method and the little peptide of CTB link coupled, the result shows that the existence of CTB can make little peptide that the activation capability of T cell is improved 10000 times, independent little peptide or the simple mixtures of little peptide and CTB but do not reach this effect, he and then discovery CTB can increase the rise effect of antigen presenting cell surface C D40 and CD86, thereby promote the secretion of IL-12 and r-IFN.The research of Maeyama has also confirmed can stimulate IL-2, IL-4, the secretion of IL-5 and IL-10 with behind rCTB and the OVA conjugate immune mouse.

About security of CTB oral vaccine and immune efficacy problem, Pitisuttithum takes the vibrio cholerae vaccine of CTB and deactivation for Thailand and U.S. volunteer, finds that two groups of volunteers' antibody generation level does not have difference; Twice of the vibrio cholerae vaccine (10 months at interval) that Jertborn, M take CTB and deactivation for the Sweden volunteer equally, serum IgA and IgG antibody test show that the volunteer can excite the antitoxic immunity to cholera.Lewis takes CTB separately for the Britain volunteer, and behind the booster immunization, cell also can produce IgM except spontaneous generation IgA and IgG.He took CTB for again Britain and Kenya HIV volunteer afterwards, and he finds that CTB can excite early and middle portion HIV patient's mucosal immune response, and research in the HIV mucosa immune vaccines provides thinking to CTB for this.Afterwards, Backstrom in intestinal bacteria amalgamation and expression the gp120 fragment and the CTB of HIV of different lengths, can produce high-titer antibody behind the immune mouse at gp120.Lehner is p27gag expression product and the CTB coupling of SIV, immune rhesus monkey, and the result shows that rhesus monkey can resist SIV to the release at lymphoglandula of the invasion and attack of mucous membrane and virus.Lian has studied the mucosal immunity effect of CTB to the HIV envelope protein, and he finds that CTB can be with HIV envelope protein submission to GM1, and significantly increases the antibody horizontal of IgA, IgG1 and IgG2a.Recent years, malicious type enterotoxins of Escherichia coli of the product that Hall and Savarino inoculate deactivation for the Egyptian (adult, pupil and baby) of different ages and rCTB (ETEC-rCTB) assess the security of ETEC-rCTB, the result does not find untoward reaction, and the serology monitoring data shows this vaccine safety, reliable and can be used as the popular geographic vaccine of diarrhoea and use.

CTB is used by FDA (Food and Drug Adminstration) (FDA) and The World Health Organization's approval as anticholera injection at present, and enter the III clinical trial phase, simultaneously it also be find up to now the strongest, can stimulate the body mucosa reaction, use maximum gi tract immunogens.The method that obtains CTB at present has two kinds: a kind of method is to separate to obtain from natural vibrio cholerae, but this method has the risk of polluting CTA, can't industrialization.Another kind method is to utilize genetic engineering bacterium production recombinant C TB (rCTB); The genetic engineering bacterium of the production rCTB of report only has intestinal bacteria at present, but expression amount is lower, and cost is higher.The price of rCTB is 40$/mg on the current international community.Therefore how more can be cheap obtain the common concern that rCTB has caused the researchist.

Summary of the invention

Multiple-shaped nuohan inferior yeast (Hansenula polymorpha) belongs to methanol yeast, is called Pichiaaugusta again.Its optimum growth temperature height, growth velocity is fast, is beneficial to large scale fermentation production, can efficiently express many genes of efficiently expressing of being difficult in other systems.Because multiple-shaped nuohan inferior yeast can be integrated a plurality of genes than substep by certain gene dosage, the reorganization bacterium can be by the required gene of the ratio expression of the best, and this does not appear in the newspapers in other methanol yeast.In addition, multiple-shaped nuohan inferior yeast has that genetic manipulation is simple, copy number of foreign gene is high, foreign protein output height, be easy to advantages such as suitability for industrialized production, be a heterologous gene expression system that is better than intestinal bacteria and other yeast (as pichia pastoris phaff and cereuisiae fermentum etc.), obtained extensive concern.Especially in secretion type expression, foreign protein can be finished translation post-treatment processes such as proteolysis maturation, glycosylation modified and disulfide linkage formation by Secretory Pathway, make the more approaching native protein form of expressed albumen, avoided the mistake glycosylation phenomenon in the cereuisiae fermentum again with biologic activity.But vibrio cholerae and yeast relationship on evolutionary relationship is far away, and belongs to prokaryotic organism, and its codon usage and debaryomyces hansenii difference are very big in the protein translation process, thereby causes translating unusual pause.So far do not see the report that utilizes debaryomyces hansenii system expression CTB as yet.When we utilize multiple-shaped nuohan inferior yeast AS 2.2412 (Chinese microorganism strain contains the common micro-organisms center C GMCC of management committee) to express CTB, in order to improve expression amount, redesigned the encoding gene of CTB according to the codon usage of multiple-shaped nuohan inferior yeast cance high-expression gene.Make up in the born of the same parents then and secreted expression carrier, and in multiple-shaped nuohan inferior yeast, efficiently express with expression and secretion type expression mode in the born of the same parents respectively.

For this reason, the object of the present invention is to provide the multiple-shaped nuohan inferior yeast that a kind of recombinant cholera toxin b subunit (CTB) gene and utilization contain this gene to efficiently express CTB as cell factory and utilize CTB and the application of fusion rotein in preparation cholera vaccine and adjuvant.The nucleotide sequence of multiple-shaped nuohan inferior yeast recombinant cholera toxin b subunit encoding gene of the present invention is shown in SEQ ID NO:1, and the aminoacid sequence of its coding CTB is shown in (SEQ ID NO:2).For reaching this purpose, technological line of the present invention is by login Genbank website Http:// www.ncbi.nlm.nih.govRetrieval obtains Hansenula porlymorpha dna sequence dna.Login then Http:// bioweb.pasteur.fr/seqanal/interfaces/codonw.htmlChangeable different statistical analysis technique is adopted in the website, utilizes Codon W to carry out Correspondenceanalysis (CA) at sequence of threads; Obtain debaryomyces hansenii cance high-expression gene codon usage table.EditSeq program with debaryomyces hansenii cance high-expression gene codon usage table intercalation of DNA Star software.(accession number: the nucleotide sequence of encoding gene M23050) as shown in Figure 1 to go up the natural choleratoxin B subunit of reporting according to Genbank, its amino acid sequence coded is shown in SEQ ID NO:2, codon usage according to the debaryomyces hansenii cance high-expression gene is transformed into nucleotide sequence with the CTB aminoacid sequence, promptly obtains the CTB encoding gene (SEQ IDNO:1) according to the design of debaryomyces hansenii cance high-expression gene codon usage.This gene (SEQ ID NO:1) is 100% with the encoding gene amino acids coding homology of natural choleratoxin B subunit, its amino acid sequence coded is shown in (SEQ ID NO:2), nucleotide sequence homology is 73.8%, consider of the swing of debaryomyces hansenii cance high-expression gene to indivedual synonym preferences, therefore, the nucleotide sequence of coding recombinant cholera toxin b subunit (CTB) aminoacid sequence (removing Fig. 1 sequence) has 75% homology at least with it.The CTB encoding gene that the codon usage according to the debaryomyces hansenii cance high-expression gene that is obtained by the present invention designs, it has overcome the inadaptability of natural CTB encoding gene in the expressed by Hansenula yeast system, thereby makes this gene can efficiently express choleratoxin B subunit in debaryomyces hansenii.The CTB that expresses in debaryomyces hansenii is safe and reliable, can directly be used to the diarrhoea of preventing vibrio cholerae to cause after purified clinically, perhaps increases immune effect by the multiple vaccine of mucosal route inoculation as mucosal adjuvant.A kind of method of utilizing debaryomyces hansenii cance high-expression gene codon usage to prepare CTB and fusion rotein thereof may further comprise the steps:

1. the foundation of multiple-shaped nuohan inferior yeast cance high-expression gene codon usage;

2. the codon usage according to the multiple-shaped nuohan inferior yeast cance high-expression gene designs the CTB encoding gene;

3. synthetic by gene, the newly-designed CTB encoding gene of synthetic;

4. can utilize the BspHI restriction enzyme site to insert foreign genes such as HIV, HBV, HCV and FMDV, express the CTB fusion rotein that has the specific antigens determinant at CTB 5 ' end (perhaps utilizing Nco I site) at CTB 3 ' end;

5. make up expressed by Hansenula yeast carrier: pHMOXZ-A (expressing in the born of the same parents), pHFMDHZ-A (expressing in the born of the same parents), pHMOXZ α-A (secretion type expression), pHFMDHZ α-A (secretion type expression);

6. structure recombinant expression vector: pHMOXZ-CTB (born of the same parents in express), pHFMDHZ-CTB (expressing in the born of the same parents), pHMOXZ α-CTB (secretion type expression) and pHFMDHZ α-CTB (secretion type expression); The screening of conversion and recon;

7. the abduction delivering of recombinant C TB;

8. expression product is identified and the biologic activity evaluation.

Different with other methanol yeast (as pichia pastoris phaff), the recombinant expression vector that the present invention makes up is without linearize, directly adopt the electroporation conversion method to transform multiple-shaped nuohan inferior yeast, electroporation conversion method among the present invention is that the contriver is to (Faber, Haima et al., 1994) improvement, step obtains debaryomyces hansenii (Hansenula polymorpha) and efficiently expresses the choleratoxin B subunit recon according to the method described above, and preparation CTB and fusion rotein thereof are that those skilled in the art can realize.

Up to now, the relevant expression of CTB in debaryomyces hansenii both at home and abroad yet there are no report.The expression of CTB in debaryomyces hansenii of the present invention's design expressed biomass and extracted preparation process and all be better than other expression system, and industrialization production and the application of recombinant C TB are become a reality.

Description of drawings

Fig. 1: natural choleratoxin B subunit (accession number: the nucleotide sequence of encoding gene M23050)

Fig. 2: debaryomyces hansenii cance high-expression gene and low expressing gene synonym usage are relatively.High: the expression cance high-expression gene; Low: the low expressing gene of expression.N is that Codon W program is carried out the branch time-like to high and low expressing gene, each amino acid whose codon number.RSCU (Relative synonymouscodon usage, relative synonym usage), RSCU reflection be the service condition of each synonym in the gene, the ratio between the expected value when its numerical value equals the actual observed value of certain codon in gene and occurs with identical frequency with each codon; Carry out codon usage relatively between the database that introducing RSCU value can make different aminoacids form.* ^@The superior codon of expression cance high-expression gene correspondence, wherein * is that difference is extremely remarkable after chi square test; ^@Be significant difference after chi square test.

Be the codon of selecting for use during design CTB gene among the present invention

Fig. 3: the structure route of recombinant expression vector

Embodiment

Following embodiment can make the technician of this professional skill field more fully understand the present invention, but does not limit the present invention in any way.

Embodiment

1. the foundation of debaryomyces hansenii cance high-expression gene codon usage

Login Genbank website Http:// www.ncbi.nlm.nih.govRetrieval obtains the Hansenulaporlymorpha dna sequence dna, gets rid of base map and other nonessential sequence.Download to behind the local hard drive 5 ' and the 3 ' non-translational region of removing each sequence with DNAStar software and obtain encoding sequence (CDS), then all gene ORF (open reading frame) are utilized DNAStar software MegAlign program to draw evolutionary tree, file saves as hansenula.pau.Login then Http:// bioweb.pasteur.fr/seqanal/interfaces/codonw.htmlThe website enters the Codonw program entry, calls in the hansenula.pau file, utilizes Codon W program to carry out correspondence analysis (CA).Program is classified to debaryomyces hansenii cance high-expression gene and low expressing gene automatically, and wherein the number of samples of cance high-expression gene and low expressing gene respectively accounts for 5% of conceptual data, obtains debaryomyces hansenii cance high-expression gene codon usage table (see figure 2).EditSeq program with debaryomyces hansenii cance high-expression gene codon usage table intercalation of DNA Star software.

2. according to multiple-shaped nuohan inferior yeast cance high-expression gene codon usage design CTB gene

Go up the natural choleratoxin B subunit (accession number: the amino acid (SEQ ID NO:2) that encoding gene M23050) (Fig. 1) is corresponding of report according to Genbank, according to the codon usage of debaryomyces hansenii cance high-expression gene the CTB aminoacid sequence is transformed into nucleotide sequence by the DNAStar program, promptly obtains CTB encoding gene (SEQ ID NO:1) according to the codon usage design of debaryomyces hansenii cance high-expression gene.

3. the structure of recombinant expression vector

For easy to connect, the 5 ' end of synthetic CTB has added EcoR I, BspH I restriction enzyme site, and 3 ' end has added the XbaI enzyme cutting site.After enzyme is cut, be connected with pHMOXZ-A (expressing in the born of the same parents), pHFMDHZ-A (expressing in the born of the same parents), pHMOXZo-A (secretion type expression), four expression vectors of pHFMDHZ α-A (secretion type expression) respectively.Obtain pHMOXZ-CTB (expressing in the born of the same parents), pHFMDHZ-CTB (expressing in the born of the same parents), pHMOXZ α-CTB (secretion type expression) and pHFMDHZ α-CTB (secretion type expression) recombinant expression vector.The structure route of recombinant expression vector is seen Fig. 3.Above-mentioned any recombinant expression vector all can efficiently express in multiple-shaped nuohan inferior yeast.

4. the conversion of recombinant expression vector and screening

Different with other methanol yeast (as pichia pastoris phaff), the recombinant expression vector that the contriver makes up directly adopts the electroporation conversion method to transform multiple-shaped nuohan inferior yeast without linearize.Transformant large, medium and small three kinds of dissimilar bacterium colonies can occur two days later scribbling growth on the antibiotic YPD of Zeocin (1% yeast extract, 1% peptone, the 2% glucose) flat board on flat board.Big bacterium colony generally all is the high clone of copy number.Whether extract macrocolony zymic DNA utilizes PCR method detection CTB to insert.Picking ferments through the correct clone of PCR evaluation.

5. the electroporation conversion method transforms the method for multiple-shaped nuohan inferior yeast AS 2.2412 (Leu defective typies)

Electroporation conversion method among the present invention is the improvement of contriver to (Faber, Haima et al., 1994), and (transformation efficiency is up to 10 promptly to have avoided short circuit phenomenon in the electric shock process to improve transformation efficiency again ⁶).Step is as follows:

1) chooses multiple-shaped nuohan inferior yeast NCYC495 mono-clonal in the 5mlYPD liquid nutrient medium, 37 ℃ of incubated overnight

2) get 2ml bacterium liquid and add among the pre-warm YPD of 200ml, 37 ℃ are cultured to (about 6h) between the OD600=1.2-1.5

3) the centrifugal 5min of room temperature 6000rpm abandons supernatant

4) with 500mlTED (100mM Tris-HCl; 50mM EDTA; 25mM DTT; PH=8.0) suspension cell

5) 37 ℃ of shaking tables, 200rpm shakes 15min

6) 4 ℃ of centrifugal 6000rpm * 5min abandon supernatant

7) with the sucrose of the 270mM of 200ml precooling suspension cell gently

8) 4 ℃ of centrifugal 6000rpm * 5min abandon supernatant

9) with the sucrose of the 270mM of 100ml precooling suspension cell gently

10) 4 ℃ of centrifugal 6000rpm * 5min abandon supernatant

11) with the sucrose of the 270mM of 1ml precooling suspension cell gently, be distributed into the 60ul/ pipe, liquid nitrogen or refrigerator below-80 ℃ are preserved standby.

12) in the competent cell of 60ul, add 5ul plasmid DNA (about 100-500ng), add behind the mixing in the electric shock cup in 2mm aperture gently

13) shock parameters: 50uF, 100 Ω, 1.5KV

14) add YPDTM (1% yeast extract of 940ul after the electric shock immediately; 1% peptone; 2% glucose; 1mM Tris-HCl; 1mM MgCl ₂), be drawn in the tubule of 2-5ml, 37 ℃ of shaking tables, 200rpm shakes 1h

15) get 100ul and be coated with the YPD flat board that contains Zeocin microbiotic (final concentration 100ug/ml), cultivate after 2 days for 37 ℃, occur the bacterium colony of large, medium and small three kinds of forms on the flat board, choose clone PCR and identify recon, identify copy number with Southern hybridization

6. fermentation (preparation recombinant C TB subunit)

The positive recombinant yeast clone that will contain multi-copy gene in the YPD substratum 37 ℃ cultivate after 12 hours, inoculate in the fresh YPD fermention medium (containing 1.5% glycerine) according to 1: 20 ratio, keep leavening temperature 30-37 ℃, pH3-5, dissolved oxygen amount 20%, air velocity 5-10L/min.After 24 hours when O2 pressure sharply raises (glycerine of prompting in the substratum is exhausted), begin to feed in raw material (YPD that contains 50% glycerine), strict control feed rate makes that the final concentration of glycerine maintains between the 0.05%-0.4% in the fermented liquid.Ferment after 24-48 hour, can gather in the crops.For the fermented liquid of expressing in the born of the same parents, 4 ℃ of centrifugal results thalline of 12000rpm * 2min; For the fermented liquid of secretion type expression, 4 ℃ of centrifugal results supernatants.Carrying out SDS-PAGE and Western-blotting analyzes.In addition, except that inducing the fermentation with glycerine, the multiple-shaped nuohan inferior yeast expression system also can ferment with 1% methyl alcohol and 1% glucose induction.

7. the biologic activity of expression product is identified

Use the ELISA method, identify the CTB of yeast expression and the binding ability of Ganglioside GM1.Step is: use the GM1 coated elisa plate; The sealing of 1% casein food grade; The CTB that adds expression; The CT antibody that adds the HRP mark.OPD or TMB colour developing.The microplate reader interpretation.

Sequence table

＜110〉Institute of Microorganism, Academia Sinica

＜120〉a kind of multiple-shaped nuohan inferior yeast express recombinant Cholera Toxin B Subunit Gene and application thereof

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Claims (5)

1. the gene of coding recombinant cholera toxin b subunit (CTB) aminoacid sequence, the nucleotide sequence of described gene is shown in SEQ ID NO:1.

2. the preparation method of a multiple-shaped nuohan inferior yeast (Hansenula polymorpha) recombinant cholera toxin b subunit (CTB) of expressing may further comprise the steps:

A) foundation of multiple-shaped nuohan inferior yeast cance high-expression gene codon usage;

B) codon usage according to the multiple-shaped nuohan inferior yeast cance high-expression gene designs the CTB encoding gene;

C) by gene machine, the newly-designed CTB encoding gene of synthetic;

D) make up the expressed by Hansenula yeast carrier: be used for pHMOXZ-A, the pHFMDHZ-A that is used for expression in the born of the same parents, the pHMOXZ α-A that is used for secretion type expression that expresses in the born of the same parents, the pHFMDHZ α-A that is used for secretion type expression;

E) make up recombinant expression vector: the pHMOXZ-CTB that is used for expressing in the born of the same parents, the pHFMDHZ-CTB that is used for expressing in the born of the same parents, be used for the pHMOXZ α-CTB of secretion type expression and be used for the pHFMDHZ α-CTB of secretion type expression; The screening of conversion and recon;

F) abduction delivering of multiple-shaped nuohan inferior yeast recombinant C TB;

G) expression product is identified and the biologic activity evaluation.

3. method according to claim 2, wherein the abduction delivering of multiple-shaped nuohan inferior yeast recombinant C TB is that the positive recombination yeast that will contain multi-copy gene is cloned in the YPD substratum 37 ℃ and cultivates after 12 hours, inoculate in the fresh YPD fermention medium that contains 1.5% glycerine according to 1: 20 ratio, keep leavening temperature 30-37 ℃, pH3-5, dissolved oxygen amount 20%, air velocity 5-10L/min.After 24 hours when O2 pressure sharply raises, glycerine in the prompting substratum is exhausted, begin the reinforced YPD that contains 50% glycerine, strict control feed rate, make that the final concentration of glycerine maintains between the 0.05%-0.4% in the fermented liquid, ferment after 24-48 hour, for expressing the 4 ℃ of centrifugal fermented liquid results of 12000rpm * 2min thalline in the born of the same parents; For the fermented liquid of secretion type expression through 4 ℃ of centrifugal results supernatants.

4. the application of gene in preparation cholera therapeutic vaccine and other antigen adjuvant of coding CTB aminoacid sequence according to claim 1.

5. the gene of coding CTB aminoacid sequence according to claim 1 connects other antigen gene at its 5 ' or 3 ' end, becomes fusion gene, and expresses in multiple-shaped nuohan inferior yeast, and expression product is the application of fusion rotein in the preparation oral vaccine.

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