CN101560247A - Mucous membrane immunologic adjuvant using heat-sensitive colitoxin dual-mutant as vaccine - Google Patents
Mucous membrane immunologic adjuvant using heat-sensitive colitoxin dual-mutant as vaccine Download PDFInfo
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Abstract
The invention discloses a separated heat-sensitive colitoxin mutant protein. Compared with A subunit of wild type heat-sensitive colitoxin, A subunit of the protein has the 63th position of the amino acid sequence being K, the 192nd position being G, or the 72nd position being R, and the 192nd position being G. The invention also discloses a coding gene of the protein, a carrier or cell containing the gene, an adjuvant composite containing the protein, and a production method of the protein.
Description
Technical field
The invention belongs to biological technical field, more specifically, the present invention relates to coli heat-sensitive enterotoxin double-mutant, its encoding gene contains the carrier or the cell of this gene, contains this proteic adjunvant composition, and this proteic production method.
Background technology
Mucosa-immune is a ring important in the immunity of organism defense system, carrying out immunoprophylaxis by mucous membrane can not only induce the mucous membrane position to produce immune antibody, also can stimulate the immunne response of general, so the mucosa-immune inoculation is the best immunization route of prevention respiratory tract, gi system disease.But because many vaccines induce the antibody horizontal of generation low, can not produce enough immunoprotections when mucous membrane is inoculated, therefore need suitable mucous membrane adjuvant to cooperate, just can induce the mucous membrane IgA antibody of providing higher level.The mucosa-immune adjuvant of present research has a variety of, and wherein by opsonigenous substances, thermo-sensitivity enterotoxin and choiera toxin particularly attract people's attention as the research of vaccine mucosa-immune adjuvant.
(heat-labile enterotoxin is to cause one of toxin that people and Mammals are suffered from diarrhoea LT) to the thermo-sensitivity enterotoxin that Enterotoxic Escherichia coli (ETEC) is produced, and is made up of 1 A subunit and 5 B subunits.The main adjustable LT of B subunit combines with GM1 on the cytolemma, and the A subunit has ADP-ribose transferase active, is main virulence factor.Complete LT A subunit can be cracked into A1 and A2 by pancreatin, and A2 is positioned at the C-terminal of A1, and the two links to each other with disulfide linkage, and wherein A1 is the toxicity position, and A2 links to each other with LT B subunit.After entering target cell, disulfide linkage is reduced, and LT shows toxicity.
Many studies show that, LT is a kind of potential mucosa-immune adjuvant, but has limited its application clinically owing to LT has very strong toxicity.People are just attempting by site-directed mutagenesis technique at present, reach the purpose that reduces LT toxicity, keeps its adjuvant function simultaneously again.Discover that the site relevant with the toxicity of LT mainly concentrates on the A subunit, can reach the toxic purpose of reduction LT by changing amino acid sites.A lot of site mutation all can influence its ADP-ribose transferase active, the more common mutant that sites such as the 7th, 44,49,50,63,72,110,112,146 are arranged, these mutant show respectively in various degree residual toxicity and adjuvanticity in various degree, conclusion differs.
LT mutant of the prior art research mainly concentrates on the single site mutation body and makes up, and single site mutation body toxicity may not completely lose, and reverse mutation occurs and to regain the probability of virulence bigger.And the LT mutant of structure multidigit point, the possibility of its reverse mutation will reduce in theory, yet whether many site mutation can influence LT as the activity of mucosa-immune adjuvant also not as can be known, and also need to seek suitable multimutation position grouping, so that LT drops to the strong as far as possible immunological adjuvant activity of performance under the minimum situation in toxicity.
Summary of the invention
The object of the present invention is to provide that a kind of toxicity is low, the coli heat-sensitive enterotoxin double-mutant of immunological adjuvant superior activity, its encoding gene contains the carrier or the cell of this gene, contains this proteic adjunvant composition, and this proteic production method.
In a first aspect of the present invention, a kind of isolating coli heat-sensitive enterotoxin mutant protein is provided, described albumen comprises proteic A subunit of coli heat-sensitive enterotoxin and B subunit, and the aminoacid sequence of this A subunit is with respect to the aminoacid sequence of the A subunit of wild-type e. coli thermo-sensitivity enterotoxin
Its 63rd is K, and the 192nd is G;
Its 72nd is R, and the 192nd is G; Or
Its 63rd is K, and the 72nd is R, and the 192nd is G.
In another preference, described albumen contains 1 proteic A subunit of coli heat-sensitive enterotoxin and 5 proteic B subunits of coli heat-sensitive enterotoxin.
In another preference, described albumen is made of coli heat-sensitive enterotoxin A subunit and B subunit basically.
In another preference, described albumen is composited by 1 A subunit and 5 B subunits basically.
In a second aspect of the present invention, provide a kind of isolating nucleic acid, the described albumen of described nucleic acid encoding.
In a third aspect of the present invention, a kind of carrier is provided, described carrier contains described nucleic acid.
In a fourth aspect of the present invention, a kind of cell is provided, described cell contains described carrier, or is integrated with described nucleic acid in its genome.
In a fifth aspect of the present invention, provide a kind of production described proteic method, comprising: under the condition that is suitable for expressing, cultivate described cell, thereby express described albumen.
In another preference, described method comprises:
(1) at the A subunit and the B subunit of cell inner expression coli heat-sensitive enterotoxin mutant protein;
(2) collect the cell of (1), with denaturing agent resuspended after with cytoclasis;
(3) with the cytoclasis product mixing of (2), centrifugal collection supernatant;
(4) separate the albumen that contains A subunit and B subunit from the supernatant that (3) obtain, renaturation obtains desirable proteins.
In another preference, described denaturing agent is NTA0.
In another preference, described NTA0 contains: Tris-HCl, NaCl, and glycerine.
In another preference, the prescription of described NTA0 is: 20mmol/L Tris-HCl, 0.5mol/LNaCl, 10% glycerine.
In another preference, described cell is a Bacillus coli cells.
In a sixth aspect of the present invention, described proteic purposes is provided, be used to prepare the adjuvant of mucosa-immune.
In a seventh aspect of the present invention, a kind of composition that is used for mucosa-immune is provided, described composition contains: the described albumen of significant quantity, as the adjuvant of mucosa-immune.
In another preference, containing described proteic significant quantity is 0.0001-20wt% (that better is 0.001-10wt%).
In another preference, also contain in the described composition: significant quantity is (as 0.0001-20wt%; That better is 0.001-10wt%) the vaccine of mucosa-immune.
Description of drawings
Fig. 1 has shown the agarose gel electrophoresis analysis of pET30a-LTK63/G192.
Wherein, 1:LTK63/G192I; 2:LTK63/G192II; 3:PCR amplification LTK63/G192 gene; 4:DL 2000; 5: the recombinant plasmid double digestion is identified; 6:pET30a-LTK63/G192 recombinant plasmid PCR identifies.
Fig. 2 has shown the agarose gel electrophoresis analysis of pET30a-LTR72/G192.
Wherein, 1:LTR72/G192I; 2:LTR72/G192II; 3:LTR72/G192; 4:PCR amplification LTR72/G192 gene; 5: the recombinant plasmid double digestion is identified; 6:DL 2000.
Fig. 3 A has shown the LTK63/G192 abduction delivering.Wherein, 1: after inducing; 2: before inducing; 3: albumen marker.
Fig. 3 B has shown the LTK63/G192 protein purification.Wherein, 1: albumen marker; Behind the 2:LTK63/G192 protein purification; 3: foreign protein.
Fig. 3 C has shown the Western-Blot detection.Wherein, 1:LTK63/G192; 2:LTR72/G192; 3: albumen marker.
Fig. 4 has shown the Patent-mouse live body toxicity detected result of LT and each mutant of LT.
The G/C of PBS group (intestines weight/corpse is heavy) value is 0.071 ± 0.0057; LT, 0.101 ± 0.0035; LTK63,0.073 ± 0.0057; LTR72,0.081 ± 0.0084; LTG192,0.091 ± 0.0053, LTK63/R72,0.075 ± 0.0031; LTK63/G192,0.080 ± 0.0027; LTR72/G192,0.081 ± 0.0027 error is ± s s n=5.
Fig. 5 has shown that ELISA detects the result of antibody horizontal.Fig. 5 A has shown that serum Anti-NDV IgG detects; Fig. 5 B has shown that nose washing lotion Anti-NDV IgA detects; Wherein,
a：NDV；b：NDV+LT；c：NDV+LTK63；d：NDV+LTR72；e：NDV+LTG192；f：NDV+LTK63/G192；g：NDV+LTR72/G192；h：NDV+LTK63/R72。
" " sampling in the 0th day; " ■ " sampling in the 11st day;
Sampling in the 25th day;
Sampling in the 35th day.
Embodiment
The inventor is surprised to find that under study for action, the 63rd of coli heat-sensitive enterotoxin sported K and the 192nd sport G (LTK63/G192), after perhaps the 72nd of coli heat-sensitive enterotoxin being sported R and the 192nd and sporting G (LTR72/G192), the toxicity of the coli heat-sensitive enterotoxin mutant that obtains significantly reduces, and has excellent immunological adjuvant activity.In addition, because the coli heat-sensitive enterotoxin has been carried out the dibit point mutation, the mutant of acquisition is difficult for reverse mutation takes place, and is more stable as adjuvant effect.
The inventor has designed multiple unit point sudden change or multisite mutation form at coli heat-sensitive enterotoxin albumin A subunit, and these sudden change modes have been carried out deep research.Through oxicity analysis and enzymic activity test, biologic activity to each mutant detects, confirm that LTK63/G192 and these two mutant toxicity of LTR72/G192 significantly reduce, and mucous membrane adjuvanticity excellence, nothing to do with antigen is through collunarium approach immunized mice, confirmation can significantly improve serum antibody and merocrine secretion's type antibody, so mutant LTK63/G192, LTR72/G192 can be developed as the mucous membrane adjuvant of animal vaccine.
In the present invention, unless otherwise indicated, term " coli heat-sensitive enterotoxin mutant protein ", " coli heat-sensitive enterotoxin mutant polypeptide ", " coli heat-sensitive enterotoxin mutant ", " E.coli LT double-mutant " or " LT mutant " is used interchangeably, all refer to have aminoacid sequence corresponding to wild-type e. coli thermo-sensitivity enterotoxin, the 63rd sports K, the 192nd sequence that sports G; Or corresponding to the aminoacid sequence of wild-type e. coli thermo-sensitivity enterotoxin, the 72nd sports R, the 192nd albumen that sports the sequence of G; Or corresponding to the aminoacid sequence of wild-type e. coli thermo-sensitivity enterotoxin, the 63rd sports K, and the 72nd sports R and the 192nd sequence that sports G.Certainly, the amino acid whose replacement that is equal to can take place also on some nonactive related locus, for example some amino acid is replaced by similar performance or close amino acid.
As used herein, " isolating " is meant that material separates (if natural substance, primal environment promptly is a natural surroundings) from its primal environment.Do not have separation and purification as polynucleotide under the native state in the active somatic cell and polypeptide, but same polynucleotide or polypeptide as from native state with in other materials that exist separately, then for separation and purification.
As used herein, " coli heat-sensitive enterotoxin mutant protein or polypeptide " is meant that coli heat-sensitive enterotoxin mutant protein is substantially free of natural relative other albumen, lipid, carbohydrate or other material.Those skilled in the art can use this albumen of purified technology of protein purifying of standard.Albumen of the present invention can use recombinant technology, produces from protokaryon or eucaryon host (for example, bacterium, yeast).
As optimal way of the present invention, described coli heat-sensitive enterotoxin mutant protein comprises the A subunit, and the aminoacid sequence of this subunit is with respect to the sequence shown in the SEQ ID NO:2 (its encoding sequence such as SEQ IDNO:1), and its 63rd is K, and the 192nd is G; Or its 72nd be R, and the 192nd is G.
As preferred mode, described coli heat-sensitive enterotoxin mutant protein also comprises B subunit (SEQ ID NO:4, its encoding sequence such as SEQ ID NO:3).More preferably, described albumen is made of coli heat-sensitive enterotoxin A subunit and B subunit basically.Best, described albumen is composited by 1 A subunit and 5 B subunits basically, and the albumen of this structure has better immunological adjuvant effect.
The method that sudden change is incorporated in the wild-type e. coli thermo-sensitivity enterotoxin protein sequence is that those skilled in the art are known.For example, the primer that contains the mutational site by design comes the proteic encoding sequence of coli heat-sensitive enterotoxin of pcr amplification wild-type, filters out the gene product that is introduced into sudden change from amplified production, utilizes this gene product to generate the albumen of mutant.In addition, also can utilize the albumen that contains the mutational site in the mode composition sequence of synthetic.
The present invention also comprises the polynucleotide of encoding said proteins.Polynucleotide of the present invention can be dna form or rna form.DNA can be strand or double-stranded.Term " polynucleotide of proteins encoded " can be the polynucleotide that comprise this polypeptide of encoding, and also can be the polynucleotide that also comprise additional code and/or non-coding sequence.
Polypeptide among the present invention and polynucleotide preferably provide with isolating form, more preferably are purified to homogeneous.
The Nucleotide full length sequence of coli heat-sensitive enterotoxin mutant protein of the present invention or its fragment can obtain with the method for pcr amplification method, recombination method or synthetic usually.For the pcr amplification method, can be according to the polynucleotide sequence of encoding said proteins, especially open reading frame sequence designs primer, and with commercially available cDNA storehouse or by the prepared cDNA storehouse of ordinary method well known by persons skilled in the art as template, amplification and must relevant sequence.When sequence is longer, usually needs to carry out twice or pcr amplification repeatedly, and then the fragment that each time amplifies is stitched together by proper order.
In case obtained relevant sequence, just can obtain relevant sequence in large quantity with recombination method.This normally is cloned into carrier with it, changes cell again over to, separates obtaining relevant sequence then from the host cell after the propagation by ordinary method.
In addition, also the method for available synthetic is synthesized relevant sequence, especially fragment length more in short-term.Usually, by first synthetic a plurality of small segments, and then connect and to obtain the very long fragment of sequence.
At present, can be fully obtain the dna sequence dna of code book invention albumen (or its fragment, or derivatives thereof) by chemosynthesis.This dna sequence dna can be introduced in various existing dna moleculars as known in the art (or as carrier) and the cell then.The method of using round pcr DNA amplification/RNA is optimized for and obtains polynucleotide of the present invention.
The present invention also relates to comprise the carrier of polynucleotide of the present invention, and the host cell that produces through genetically engineered with carrier of the present invention or the proteic encoding sequence of the present invention, and the method that produces polypeptide of the present invention through recombinant technology.
Recombinant DNA technology (Science, 1984 by routine; 224:1431), can utilize polymerized nucleoside acid sequence of the present invention to express or produce the coli heat-sensitive enterotoxin mutant protein of reorganization.In general following steps are arranged:
(1). with the polynucleotide (or varient) of coding coli heat-sensitive enterotoxin mutant protein of the present invention, or transform or the transduction proper host cell with the recombinant expression vector that contains these polynucleotide;
(2). the host cell of in suitable medium, cultivating;
(3). separation, protein purification from substratum or cell.
Preferably, described coli heat-sensitive enterotoxin mutant protein is at expression in escherichia coli, because the coli heat-sensitive enterotoxin itself derives from intestinal bacteria, it is good therefore to utilize intestinal bacteria to express the protein-active that is obtained.
In preferred embodiments of the present invention, the expression of mutant protein of the present invention is improved, thereby obtain with native state under LT albumin A subunit and the ratio and the essentially identical LT mutant protein of space structure of B subunit.The inventor finds that after A subunit and B subunit were expressed simultaneously, the A subunit basically formed inclusion body in bacterium, the B subunit is because less, and a part forms inclusion body, and another part is expressed as soluble form.Not separated or out of proportion for guaranteeing A, B subunit, thus select the bacterial precipitation of centrifugal gained directly to use denaturing agent (preferred NTA0) resuspended, centrifugal collection supernatant after fully stirring.The A subunit still is in the same place with the B subunit like this, and does not upset the ratio between A subunit and the B subunit; After the renaturation, the E.coli LT structure is not destroyed.And as according to existing conventional operation, behind centrifugal acquisition cell precipitation, break the bacterium processing earlier and handle inclusion body with denaturing agent again, then will make most of B subunit be removed, thereby make that the albumen configuration of follow-up acquisition is different with native state.
Among the present invention, described polynucleotide sequence can be inserted in the expression vector.Term " expression vector " refers to bacterial plasmid well known in the art, yeast plasmid or other carriers.As long as can duplicate in host and stablize, any plasmid and carrier can be used.A key character of expression vector is to contain replication orgin, promotor, marker gene and translation controlling elements usually.
Method well-known to those having ordinary skill in the art can be used to make up and contains polynucleotide sequence of the present invention and suitable transcribing/the translate recombinant expression vector of control signal.These methods comprise extracorporeal recombinant DNA technology, DNA synthetic technology, the interior recombinant technology of body etc.Described dna sequence dna can effectively be connected on the suitable promotor in the expression vector, and is synthetic to instruct mRNA.
In addition, expression vector preferably comprises one or more selected markers, being provided for selecting the phenotypic character of transformed host cells, as is used for colibacillary tsiklomitsin or amicillin resistance.
Comprise the carrier of above-mentioned suitable dna sequence dna and suitable promotor or control sequence, can be used to transform appropriate host cell, so that it can marking protein.Host cell can be a prokaryotic cell prokaryocyte, as bacterial cell.
Can carry out with routine techniques well known to those skilled in the art with the recombinant DNA transformed host cell.When the host was prokaryotic organism such as intestinal bacteria, the competent cell that can absorb DNA can be used CaCl in exponential growth after date results
2Method is handled, and used step is well-known in this area.Another kind method is to use MgCl
2If desired, transforming also the method for available electroporation carries out.
The transformant that obtains can be cultivated with ordinary method, expresses the polypeptide of coded by said gene of the present invention.According to used host cell, used substratum can be selected from various conventional substratum in the cultivation.Under the condition that is suitable for the host cell growth, cultivate.After host cell grows into suitable cell density, induce the promotor of selection with suitable method (as temperature transition or chemical induction), cell is cultivated for some time again.
Can utilize its physics, the separating by various separation methods with other characteristic and the albumen of purification of Recombinant of chemistry, these methods are well-known to those skilled in the art.The example of these methods includes, but are not limited to: conventional renaturation handles, with protein precipitant handle (salt analysis method), centrifugal, the broken bacterium of infiltration, superly handle, the combination of super centrifugal, sieve chromatography (gel-filtration), adsorption chromatography, ion exchange chromatography, high performance liquid chromatography (HPLC) and other various liquid chromatography (LC) technology and these methods.
In preferred embodiments of the present invention, the inventor utilizes overlapping extension PCR amplification technique external acquisition E.coli LT double-mutant LTK63/G192 and the full gene fragment of LTR72/G192, enzyme is cut with sequencing result and is shown that the expression vector pET30a-LTK63/G192 of structure, pET30a-LTR72/G192 reading frame are correct, and corresponding site amino acid has obtained replacement.The abduction delivering product detects with SDS-PAGE, and each mutant all gives expression to two protein bands of about 33.0Ku and 13.0Ku, matches with LTA, LTB molecular weight subunit; Western-blot detects, two protein bands all can with anti-His antibody generation specific immune response.ADP-ribose transferase active analysis of experiments, two mutant protein enzymic activitys all are lower than wild-type LT albumen, but still keep some enzymic activitys.The Patent-mouse toxicity test detects, and the toxicity of LTK63/G192 and LTR72/G192 mutant protein is all very low.With these two albumen as the adjuvant of NDV through collunarium approach immune mouse, the result shows that LTK63/G192 and LTR72/G192 all have higher adjuvanticity.
The present invention also provides the purposes of described coli heat-sensitive enterotoxin mutant protein, is used to prepare the adjuvant of mucosa-immune, and it has stable performance, can induce body to produce high antibody horizontal, and the low characteristics of toxicity.
In described coli heat-sensitive enterotoxin mutant, the B subunit since adjustable LT combine with GM1 on the cytolemma, thereby activity with immunological adjuvant, yet because of the molecular weight of B subunit own little, be used for separately as adjuvant, be easy to cause after in entering body immunogenicity to reduce or disappear, effect is undesirable, and the B subunit is used for having good activity as immunological adjuvant, the immunogenicity height with the combination that reduces toxic A subunit.
Therefore, the present invention also provides a kind of composition that is used for mucosa-immune, and it contains the coli heat-sensitive enterotoxin mutant protein of the present invention of safe and effective amount, and pharmaceutically acceptable carrier or vehicle.This class carrier comprises (but being not limited to): salt solution, damping fluid, glucose, water, glycerine, ethanol and combination thereof.Composition of the present invention can be made into to be suitable for any form of mucosal drug delivery.The content of coli heat-sensitive enterotoxin mutant protein in composition for example can be 0.0001-20wt%; That better is 0.001-10wt%.
When using said composition, be that coli heat-sensitive enterotoxin mutant protein with safe and effective amount is applied to Mammals, wherein this safe and effective amount is usually at least about 1 microgram/kg body weight, and in most of the cases be no more than about 8 mg/kg body weight, preferably this dosage is about 1 microgram/kg body weight-Yue 1 mg/kg body weight.Certainly, concrete dosage also should be considered administering mode, factors such as experimenter's physical appearance, and these all are within the skilled practitioners skill.
In addition, also contain in the described composition: the vaccine of significant quantity, its content in composition is 0.0001-20wt% for example; That better is 0.001-10wt%.Thereby under the assisting a ruler in governing a country of coli heat-sensitive enterotoxin mutant protein of the present invention, produce good immune effect of vaccine.
Major advantage of the present invention is:
(1) confirmed that these two mutant toxicity of LTK63/G192 and LTR72/G192 significantly reduce, and mucous membrane adjuvanticity excellence, obviously be better than LTK63/G72.
(2) expression process of LT mutant is optimized, thereby the albumen that obtains more approaches its natural existing way, has good activity.
Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiment only to be used to the present invention is described and be not used in and limit the scope of the invention.The experimental technique of unreceipted actual conditions in the following example, usually according to people such as normal condition such as Sambrook, molecular cloning: lab guide (New York:Cold Spring Harbor Laboratory Press, 1989) condition described in, or the condition of advising according to manufacturer.Unless otherwise indicated, otherwise per-cent and umber calculate by weight.
Materials and methods
1. bacterial strain and plasmid
Bacterial strain and plasmid: with P1/P2 is primer, intestinal bacteria with generation coli heat-sensitive enterotoxin are template, pcr amplification obtains wild LT gene, connects into after cutting with NcoI and SalI enzyme among the pET30a (Novagen) that cuts through same enzyme, obtains plasmid pET30a-LT.
Bacterial strain adopts conventional bacillus coli DH 5 alpha, BL21 (DE3).
2. laboratory animal
6～8 age in week Kunming be that female small white mouse is provided by laboratory animal portion of Medical Center of Fudan University, the low virulence living vaccine (Lasota) of newcastle disease is available from Songjiang, Shanghai biologics company limited.
3. mutant expression vector establishment
(1) design of primers
With reference to logining the LT complete genome sequence, design primer to P1/P2, the full gene of LT that is used to increase, the primer two ends are added NcoI and SalI restriction enzyme site (the frame line is partly represented) and protection base respectively; Design primer P3/P4 with its 63rd amino acid sites for the center, make its 63rd amino acids codon be converted to Lys codon AAA (underscore is represented) by Ser codon TCT; With the 72nd amino acids of LT is that the nucleotides sequence at center is classified as according to the design mutant primer P5/P6, and 72 GCA are replaced with CGT (underscore is represented); It is right to be with the 192nd amino acids of LT that the nucleotides sequence at center is classified as according to design P7/P8 mutant primer, and 192 AGA are replaced with GGA (underscore is represented), and it is synthetic that primer is given birth to the worker by Shanghai.
P1：5′-AG
3CAATGGCGACAGATTATACC-3′(SEQ ID NO：5)；
P2：5′-AC
3TTTTTCATACTGATTGCCGC-3′(SEQ ID NO：6)；
P3：5′-CGGATATGTTTCCACT
AAACTTAGTTTGAGAAGTGCTC-3′(SEQ ID NO：7)；
P4：5′-GAGCACTTCTCAAACTAAG
TTTAGTGGAAACATATCCG-3′(SEQ ID NO：8)；
P5：5′-GAGAAGTGCTCACTTA
CGTGGACAGTCTATATTATCAGG-3′(SEQ ID NO：9)；
P6：5′-CCTGATAATATAGACTGTCC
ACGTAAGTGAGCACTTCTC-3′(SEQ ID NO：10)；
P7：5′-GGTTGTGGAAATTCATCA
GGAACAATCACAGGTGATACTTG-3′(SEQ ID NO：11)；
P8：5′-CAAGTATCACCTGTGATTGTTC
CTGATGAATTTCCACAACC-3(SEQ ID NO：12)。
(2) LTK63 mutator gene expression vector establishment
A. the preparation of mutant gene fragment I and II
In 100 μ l reaction systems, add dNTPs, Ex Taq, 10 * damping fluid, corresponding primer to, sterilized water, template pET30a-LT.Wherein LTK63 gene fragment I amplification the primer is to being P1/P4, and fragment II amplification the primer is to being P2/P3.Reaction conditions finishes, and reclaims the PCR product.
B. the preparation of mutant LTK63 gene
Get PCR product mixing among a., adding dNTPs, Ex Taq, 10 * damping fluid, sterilized water carry out 8 PCR circulations earlier, add dNTPs, Ex Taq, 10 * damping fluid, sterilized water and primer again P1/P2 is carried out new PCR circulation, and reaction finishes, and reclaims the PCR product.
C. the evaluation of expression vector establishment and recombinant plasmid
Above-mentioned PCR product and pET30a carry out double digestion with NcoI and SalI respectively, and rubber tapping is reclaimed, and add DNA Ligation Kit with suitable mol ratio and connect.Connect product and transform DH5 α.Choose the clone and extract plasmid, make PCR and enzyme is cut evaluation.Positive recombinant plasmid order-checking, whether checking reading frame and mutational site be correct.
(3) LTR72 mutator gene expression vector establishment
Fragment I amplification the primer is to being P1/P5, and fragment II amplification the primer is to being P2/P6, and all the other are operated as LTK63 mutator gene expression vector establishment.
(4) LTK63/G192 mutator gene expression vector establishment
A. the preparation of mutant gene fragment I and II
In 100 μ l reaction systems, add dNTPs, Ex Taq, 10 * damping fluid, corresponding primer to, sterilized water, template pET30a-LTK63.Wherein LTK63/G192 gene fragment I amplification the primer is to being P1/P8, and fragment II amplification the primer is to being P2/P7.Reaction finishes, and reclaims the PCR product.
B. the preparation of the 63rd and 192 mutator genes (LTK63/G192)
The PCR product of getting aforementioned acquisition mixes, adding dNTPs, Ex Taq, 10 * damping fluid, sterilized water carry out 8 PCR circulations earlier, add dNTPs, Ex Taq, 10 * damping fluid, sterilized water and primer again P1/P2 is carried out new PCR circulation, reaction finishes, and reclaims the PCR product.
C. the evaluation of expression vector establishment and recombinant plasmid
Above-mentioned PCR product and pET30a carry out double digestion with NcoI and SalI respectively, and rubber tapping is reclaimed, and add conventional dna ligation kit and connect.Connect product transformed into escherichia coli DH5 α.The picking mono-clonal also extracts plasmid, and ordinary method is carried out PCR and enzyme is cut evaluation.Positive recombinant plasmid order-checking, checking reading frame and mutational site, thus acquisition has the recombinant expression vector pET30a-LTK63/G192 in correct mutational site.
(5) LTR72/G192 mutator gene expression vector establishment
Except template and primer generation respective change, all the other are operated as LTK63/G192 mutator gene expression vector establishment.
4. Recombinant Protein Expression, purifying and Western Blot detect
Express
Recombinant plasmid transformed BL21 (DE3), after the PCR evaluation, with IPTG abduction delivering albumen, the centrifugal culture supernatant of going of bacterium liquid is used NTA0 (20mmol/L Tris-HCl, 0.5mol/L NaCl, 10% glycerine) resuspended bacterial precipitation, the ultrasonic disruption thalline, 4 ℃ of magnetic stirrer are spent the night, centrifugal collection supernatant.
This features of experiment is: LT is that A subunit and B subunit are formed, and is combined into complete LT albumen behind A, the B subunit expression.So this bacterial precipitation of testing centrifugal gained directly uses Urea-NTAO resuspended, 4 ℃ of magnetic stirrer are spent the night, centrifugal collection supernatant.The A subunit still is in the same place with the B subunit like this, and does not upset the ratio between A subunit and the B subunit, and by after the renaturation, the E.coli LT structure is not destroyed like this.
Renaturation adopts ordinary method, dialyses in the NTA0 that contains 2M urea.
Purifying
With Ni-NTA Resin dress post, carry out purifying with the FPLC system.The albumen of purifying is in 4 ℃ of dialysis renaturation 24 hours, PBS dialysis, BCA standard measure.
Western blot detects
Purifying protein is carried out conventional SDS-PAGE electrophoresis, and be transferred on the nitrocellulose filter, it is one anti-using anti-CT antibody and His-tag antibody (all available from NEWLANBORS company) respectively, the albumen of test sample.
5. the ADP-ribose transferase active of recombinant protein detects
ADP-ribose transferase active detects according to document Soman G etc., Use of Substituted (Benzylidineamino) guanidines in the Study of Guanidino Group SpecificADP-ribosyltransferase[J]; Biochemistry, 1986,25:4113-4119 and De Haan etc., Mucosal immunogenicity and adjuvant activity of the recombinant Asubunit of the Escherichia coli heat-labile enterotoxin[J].Immunology, 1999,97 (4): the method for 706-713 is carried out.
Generate DEA-BAG (p-diethylamino-(benzylidineamino) guanidine) with the reaction of aminoguanidine bicarbonate and paradiethylaminobenzaldehyde, detect the photoabsorption of 355nm, the concentration of the standard of drawing-absorbance value curve with the DEA-BAG of known 8 different concns.Respectively get 750ng albumen trypsin treatment, 37 ℃ of water-baths 1 hour.Use the Trypsin inhibitor SBTI inhibited reaction.The DEA-BAG solution that adds equivalent starts reaction with NAD, 30 ℃ of water-baths 2 hours.Each DOWEX-50W resin absorption with equivalent goes out unreacted DEA-BAG.Filter, the photoabsorption of each reaction filtrate is detected at the 355nm place, and each albumen is done 3 each repetition.According to the concentration of DEA-BAG in the typical curve calculating reaction solution, calculate the amount of the DEA-BAG that is consumed.Amount with the DEA-BAG that LT was consumed is 100% enzymic activity, calculates each proteic relative activity.
6. the toxicity of recombinant protein detects
Patent-mouse toxicity detects test, according to Feng Qiang etc., and the expression of E.coli LT and purification storage strategy [J]; The biotechnology journal, 2003,19 (5): the method for 532-537 is carried out.The gained data are measured significant difference with the t-check, and P＞0.05 is not for remarkable, and P≤0.01 is for extremely remarkable, and 0.01＜P≤0.05 is remarkable.
7. the detection of albumen adjuvanticity
A. mouse immune experiment
5-6 female mice in age in week is divided into 8 groups, 5/group at random.One group of newcastle disease vaccine (being dissolved among the 0.1M PBS) collunarium immunity of only using 5 plumage parts (with reference to the vaccine working instructions), all the other respectively organize newcastle disease vaccine and various toxin protein 4 μ g (being dissolved among the 0.1M PBS) immunity with 5 plumage parts.Respectively 1,12, carried out first, second and third time immunity in 26 days.0,11, docking blood sampling in 25,35 days uses 500 μ l PBS (0.1%BSA) to wash nose simultaneously.Blood sample spends the night for 4 ℃, and the centrifugal 5min of 5000rpm/min collects serum and is used for detecting.-20 ℃ of preservations.
B. indirect ELISA detects different treatment group antibody horizontal
By ELISA Sptting plate (100 μ l/ hole), ordinary method detects IgA antibody horizontal in serum IgG, the nose washing lotion with newcastle disease LaSota strain virus bag.
Embodiment
The pcr amplification and the restriction analysis of embodiment 1 expression vector establishment
Mutant expression vector pET30a-LTK63 and pET30a-LTR72 with aforementioned structure is template respectively, is that primer amplification goes out LTK63/G192I and the LTR72/G192I gene that size is about 600bp with P1/P4; With P2/P3 is that primer amplification goes out LTK63/G192II and the LTR72/G192II gene that size is about 530bp.Is template to fragment I and II with each, and being primer with P1/P2 amplifies LTK63/G192 and the LTR72/G192 of about 1100bp with gene splicing by overlap extension, size all with expect suitable.
Sequencing result shows that the 63rd amino acids of LT gene fragment is Lys by the Ser codon mutation among the pET30a-LTK63/G192, and the 192nd amino acid sports Gly by Arg; The 72nd amino acid code of LT gene fragment sports Arg by Ala among the pET30a-LTR72/G192, and the 192nd amino acid code sports Gly by Arg.
The agarose gel electrophoresis analysis that expression vector pET30a-LTK63/G192 and pET30a-LTR72/G192 make up as depicted in figs. 1 and 2.
Abduction delivering, purifying and the Western-Blot of embodiment 2 reorganization bacterium detect
Being inoculated into respectively in the liquid nutrient medium through each correct mutants which had of order-checking reading frame, is the IPTG abduction delivering of 1mmol/L with final concentration, and two mutant all give expression to two protein bands of about 33.0Ku and 13.0Ku; Recombinant expression protein carries out purifying with conventional Ni-NTA resin, uses the imidazoles wash-out, and electrophoresis detection obtains two bands of about 33.0Ku and 13.0Ku, and is anti-as one with anti-His-tag antibody, detects recombinant protein.LTK63/G192 protein expression situation is seen Fig. 3 A, and the SDS-PAGE of LTK63/G192 protein purification sees Fig. 3 B, and two proteic Western-Blot of LTK63/G192 albumen and LTR72/G192 detect and see Fig. 3 C.
Embodiment 3ADP-ribose transferase active detects
With DEA-BAG as substrate, start reaction with NAD, survey the absorbance value at 355nm place behind the reaction terminating, calculate the consumption of substrate according to normalized form, with amount of substrate that LT was consumed as enzymic activity 100%, calculate other each proteic relative activity, in contrast with isopyknic not protein-contg PBS.Result such as table 1 show.
Table 1 ADP-ribose transferase active detected result
| Toxin protein | ADP-ribose transferring enzyme (%) |
| Contrast (PBS) | 6.54 |
| LT | 100 |
| LTK63 | 11.71 |
| LTR72 | 12.06 |
| LTG192 | 8.71 |
| LTK63/G192 | 8.76 |
| LTR72/G192 | 8.78 |
As seen from the above table, the ADP-ribose transferase active of LTK63/G192 and LTR72/G192 is lower than LTK63 and LTR72.
The toxicity of embodiment 4LT mutant detects
Respectively with PBS, wild-type LT albumen and each mutant protein mouse (Patent-mouse) of feeding, get the heavy ratio heavy of its intestines with corpse, calculate each class mean and standard deviation, each mutain group all is t-check, result such as table 2 and Fig. 4 with PBS, wild-type LT.
The t-assay that table 2Patent-mouse live body toxicity detects
The result shows that each mutant is compared with wild-type LT, and toxicity all significantly reduces.
The research of embodiment 5 adjuvanticities
Each is organized mouse and uses newcastle disease vaccine (NDV) or newcastle disease vaccine and each toxin protein to carry out first, second and third time immunity at 1,12,26 day respectively.Collect serum docking blood sampling in 0,11,25,35 day, use 500 μ l PBS (0.1%BSA) to wash nose simultaneously.Detect the IgG of each serum sample and the IgA of nose washing lotion sample with ELISA, under the 450nm light wave, read the OD value.Getting three exempts from reading that each group of back adds toxin protein and only carries out the t-value with the reading of NDV immune group and check.P＞0.05 difference is not remarkable, 0.01＜P＜0.05 significant difference, and P＜0.01 difference is extremely remarkable.The result is shown in Fig. 5 and Biao 3-table 4.
Table 3: three add toxin protein+NDV group after exempting from checks with the t-value of the IgG level of NDV group
Table 4: three exempt from the back, and each adds the t-value check of the IgA level of toxin protein+NDV group and NDV group
The result shows, compares with NDV with single, can significantly improve IgG and IgA level in the animal body with LTK63/G192 or LTR72/G192 simultaneously, and promptly they have good immunological adjuvant activity; And LTK63/R72 is not remarkable.
A kind of composition that is used for mucosa-immune, described composition contains: the LT mutant protein LTR72/G192 of the aforementioned purifying of 4 μ g, as the adjuvant of mucosa-immune; The newcastle disease vaccine of 1-2 head part (is dissolved among the PBS, 0.5ml); And the 0.1M PBS of 0.5mL.
All quote in this application as a reference at all documents that the present invention mentions, just quoted as a reference separately as each piece document.Should be understood that in addition those skilled in the art can make various changes or modifications the present invention after having read above-mentioned teachings of the present invention, these equivalent form of values fall within the application's appended claims institute restricted portion equally.
Sequence table
＜110〉Academy of Agricultural Sciences, Shanghai City
＜120〉coli heat-sensitive enterotoxin double-mutant is as vaccine mucosa-immune adjuvant
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Claims (10)
1. isolating coli heat-sensitive enterotoxin mutant protein, it is characterized in that, described albumen comprises proteic A subunit of coli heat-sensitive enterotoxin and B subunit, and the aminoacid sequence of this A subunit is with respect to the aminoacid sequence of the A subunit of wild-type e. coli thermo-sensitivity enterotoxin
Its 63rd is K, and the 192nd is G;
Its 72nd is R, and the 192nd is G; Or
Its 63rd is K, and the 72nd is R, and the 192nd is G.
2. albumen as claimed in claim 1 is characterized in that, described albumen contains 1 proteic A subunit of coli heat-sensitive enterotoxin and 5 proteic B subunits of coli heat-sensitive enterotoxin.
3. an isolating nucleic acid is characterized in that, described nucleic acid encoding claim 1 or 2 described albumen.
4. a carrier is characterized in that, described carrier contains the described nucleic acid of claim 3.
5. a cell is characterized in that, described cell contains the described carrier of claim 4, or is integrated with the described nucleic acid of claim 3 in its genome.
6. production claim 1 or 2 described proteic methods is characterized in that, comprising: under the condition that is suitable for expressing, cultivate the described cell of claim 5, thereby express claim 1 or 2 described albumen.
7. method as claimed in claim 6 is characterized in that, comprising:
(1) at the A subunit and the B subunit of cell inner expression coli heat-sensitive enterotoxin mutant protein;
(2) collect the cell of (1), with denaturing agent resuspended after with cytoclasis;
(3) with the cytoclasis product mixing of (2), centrifugal collection supernatant;
(4) separate the albumen that contains A subunit and B subunit from the supernatant that (3) obtain, renaturation obtains desirable proteins.
8. claim 1 or 2 described proteic purposes is characterized in that, are used to prepare the adjuvant of mucosa-immune.
9. a composition that is used for mucosa-immune is characterized in that, described composition contains: the claim 1 of significant quantity or 2 described albumen, and as the adjuvant of mucosa-immune.
10. composition as claimed in claim 9 is characterized in that, also contains in the described composition: the vaccine of the mucosa-immune of significant quantity.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104498520A (en) * | 2014-12-25 | 2015-04-08 | 华东理工大学 | In-vitro preparation method of escherichia coli heat-sensitive toxin mutant LTm |
| CN105238805A (en) * | 2014-07-11 | 2016-01-13 | 上海市农业科学院 | Expression vector and preparation method of recombinant escherichia coli heat-labile enterotoxin mutant protein |
| CN110075290A (en) * | 2018-01-25 | 2019-08-02 | 吴夙钦 | Influenza mucosal vaccine composition and the preparation method and application thereof |
| CN117625505A (en) * | 2023-11-27 | 2024-03-01 | 华中农业大学 | Lactococcus lactis vector oral vaccine for producing enterotoxin escherichia coli K88 genotype and application |
| CN117625505B (en) * | 2023-11-27 | 2024-05-03 | 华中农业大学 | Lactococcus lactis vector oral vaccine for producing enterotoxin escherichia coli K88 genotype and application |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1696291A (en) * | 2004-07-15 | 2005-11-16 | 中国人民解放军第三军医大学 | New type mutant of heatlabile enterotoxin from bacteria coli, and preparation method |
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2008
- 2008-04-15 CN CN2008100360321A patent/CN101560247B/en not_active Expired - Fee Related
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105238805A (en) * | 2014-07-11 | 2016-01-13 | 上海市农业科学院 | Expression vector and preparation method of recombinant escherichia coli heat-labile enterotoxin mutant protein |
| CN104498520A (en) * | 2014-12-25 | 2015-04-08 | 华东理工大学 | In-vitro preparation method of escherichia coli heat-sensitive toxin mutant LTm |
| CN110075290A (en) * | 2018-01-25 | 2019-08-02 | 吴夙钦 | Influenza mucosal vaccine composition and the preparation method and application thereof |
| CN117625505A (en) * | 2023-11-27 | 2024-03-01 | 华中农业大学 | Lactococcus lactis vector oral vaccine for producing enterotoxin escherichia coli K88 genotype and application |
| CN117625505B (en) * | 2023-11-27 | 2024-05-03 | 华中农业大学 | Lactococcus lactis vector oral vaccine for producing enterotoxin escherichia coli K88 genotype and application |
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