CN103667164A - Food-grade recombinant lactobacillus for production and delivery of protein or active peptide, and preparation method thereof - Google Patents
Food-grade recombinant lactobacillus for production and delivery of protein or active peptide, and preparation method thereof Download PDFInfo
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Abstract
The invention relates to a food-grade recombinant lactobacillus for the production and delivery of a protein or an active peptide, and preparation method thereof. The food-grade recombinant lactobacillus is characterized by being composed of a food-grade thyA gene (a thymidylate synthase gene) deficient lactobacillus strain, and an expression frame of a foreign protein or a foreign active peptide integrated with the chromosome of the food-grade thyA gene deficient lactobacillus strain. The food-grade recombinant lactobacillus is further characterized in that the complete expression frame of the foreign protein or peptide is composed of a constitutive promoter sequence, a signal peptide sequence, and an anchor stator sequence (which is not included for secretory expression). The food-grade recombinant lactobacillus disclosed by the invention contains no any new introduced antibiotic resistance gene. The food-grade recombinant lactobacillus is a live vector technology platform and has excellent universality; and the food-grade recombinant lactobacillus can be used for developing live vector vaccines, live vector nano antibodies, nutritional proteins, active peptides, protide hormones, cytokines and the like, and thus can be applied to many industries such as medicine, food and feed.
Description
Technical field
The present invention relates to a kind of food grade milk-acid bacteria live vector of producing and send for protein or active polypeptide and preparation method thereof.More particularly, the present invention relates to a kind of for the production of the food-grade recombination lactic acid bacterium with the protein such as delivery of antigens protein, antibody protein, trophicity protein, active polypeptide, protein hormone, cytokine or active polypeptide, and preparation method thereof.
Technical background
Human or animal's gastrointestinal tract mucosa, respiratory mucosa and reproductive tract mucous membrane exist rich and varied medicine associated receptor, are the targets of numerous protein or polypeptide drugs.Wherein, gastrointestinal system is again a complicated large-scale organ simultaneously, is exercising very important physiological function, comprises absorption, immunomodulatory, energy balance and the metabolism etc. of food.In gi tract, respiratory tract and reproductive tract, exist a large amount of microorganisms, both comprised probiotics, as milk-acid bacteria, also comprise pathogenic micro-organism.
Milk-acid bacteria is gram-positive microorganism, belongs to probiotic bacterium.In recent years, along with the development of milk-acid bacteria genomics and enriching constantly of genetic manipulation instrument, milk-acid bacteria is developed into gradually the cell factory of desirable production protein or active polypeptide and sends live vector.The expression system that is applied to the milk-acid bacteria in food, beverage and feeds product must be food grade.Be that applied Host Strains, inductor and carrier three must be food grade.First, the Host Strains that expression of recombinant proteins is used must be the clear and stable food-grade microorganisms of safe, feature; Secondly, inductor must be food grade, can be eaten by people or the material harmless to people or the harmless condition such as temperature, acid induction; In addition, carrier must be also food grade, can not have the functional DNA of nonfood grade to exist, and must derive from food-grade microorganisms with food grade expression system relative dna, and not containing antibiotics resistance gene, does not have hazardous compound to produce.Such expression system is just applicable to large-scale commercial production.
Use milk-acid bacteria to produce and delivery vector as protein or active polypeptide, first lactic bacterium strains to be transform as to " cell factory " of a pharmaceutical protein or polypeptide, comprise: the auxotroph of a) setting up milk-acid bacteria, be about to a milk-acid bacteria necessary gene of surviving and knock out (being generally thymidylate synthase gene thyA) from karyomit(e), like this, when milk-acid bacteria is discharged in environment by human or animal, lactic-acid bacteria cells can be dead rapidly, to avoid the diffusion of gene in environment; B) build suitable expression cassette, to guarantee that drug target protein or polypeptide can express in suitable level, effectively be secreted into extracellular or be illustrated in lactic-acid bacteria cells wall; C) to guarantee the correct folding of protein, keep correct three-dimensional structure and biological activity; D) whole expression cassette is inserted into the karyomit(e) of milk-acid bacteria, and removes all antibiotics resistance genes of introducing in genetic manipulation process, to guarantee the biological safety of product.Then, recombinant lactic acid bacteria can carry out fermentative production by normal fermentation technology, through frozen dried, can be prepared into capsule, tablet or electuary, for directly oral.
United States Patent (USP) " Lactobacilli harboring aggregation gene as a vaccine delivery vehicle (US006100388A; 2000) " has been announced a kind of by exogenous antigen protein and the method for condensing gene (aggregation gene) or Saliva Orthana binding factor amalgamation and expression, and lactobacillus reuteri (Lactobacillus reuteri) is developed as to vaccine carrier.United States Patent (USP) " Bacterial delivery ofbiologically active polypeptides (US8066987B2,2011) " has been announced a kind of method of using Gram-negative bacteria to send biologically active polypeptides.Milk-acid bacteria used in the present invention is gram-positive microorganism, and meanwhile, the genetic regulation element using is not all at above-mentioned patent institute protection domain.
Use food grade milk-acid bacteria as cell factory and send live vector and produce and send protein or active polypeptide has following advantage:
1) owing to thering is good security, the food-grade recombination lactic acid bacterium of expressing exogenous protein or active polypeptide can be for directly oral, with respect to protein or the polypeptide drugs of needs injection, oral administration is easier to be accepted by patient, and treatment cost is lower.This product also can directly apply to feed fermentation, feed interpolation etc.
2) can be by protein or polypeptide along with the efficient target of milk-acid bacteria be in alimentary canal mucous membrane, respiratory mucosa, or reproductive tract mucous membrane.Because milk-acid bacteria live vector protein or polypeptide are synthetic and discharge at human body or animal internal in-situ by food-grade recombination lactic acid bacterium, these protein or polypeptide can only accumulate relatively high concentration in target position, and for non-target position, the concentration of accumulation is extremely low, so the side effect causing is also extremely low.By contrast, traditional protein or polypeptide need to be injected or vein enters human body or animal body, then by spreading all over the recycle system of whole body, arrive target, but are also distributed in a large number non-target tissue and organ simultaneously, both having brought certain side effect, is also a kind of waste.
3) good dynamic metabolism.Food-grade recombination lactic acid bacterium, surely grow in alimentary canal mucous membrane, respiratory mucosa, or during reproductive tract mucous membrane, exogenous protein or polypeptide are along with the growth of lactic-acid bacteria cells and division discharge gradually.This has played the slow releasing function of medicine, has also simulated the synthetic and dispose procedure of the cytokine of animal body self, meets optimal pharmacokinetics requirement.And traditional protein or polypeptide drugs are owing to being one to cross property administration, medicine reaches rapidly peak value after administration, then decay rapidly.
4) produce very convenient.Food grade milk-acid bacteria live vector protein or active polypeptide can be produced by conventional fermentation using bacteria and freeze drying technology.The bacterium powder of freeze-drying can be made into tablet, capsule or electuary.Whole manufacturing process is very simple, with low cost.
Summary of the invention
The object of this invention is to provide a kind of food grade milk-acid bacteria live vector of producing and send for protein or polypeptide and preparation method thereof.
The invention is characterized in, host milk-acid bacteria is thyA gene (thymine synthetase gene) defective type; The expressed intact frame of exogenous protein or polypeptide forms (when for secreting, expressing, not comprising grappling subsequence) by constitutive promoter sequence, signal peptide sequence and grappling subsequence.ThyA gene on the expression cassette of exogenous protein or active polypeptide and milk-acid bacteria karyomit(e) is incorporated on milk-acid bacteria karyomit(e) by recombinate producer displacement of locus specificity.The antibiotics resistance gene that genetic manipulation is introduced is finally knocked.
The present invention is achieved by following technical proposals:
Milk-acid bacteria starting strain of the present invention refers to that < < that the Ministry of Health issues can be used for all bacterial strains of all genus lactubacillus that the bacterial classification list > > of food (defend do supervision send out (2010) No. 65) comprises, that is: Lactobacterium acidophilum (Lactobacillus acidophilus), lactobacterium casei (Lactobacillus casei), lactobacillus crispatus (Lactobacillus crispatus), lactobacillus bulgaricus (Lactobacillus bulgaricus), lactobacillus delbruckii (Lactobacillus delbrueckii), lactobacillus fermentum (Lactobacillus fermentium), lactobacillus gasseri (Lactobacillus gasseri), lactobacterium helveticus (Lactobacillus helveticus), Lactobacillus johnsonii (Lactobacillus johnsonii), lactobacillus paraceasi (Lactobacillus paracasei), plant lactobacillus (Lactobacillus plantarum), lactobacillus reuteri (Lactobacillus reuteri), lactobacillus rhamnosus (Lactobacillus rhamnosus), lactobacillus salivarius (Lactobacillus salivarius), and Lactococcus lactis subsp.lactis (Lactococcus lactis subspecies lactis) etc.
Exogenous protein of the present invention or polypeptide, comprise antigen protein, antibody protein, trophicity protein, active polypeptide, protein hormone, cytokine etc.Wherein cytokine comprises IL-1 β, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-15, IL-16, IL-17, IL-18, IL-19, IL-20, IL-21, IL-23, IL-24, IL-25, IL-26, IL-32, cMGF, LT, GM-CSF, M-CSF, SCF, IFN-γ, IFN λ, EPO, G-CSF, LIF, OSM, CNTF, GH, PRL, IFN α, IFN β etc.
Exogenous protein of the present invention or polypeptide, its nucleotide sequence all needs to be optimized according to the codon usage bias of milk-acid bacteria, is beneficial to express at lactic-acid bacteria cells.
Promotor of the present invention refers to constitutive promoter.This constitutive promoter is the similar sequence of Bacterium lacticum rRNA promotor.Gene Replacement of the present invention, realizes by the restructuring of Cre/lox locus specificity.When knocking out thyA gene, insert in situ the expressed intact frame of exogenous protein or polypeptide, remove antibiotics resistance gene simultaneously.
Exogenous protein of the present invention or polypeptide, final secreted expression is to outside host's lactic-acid bacteria cells, or grappling is illustrated on lactic-acid bacteria cells wall, is to realize by the signal peptide from different and the sub-amalgamation and expression of grappling.
Compared with prior art, the present invention has following beneficial effect:
1) because used expression regulation element all comes from lactic-acid bacteria cells self, the expression cassette of exogenous protein or polypeptide has been incorporated on milk-acid bacteria karyomit(e), and the antibiotics resistance gene of introducing in genetic manipulation process is finally all removed, so final milk-acid bacteria live vector protein or polypeptide are real food grade products, meet Biosafety requirement.
2) milk-acid bacteria live vector protein of the present invention or polypeptide, can use by oral, collunarium, reproductive tract, direct target digestive tube, respiratory tract or reproductive tract mucous membrane, and synthesize in position and discharge protein or polypeptide, thereby it is produced and use cost is very cheap.
3) expression cassette due to exogenous protein or polypeptide is present on karyomit(e) rather than plasmid, recombinant lactic acid bacteria proterties when succeeding transfer culture is more stable, can not produce the spawn degeneration problem causing due to plasmid loss, again owing to using non-inducible promoter, during fermentation, do not need to use inductor, thereby produce very easy.
4) food grade milk-acid bacteria live vector belongs to a technology platform, there is good versatility, can be used for developing antigen protein, antibody protein, trophicity protein, active polypeptide, protein hormone, cytokine etc., be applied to all conglomeraties such as medicine, food, feed.
Accompanying drawing explanation
Accompanying drawing 1: the secreting, expressing frame of exogenous protein or active polypeptide.
Accompanying drawing 2: the cell walls presenting and expressing frame of exogenous protein or active polypeptide.
Embodiment
Below in conjunction with specific examples, the present invention will be further described.Protection scope of the present invention is not limited to following example.
Embodiment 1: the encoding gene nucleotide sequence of exogenous protein or polypeptide is optimized.
For make exogenous protein or polypeptide can be milk-acid bacteria smooth and high efficient expression, need to according to milk-acid bacteria, to the use Preference of codon, be optimized the nucleotide sequence of its encoding gene, to remove rare codon.
The principle of optimizing is: TTC → TTT; GTA → GTT; TCC → TCT; GAG → GAA; TGC → TGT; AGA → CGT; AGG → CGT
Embodiment 2: protein
or the secretion of polypeptide
The structure of expression cassette
The combination that the secreting, expressing frame of protein or polypeptide comprises following expression regulation element, i.e. catenation sequence between promotor, signal peptide sequence, antigen encoding gene sequence and each expression regulation element (seeing accompanying drawing 1).
In the present embodiment, the expression of protein or polypeptide is controlled by constitutive promoter.This constitutive promoter is a kind of in following sequence, or surpasses a kind of in 80% sequence with the similarity of following sequence:
1)GTC?GGT?CGA?GTT?GTT?GAC?AGG?ATA?AAG?GTC?GCC?TGG?TAT?GGT?CTC?AAT?ATA?GCG
2)GTC?GTG?GCA?GTT?GTT?GAC?AAC?CTG?TGG?GCG?GTT?TGA?TTT?GTT?CTT?GCT?ATA?GCG
3)ACC?GAG?CCA?GTT?GTA?GAC?AGG?GCT?GTG?CTG?ACC?TGG?TAA?GGT?ATA?TTA?TAG?CG
4)GAG?CTG?CGA?GTT?GTT?GAC?ATT?GTT?AAT?GGC?CCC?TGA?TAT?ATT?TGG?CGT?ATA?GCG
5)GGG?GTA?GTT?GTT?GAC?AGC?GTG?GGT?TGG?TGC?TGG?TAA?TTT?TGC?GCT?ATA?GCG
6)GAG?TCT?TGC?GCT?ATA?GTT?GTT?GTC?AGA?ATG?GAG?ATA?CTA?TGA?TAT?AAT?GTT?GCT?ATA?GCG
7)AGG?GAG?TGA?GTT?GTG?ACA?GGG?CTG?TGA?TGG?TGT?GGT?ATT?GTT?TTG?GTA?TAG?CG
8)ACG?AGT?TGT?TGG?TAT?TGT?GGC?GGT?ATA?GCG
9)AGG?GGT?TGA?GTT?TGA?CAC?TGA?TCC?CGG?CTG?GTG?GTA?AAT?TTT?CGT?TAT?AGC?G
In the present embodiment, between promoter sequence and signal peptide sequence, with CAT ATG, the specific enzymes of endonuclease NdeI is cut site sequence connection, introduces thus atg start codon ATG, facilitates genetic manipulation.
In the present embodiment, signal peptide sequence is a kind of in following sequence, or surpasses a kind of in 80% sequence with the similarity of following sequence:
1)ATT?AAT?GAT?TCA?ACC?ACG?GTT?GAA?CCA?GTG?CTG?GAT?GGC?CCG?TAT?CAGCCA?ACT?ACA?TTT?AAA?CCG?CCA?AAC?GAT?TAT?TGG?TTG?CTG?ATT?AGT?AGC?AAT?ACC?GAT?GGT?GTT?GTG?TAC
2)AAA?CCG?CCA?AAC?GAT?TAT?TGG?TTG?CTG?ATT?AGT?AGC?AAT?ACC?GAT?GGT?GTT?GTG?TAC?GAA?AGC?ACT?AAC?AAC?TCA?GAT?TTC?TGG?ACA?GCG?GTT?ATT?GCC?GTG?GAA?CCG?CGT?GTT?TCA
3)TAA?ACG?GAT?AAC?CAA?AAT?ACG?GGC?TAA?CAT?CAT?CGT?CAT?GGT?TTG?CCA?TTA?CTG?CCA?GTT?TGG?CCG?CTC?GTC?GTC?ATC?TCT?TTC?GGG?GGT?GTG?TCA?ATC?TGG?GTG?GTT?GAT?GCT?TGA?TCT
4)TTT?AGC?AAT?CGT?CGC?ACC?TTA?ACG?AGT?AAC?AAC?CGT?TTA?GTT?GGT?ATG?TTG?AAG?TAT?GGT?GGC?CGC?GTG?TGG?ACC?TTT?CAT?GGC?GAA?ACG?CCA?CGT?GCG?ACC?ACG?GAT?AGT?AGC?AAC
5)ATT?TAT?CGT?CAG?CTG?TTA?ACC?AAT?TCA?TAT?TCT?GTT?GAT?CTG?CAC?GAT?GAA?ATC?GAA?CAA?ATC?GGT?AGC?GAA?AAG?ACC?CAG?AAC?GTG?ACG?ATT?AAT?CCG?GGC?CCA?TTT?GCG?CAAACC?CGT
6)AAA?TTG?ATG?ATC?TTA?GGC?ATG?CTC?GTT?TTT?AAA?ATT?AAT?AAG?GGG?GTA?ACG?GGG?GCA?ACA?ATG?GCG?CAT?GCT?AGC?ATC?AAT?CCT?GAA?ATG?ACG?ACC?GCA?GCC
7)TTA?ACG?AGT?AAC?AAC?CGT?TTA?GTT?GGT?ATG?TTG?AAG?TAT?GGT?GGC?CGC?GTG?TGG?ACC?TTT?CAT?GGC?GAA?ACG?CCA?CGT?GCG?ACC?ACG?GAT?AGT?AGC?AAC?ACT?GCC?GAT?CTG?AAT?AAC?ATT
8)GCC?GTG?GAA?CCG?CGT?GTT?TCA?CAA?ACG?AAT?CGC?CAG?TAT?ATT?CTG?TTT?GGT?GAA?AAC?AAG?CAA?TTC?AAC?ATC?GAA?AAC?AAC?AGT?GAT?AAA?TGG?AAG?TTT?TTC?GAA?ATG?TTC?AAG
In the present embodiment, between signal peptide sequence and antigen encoding gene sequence, with GTC GAC, the specific enzymes of endonuclease SalI is cut site sequence connection, to facilitate genetic manipulation.
In the present embodiment, gene is synthetic to be completed by the nucleic acid Composite service business on market, is dry powder DNA.Synthetic gene is present on the cloned plasmids that nucleic acid Composite service business provides.DNA is dissolved in to 100 microlitre ultrapure waters, as the template of the PCR step in embodiment 4.
Embodiment 3: the structure of the cell walls presenting and expressing frame of protein or polypeptide
The combination that the cell walls grappling expression cassette of protein or polypeptide comprises following expression regulation element, i.e. catenation sequence between promotor, signal peptide sequence, antigen encoding gene sequence, grappling subsequence and each expression regulation element (seeing accompanying drawing 2).
In the present embodiment, the catenation sequence between promotor, signal peptide sequence, antigen encoding gene sequence and this 3 elements is with identical described in embodiment 2.
In the present embodiment, the grappling subsequence that cell walls surface display is used, come from one of LPXT-block cell walls anchoring structure territory (LPXTG-motif cell wall anchor domain) of following protein, these protein are as follows at the coding of GenBank:
YP_005872271、YP_005873461、CCC77736、CCC77890、CCC78260、CCC78361、
CCC78381、CCC78520、CCC78612、CCC78778、CCC79729、YP_005873877、
YP_005873931, YP_005873973, YP_005874035, YP_005861438 etc.
Take protein YP_005861438 as example, and the aminoacid sequence of its grappling is:
TTNKLPQTGAKNELIAALSGLAVAGTTLVSYLGINRKKKNN
In the present embodiment, between antigen encoding gene sequence and grappling, with GGT GGA, the encoding sequence of two glycosides propylhomoserins connects.
In the present embodiment, gene is synthetic to be completed by the nucleic acid Composite service business on market, is dry powder DNA.Synthetic gene is present on the cloned plasmids that nucleic acid Composite service business provides.DNA is dissolved in to 100 microlitre ultrapure waters, as the template of the PCR step in embodiment 4.
Embodiment 4: the expression cassette of protein or polypeptide is replaced the thyA gene on Bacterium lacticum karyomit(e), and remove the antibiotics resistance gene of introducing
Bacterium lacticum described in the present embodiment refers in particular to lactobacillus fermentum (Lactobacillus fermentium).
Experiment completes in two steps, first a DNA segment that contains exogenous protein or expression of polypeptides frame, lox site, upstream, chloramphenicol resistance gene, lox site, downstream is recombinated and is inserted into the chromosomal thyA gene location of lactobacillus fermentum by locus specificity, replace thyA gene; And then use Cre enzyme to remove chloramphenicol resistance gene.
Wherein, insert segment design as follows: from DNA segment-exogenous protein of the 1000bp of lactobacillus fermentum Chromosome t hyA upstream region of gene or expression of polypeptides frame-lox site sequence-chloramphenicol resistance gene-lox site sequence-from the DNA segment of the 1000bp in lactobacillus fermentum Chromosome t hyA gene downstream.
Wherein, from the chromosomal DNA segment of lactobacillus fermentum, take lactobacillus fermentum genomic dna as template, by PCR, obtain.Take the exogenous protein described in embodiment 3 or expression of polypeptides frame is template, introduces lox site sequence by PCR at two ends.3 PCR segments head and the tail are overlapping 25bp in succession, and the DNA that is connected to a total length by overlapping extension PCR inserts segment.This DNA is inserted to segment to be connected with pNZ plasmid, and transform intestinal bacteria Top10 competent cell, on the BHI solid medium flat board that contains 5 mcg/ml paraxin, screen positive recombinant, and further in the BHI liquid nutrient medium that contains 5 mcg/ml paraxin, expand numerous, large quantity extracting plasmid DNA sequence verification.Extracted plasmid is converted into lactobacillus fermentum by electric shock, and the bacterium colony of only not growing on erythromycin flat board at paraxin flat board by parallel coated plate screening, further verifies by bacterium colony PCR, to obtain double exchange recon.
The plasmid that contains Cre recombinase encoding gene is transformed above-mentioned by the double exchange recon (need be prepared as in advance competent cell) of having verified, 37 ℃ are incubated at containing the MRS of 10 mcg/ml erythromycin dull and stereotyped, bacterium colony occurs that rear parallel painting is dull and stereotyped in the substratum that contains respectively 30 mcg/ml erythromycin and 10 mcg/ml paraxin, screening is responsive but have the recon of erythromycin resistance to paraxin, uses the excision of colony polymerase chain reaction (PCR) method checking chloramphenicol resistance gene.
By 37 ℃ of incubated overnight of this recon in the 10ml antibiotic MRS substratum containing how, so that the plasmid loss that contains Cre recombinase encoding gene.Nutrient solution is coated with to MRS flat board, 37 ℃ of incubated overnight, get the parallel coated plate of bacterium colony dull and stereotyped and not dull and stereotyped containing any antibiotic MRS in the MRS that contains 30 mcg/ml erythromycin, recon with screening to erythromycin-sensitive, the loss of the plasmid that contains Cre recombinase encoding gene by bacterium colony PCR checking.
Claims (10)
1. through genetic engineering modified food grade milk-acid bacteria live vector, for protein or active polypeptide, produce and send.It is characterized in that, it comprises the lactic bacterium strains of expressing and producing the thyA genetically deficient of exogenous protein or active polypeptide, is inserted into the expression cassette of the chromosomal exogenous protein of milk-acid bacteria or active polypeptide.
2. according to the lactic bacterium strains of claim 1, comprise Lactobacterium acidophilum (Lactobacillus acidophilus), lactobacterium casei (Lactobacillus casei), lactobacillus crispatus (Lactobacillus crispatus), lactobacillus bulgaricus (Lactobacillus bulgaricus), lactobacillus delbruckii (Lactobacillus delbrueckii), lactobacillus fermentum (Lactobacillus fermentium), lactobacillus gasseri (Lactobacillus gasseri), lactobacterium helveticus (Lactobacillus helveticus), Lactobacillus johnsonii (Lactobacillusjohnsonii), lactobacillus paraceasi (Lactobacillusparacasei), plant lactobacillus (Lactobacillus plantarum), lactobacillus reuteri (Lactobacillus reuteri), lactobacillus rhamnosus (Lactobacillus rhamnosus), lactobacillus salivarius (Lactobacillus salivarius), and Lactococcus lactis subsp.lactis (Lactococcus lactis subspecies lactis) etc.
3. according to the recombinant strains of lactic acid bacteria of claim 1, the expression cassette of exogenous protein or active polypeptide is incorporated on milk-acid bacteria karyomit(e) by the displacement with thyA gene, and has knocked out antibiotics resistance gene.
4. according to the exogenous protein of claim 1 or active polypeptide, refer to antigen protein, antibody protein, trophicity protein, active polypeptide, protein hormone, cytokine etc.Wherein cytokine comprises IL-1 β, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-15, IL-16, IL-17, IL-18, IL-19, IL-20, IL-21, IL-23, IL-24, IL-25, IL-26, IL-32, cMGF, LT, GM-CSF, M-CSF, SCF, IFN-γ, IFN λ, EPO, G-CSF, LIF, OSM, CNTF, GH, PRL, IFN α, IFN β etc.
5. according to the exogenous protein of claim 1 or active polypeptide, it is characterized in that, the nucleotide sequence of its encoding gene is optimized the use Preference of codon according to milk-acid bacteria.Its optimization principles is:
TTC→TTT；GTA→GTT；TCC→TCT；GAG→GAA；TGC→TGT；AGA→CGT；AGG→CGT
6. according to the nano antibody of claim 1 or affine expression of polypeptides frame, it is characterized in that, it comprises constitutive promoter sequence, signal peptide sequence and grappling subsequence (when for secreting, expressing, not comprising grappling subsequence).
7. according to the constitutive promoter sequence of claim 6, it is characterized in that, is a kind of in following sequence, or surpasses a kind of in 85% sequence with the similarity of following sequence:
1)GTC?GGT?CGA?GTT?GTT?GAC?AGG?ATA?AAG?GTC?GCC?TGG?TAT?GGT?CTC?AATATA?GCG
2)GTC?GTG?GCA?GTT?GTT?GAC?AAC?CTG?TGG?GCG?GTT?TGA?TTT?GTT?CTT?GCTATA?GCG
3)ACC?GAG?CCA?GTT?GTA?GAC?AGG?GCT?GTG?CTG?ACC?TGG?TAA?GGT?ATA?TTATAG?CG
4)GAG?CTG?CGA?GTT?GTT?GAC?ATT?GTT?AAT?GGC?CCC?TGA?TAT?ATT?TGG?CGTATA?GCG
5)GGG?GTA?GTT?GTT?GAC?AGC?GTG?GGT?TGG?TGC?TGG?TAA?TTT?TGC?GCT?ATAGCG
6)GAG?TCT?TGC?GCT?ATA?GTT?GTT?GTC?AGA?ATG?GAG?ATA?CTA?TGA?TAT?AATGTT?GCT?ATA?GCG
7)AGG?GAG?TGA?GTT?GTG?ACA?GGG?CTG?TGA?TGG?TGT?GGT?ATT?GTT?TTG?GTATAG?CG
8)ACG?AGT?TGT?TGG?TAT?TGT?GGC?GGT?ATA?GCG
9)AGG?GGT?TGA?GTT?TGA?CAC?TGA?TCC?CGG?CTG?GTG?GTA?AAT?TTT?CGT?TATAGC?G
8. according to the signal peptide sequence of claim 6, it is characterized in that, is a kind of in following sequence, or surpasses a kind of in 85% sequence with the similarity of following sequence:
1)ATT?AAT?GAT?TCA?ACC?ACG?GTT?GAA?CCA?GTG?CTG?GAT?GGC?CCG?TAT?CAGCCA?ACT?ACA?TTT?AAA?CCG?CCA?AAC?GAT?TAT?TGG?TTG?CTG?ATT?AGT?AGCAAT?ACC?GAT?GGT?GTT?GTG?TAC
2)AAA?CCG?CCA?AAC?GAT?TAT?TGG?TTG?CTG?ATT?AGT?AGC?AAT?ACC?GAT?GGTGTT?GTG?TAC?GAA?AGC?ACT?AAC?AAC?TCA?GAT?TTC?TGG?ACA?GCG?GTT?ATTGCC?GTG?GAACCG?CGT?GTT?TCA
3)TAA?ACG?GAT?AAC?CAA?AAT?ACG?GGC?TAA?CAT?CAT?CGT?CAT?GGT?TTG?CCATTA?CTG?CCA?GTT?TGG?CCG?CTC?GTC?GTC?ATC?TCT?TTC?GGG?GGT?GTG?TCAATC?TGG?GTG?GTT?GAT?GCT?TGA?TCT
4)TTT?AGC?AAT?CGT?CGC?ACC?TTA?ACG?AGT?AAC?AAC?CGT?TTA?GTT?GGT?ATGTTG?AAG?TAT?GGT?GGC?CGC?GTG?TGG?ACC?TTT?CAT?GGC?GAA?ACG?CCA?CGTGCG?ACC?ACG?GAT?AGT?AGC?AAC
5)ATT?TAT?CGT?CAG?CTG?TTA?ACC?AAT?TCA?TAT?TCT?GTT?GAT?CTG?CAC?GATGAA?ATC?GAA?CAA?ATC?GGT?AGC?GAA?AAG?ACC?CAG?AAC?GTG?ACG?ATT?AATCCG?GGC?CCATTT?GCG?CAA?ACC?CGT
6)AAA?TTG?ATG?ATC?TTA?GGC?ATG?CTC?GTT?TTT?AAA?ATT?AAT?AAG?GGG?GTAACG?GGG?GCA?ACA?ATG?GCG?CAT?GCT?AGC?ATC?AAT?CCT?GAA?ATG?ACG?ACCGCA?GCC
7)TTA?ACG?AGT?AAC?AAC?CGT?TTA?GTT?GGT?ATG?TTG?AAG?TAT?GGT?GGC?CGCGTG?TGG?ACC?TTT?CAT?GGC?GAA?ACG?CCA?CGT?GCG?ACC?ACG?GAT?AGT?AGCAAC?ACT?GCC?GAT?CTG?AAT?AAC?ATT
8)GCC?GTG?GAA?CCG?CGT?GTT?TCA?CAA?ACG?AAT?CGC?CAG?TAT?ATT?CTG?TTTGGT?GAA?AAC?AAG?CAA?TTC?AAC?ATC?GAA?AAC?AAC?AGT?GAT?AAA?TGG?AAGTTT?TTC?GAA?ATG?TTC?AAG
9. according to the grappling subsequence of claim 6, it is characterized in that, be one of LPXT-block cell walls anchoring structure territory (LPXTG-motifcell wall anchor domain) that comes from following protein, these protein are as follows at the coding of GenBank:
YP_005872271、YP_005873461、CCC77736、CCC77890、CCC78260、CCC78361、
CCC78381、CCC78520、CCC78612、CCC78778、CCC79729、YP_005873877、
YP_005873931, YP_005873973, YP_005874035, YP_005861438 etc.
10. according to the recombinant strains of lactic acid bacteria of claim 1, can for the production of and protein or active polypeptide are delivered to digestive tube, respiratory tract or reproductive tract.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN107961373A (en) * | 2017-11-14 | 2018-04-27 | 东北农业大学 | A kind of strain of gene engineered subunit oral vaccine and its construction method and purposes for being used to prevent pig epidemic diarrhea |
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| CN1642967A (en) * | 2002-01-31 | 2005-07-20 | 圣必健公司 | Clinical grade vectors based on natural microflora for use in delivering therapeutic compositions |
| CN1648255A (en) * | 2004-12-09 | 2005-08-03 | 中国疾病预防控制中心传染病预防控制所 | Food grade carrier of lactic galactococcus |
| CN101168741A (en) * | 2007-09-28 | 2008-04-30 | 中国疾病预防控制中心传染病预防控制所 | Lactococcus lactis food-sate secretion expression carrier and its preparing method and application |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN1642967A (en) * | 2002-01-31 | 2005-07-20 | 圣必健公司 | Clinical grade vectors based on natural microflora for use in delivering therapeutic compositions |
| CN1648255A (en) * | 2004-12-09 | 2005-08-03 | 中国疾病预防控制中心传染病预防控制所 | Food grade carrier of lactic galactococcus |
| CN101168741A (en) * | 2007-09-28 | 2008-04-30 | 中国疾病预防控制中心传染病预防控制所 | Lactococcus lactis food-sate secretion expression carrier and its preparing method and application |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN107961373A (en) * | 2017-11-14 | 2018-04-27 | 东北农业大学 | A kind of strain of gene engineered subunit oral vaccine and its construction method and purposes for being used to prevent pig epidemic diarrhea |
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