CN103656633A - Food grade lactic acid bacteria active carrier Group A rotavirus vaccine and preparation method thereof - Google Patents
Food grade lactic acid bacteria active carrier Group A rotavirus vaccine and preparation method thereof Download PDFInfo
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Abstract
The invention discloses food grade lactic acid bacteria active carrier Group A rotavirus vaccine and a preparation method thereof. The food grade lactic acid bacteria active carrier Group A rotavirus vaccine is characterized in that VP6 antigen protein from Group A virus, common serotype VP7 antigen protein (P serotype) and VP4 antigen protein (G serotype-expressed separately in the form of VP5* and VP8* protein subunit), vaccine adjuvant escherichia coli thermal unstable toxin B (LTB) and cholera toxin subunit B (CTB) are expressed and secreted by thyA gene deletion lactic acid bacteria cell or shown by the cell wall. Expressions of antigen protein and vaccine adjuvant protein are controlled by inducible or constitutive promoter, protein expression cassette is integrated onto the chromosome of the expression host lactic acid bacteria strain, and external antibiotics resistance gene introduced in gene manipulation is removed. The lactic acid bacteria active carrier rotavirus vaccine has the advantages of having wide serotype covering range, being easy to produce in large scale, and being safe and convenient to use without a refrigerator and a needle tubing.
Description
Technical field
The present invention relates to a kind of food stage lactobacillus live vector A group rotavirus vaccine and preparation method thereof, more particularly, the present invention relates to the polyvalent vaccine for preventing human or animal A group rotavirus to suffer from diarrhoea of using food stage lactobacillus living cells to prepare as carrier, and vegetation method.
Technical background
Rotavirus infection diarrhoea is infant emergency treatment and the dead second cause of disease.The nearly all 3 years old following child in the whole world will experience rotavirus infection and morbidity, every year because of rotavirus diarrhea approximately 1.3 hundred million people that cause suffering from diarrhoea, severe diarrhea approximately 1,800 ten thousand people, death toll approximately 450,000 people.Death is mainly distributed in South Asia, Africa, and East Asia Region.
China has nearly 40,000 children dead because of rotavirus diarrhea every year, accounts for 12% of 5 years old following total death toll of child of China.All there is the record of outbreak of epidemic in China each province, city.All can occur the whole year, and the autumn and winter are epidemic peak.42% rotavirus infection person does not have symptom, but can transmitted virus, and Diarrhea and asymptomatic carrier are the main sources of infection.
Rotavirus diarrhea occurs in the infant at 6-24 monthly age mostly, this disease infects except causing in intestinal, and diarrhoea, dehydration, electrolyte disturbance etc., also can cause viremia, cause even more serious complication, as myocarditis, encephalitis, pneumonia, hepatitis or acute pancreatitis etc.China's CDC rotavirus diarrhea disease burden latest data shows: 168 yuan/time of 5 years old following child's rotavirus diarrhea outpatient service average costs (92-230 unit/time), 5 years old following child's rotavirus diarrhea, 3145 yuan/time of the average costs (2760-4909 unit/time) of being in hospital.
Rotavirus (Rotavirus is called for short RV) is a kind of diplornavirus, belongs to Reoviridae.It is the single main cause of baby and infantile diarrhea.Rotavirus always has seven kinds, is numbered A, B, C, D, E, F and G etc. respectively with English alphabet.The mankind are mainly the infection that is subject to rotavirus A kind, B kind and C kind, and wherein modal be the infection of rotavirus A kind, it is all that this kind causes that human rotavirus infect to surpass 90% case.And these seven kinds of rotavirus all can cause disease with it other animals.
It is one of main arch-criminal of pig virus diarrhoea too that A group rotavirus infects.According to U.S.'s data, show, 1-4 piglet group sickness rate in age in week surpasses 80%, and mortality rate 7%～20%, causes very serious economic loss to pig industry.Current any antibiotic and treatment by Chinese herbs are all invalid for rotavirus infection, and still blank for the Rotavirus Vaccine of pig.
Take Human reoviruslike agent as example, and the genome of rotavirus has comprised 11 unique ribonucleic acid Double-helical molecules, always has 18,555 nucleoside bases pair in these 11.Each spiral or segmentation are genes, and are 1 to 11 according to the descending number consecutively of molecular dimension.Each gene can be encoded into a kind of protein, and wherein the 9th gene and the 11st gene can be encoded into two kinds of protein.Ribonucleic acid periphery is to have surrounded three layers of icosahedral protein housing (seeing Fig. 2).The about diameter 76.5nm of virion, and there is no peplos (Viral envelope).
Dual categorizing system has been used in the classification of rotavirus, according to two structural protein matter of this virosomal surface, does to classify.According to VP7 (glycoprotein), defined G type; According to VP4 (to protease-sensitive protein), defined P type.
At present, G serotype has at least identified 16 kinds, and P serotype has at least been identified 28 kinds.Wherein G serotype is modal is 1-4, and recent findings 9 and 12 also causes the generation of rotavirus infection in different regions.P serotype is modal is 4,6 and 8.
11 RNA segments of rotavirus, 12 protein of encoding altogether, comprise 6 structural protein matter and 6 unstructuredness protein.Wherein 6 virus proteins (viral protein, VP) framework whole virion (virion).These structural protein matter are called as respectively VP1, VP2, VP3, VP4, VP6 and VP7.6 unstructuredness protein (nonstructural protein, NSP) are only manufactured in the cell of rotavirus infection, and do not form the structure of virion.Be called NSP1, NSP2, NSP3, NSP4, NSP5 and NSP6.
In these 12 protein, with the closely-related protein of wheel virus antigen be VP7, VP6 and VP4.VP7 protein is a kind of glycoprotein that forms the outer capsid of virion.It determines the G type of rotavirus, has very strong antigenicity.VP6 protein is the structural protein matter that forms middle level capsid.It is the protein that antigenicity is strong, and can be used to differentiate the kind of rotavirus.VP4 protein is positioned at the surface of virion, outstanding and become a furcella.It connects the acceptor molecule of host cell surface, and orders about cell entry host cell.VP4 protein has determined the P type that this is viral, the more important thing is that it has determined the toxicity (Virulence) of rotavirus, this be because, only have VP4 protein by trypsin, to be decomposed into after VP5* and VP8* protein subunit in human or animal's intestinal, rotavirus just has infectiousness.VP5* and VP8* mediate respectively absorption and the intrusion of rotavirus, and this is also why rotavirus only infects the reason of animal intestinal specifically.
Rotavirus, mainly by fecal-oral transmission, also can pass through water, soil, food, toy, medicated clothing, the apparatus of pollution, even by propagation such as respiratory tracts, and therefore more difficult prevention.The main infection site of rotavirus is the finger-like fine hair of animal small intestinal inwall.Anatomical evidence shows, very thin because infecting the small intestinal inwall of the dead infant of rotavirus.Clinical symptoms after infant infection rotavirus is severe diarrhea, and suffer from diarrhoea 10-15 every day, continues nearly one week, if treatment not in time or incorrect, as easy as rolling off a logly causes infant dehydration dead.
When rotavirus touches host's small intestinal inwall epithelial cell, the VP4 that host produces or VP7 neutrality antibody are the main barriers that stops rotavirus to continue to infect.After this step failure, under the hydrolyzate VP5* and VP8* mediation of VP4, rotavirus is invaded host's epithelial cell, and causes host's protein metabolism disorder, has improved intracellular free calcium level and cell permeability, causes dyspepsia and diarrhoea.Now, host's anti-VP6 immunoglobulin A (IgA), and the cytokine of T emiocytosis can suppress rotavirus continuing in host cell and copies.If this step failure, rotavirus is by copying, and generation NSP4 enterotoxin, causes secretory diarrhea.Rotavirus can also stimulate enteric nervous system simultaneously, increases intestinal peristalsis promoting, stimulates secretory diarrhea.At later period of infection, host cell breaks, and by rotavirus, is killed.
Between each serotype of people's rotavirus, there is no cross protection, is typical isomorphic response.Rotavirus infection is confined to small intestinal mucosa, and the immunoreation that viral infection produces is also on small intestinal mucosa surface, and the time maintaining is also very short, only has some months.If but subinfection again, symptom conventionally than first, infect ill slightly.During child to 3 year old, substantially can set up the immunity to rotavirus.People's rotavirus middle level capsid protein matter (VP6) can with the rotavirus generation cross protection of calf, mice, piglets, lamb, rabbit and monkey, but shell antigen has the specificity of type.The Oral Rotavirus vaccine of Lanzhou Institute of Biological Products (2000 granted) is exactly according to qin Na Shi smallpox vaccination principle, by the G10 type lamb rotavirus exploitation that people is to weak poison, is people's Rotavirus Vaccine, belongs to the representative of first generation vaccine.
2nd generation Rotavirus Vaccine is with pentavalent people-Niu reprovision vaccine of Merck company
unit price Human reoviruslike agent vaccine with GSK
for representative.The former belongs to viral gene reprovision Seedling, and infection cell tissue together with the use rotavirus of animal and people's rotavirus, reconfigures, and then to people, inoculates; The latter is the Human reoviruslike agent strain through attenuation.
These vaccines all belong to live virus vaccines, all exist restructuring, make a variation and return malicious risk.The Human reoviruslike agent vaccine of being prepared by animal rotavirus, there is virus disseminating risk to former host animal in its strain.Because strain is single, its protectiveness is affected by areal variation and shows unstable in addition.Although multivalence reprovision vaccine and attenuated vaccine can cover more serotype, for the child of hypoimmunity or disappearance, still there is virulence risk.
Recombinant vaccine, comprises live vector vaccine, DNA vaccination, subunit vaccine etc., becomes gradually the focus of current vaccine research and development.Wherein live vector vaccine can be divided into again weak poisonous carrier and non-toxic carrier, virus or bacteria carrier etc. according to the difference of its carrier.Conventional viral carrier is vaccinia virus, bird pox virus etc., and bacillary carrier has lactobacillus, escherichia coli, attenuation salmonella etc.
Live vector vaccine does not re-use pathogenic viral pathogen, but the antigen encoding gene of pathogen (DNA) is carried on vaccine carrier, or antigen presentation, on vaccine carrier, is delivered to antigen or DNA by vaccine carrier to host's target position.Live vector vaccine can build polyvalent vaccine easily, and applicable large-scale production and inoculation.
In recent years, along with the development of lactobacillus genomics and enriching constantly of genetic manipulation instrument, the drug delivery that the live body lactobacillus of take is delivery vector becomes a focus of lactobacillus applied research gradually.Lactobacillus is a very good Rotavirus Vaccine delivery vector, because it is the probiotic bacteria of human body, adapts to intestinal environment, the more important thing is, it determine grow site and rotavirus to infect target spot identical, be all the epithelial cell of small intestinal inwall, there is natural space site competition.Meanwhile, lactobacillus self is again natural immunological adjuvant, and human body is had to good prebiotic effect.
Use food stage lactobacillus exploitation recombinant live-vector vaccine also to there is following advantage:
-can not only stimulate body to produce humoral immunization and the cellular immunization of high-efficient and lasting, can also bring out effective mucosal immunity, produce efficient comprehensively immunne response;
-with low cost, be easy to a large amount of production;
-than other preparation, there is longer storage life and stronger stability;
-immunization route is simple, be easy to inoculation, is particularly suitable for large-scale production and immunity inoculation.
Domestic patent (publication number CN1952156A) has been announced restructuring and the expression technology of a kind of rotavirus vp 6 genes and the non-resistance expression vector of lactobacillus.This patent is relevant with lactobacillus vaccine development, but just uses non-resistance expression vector to complete the expression of VP6 gene at lactobacillus.And the expression cassette of VP6 is still present on plasmid rather than chromosome.
Lactobacillus live vector has been widely used in the vaccine development of mucosa-immune.European Union's patent " Oral recombinant lactobacilli vaccines (EP1084709A1) " has been announced a kind of method of lactobacillus surface display antigen.As can commercial lactobacillus vaccine, antigen shows to be one of a plurality of sport technique segments at lactobacillus cell wall.European Union's patent " Lactobacilli harboring aggregation and mucin binding genes as vaccine delivery vehicles (EP1066375B1) " has been announced a kind of method of Lactobacillus reuteri cell wall surface display antigen.United States Patent (USP) " Live bacterial vaccine (US2009/0324638A1) " has been announced a kind of method for lactobacillus secretion or cell wall display protein matter or polypeptide.
Yet, so far, the total solution that does not also have a kind of rotavirus lactobacillus live vector vaccine to prepare.
Summary of the invention
The object of the invention is to announce a kind of A group rotavirus vaccine that food stage lactobacillus is live vector and preparation method thereof of take.
Food stage lactobacillus live vector Rotavirus Vaccine of the present invention, it is characterized in that, in lactobacillus cell expression secretion or cell wall, shown VP7 antigen protein (P serotype) and the VP4 antigen protein (G serotype) from the VP6 antigen protein of A papova, common serotype, the wherein VP4 antigen protein VP5* after trypsin decomposes and application of VP8* protein subunit form with it.
Food stage lactobacillus live vector Rotavirus Vaccine of the present invention, is further characterized in that, except above-mentioned antigen, it also comprises two vaccine adjuvants, i.e. coli heat instability mode toxin B (LTB) and cholera toxin subunit b (CTB).
Food stage lactobacillus live vector Rotavirus Vaccine of the present invention, be further characterized in that, the expression cassette of above-mentioned antigen and vaccine adjuvant by with lactobacillus cell chromosome on thyA gene substitution, not only knocked out thyA gene, the expression cassette of antigen and vaccine adjuvant has been incorporated into lactobacillus chromosome simultaneously, has removed the external source antibiotics resistance gene of introducing in genetic manipulation process simultaneously.
Food stage lactobacillus live vector Rotavirus Vaccine of the present invention, be further characterized in that, by different Expression elements, as being used in combination of promoter, signal peptide and grappling, antigen and vaccine adjuvant are to be secreted into outside lactobacillus cell, or are illustrated in cell wall surface.
The present invention is achieved by following technical proposals:
Main technologies of the present invention comprises: the antigen and the vaccine adjuvant that use a plurality of bacterial strains of lactobacillus to express respectively different serotypes.The expression of antigen and vaccine adjuvant is controlled by constitutive promoter, the expression cassette of antigen and vaccine adjuvant passes through Cre/lox locus specificity recombination and integration to the chromosomal thyA gene location of expressive host lactobacilli strain, replace with thyA gene, and remove antibiotics resistance gene.By the control of signal peptide and grappling, antigen and vaccine adjuvant are finally secreted into outside expressive host lactobacillus cell, or grappling is illustrated on cell wall.
Lactobacillus of the present invention refers to that < < that Ministry of Public Health is issued can be used for all bacterial strains of all genus lactubacillus that the strain list > > of food (defend do supervision send out (2010) No. 65) comprises, that is: bacillus acidophilus (Lactobacillus acidophilus), lactobacillus casei (Lactobacillus casei), Lactobacillus crispatus (Lactobacillus crispatus), Lactobacillus bulgaricus (Lactobacillus bulgaricus), Deshi Lactobacillus (Lactobacillus delbrueckii), Lactobacillus fermenti (Lactobacillus fermentium), Lactobacillus gasseri (Lactobacillus gasseri), lactobacillus helveticus (Lactobacillus helveticus), Lactobacillus johnsonii (Lactobacillusjohnsonii), Lactobacillus paracasei (Lactobacillusparacasei), Lactobacillus plantarum (Lactobacillus plantarum), Lactobacillus reuteri (Lactobacillus reuteri), lactobacillus rhamnosus (Lactobacillus rhamnosus), Lactobacillus salivarius (Lactobacillus salivarius), and Lactococcus lactis subsp.lactis (Lactococcus lactis subspecies lactis) etc.
Wheel virus antigen of the present invention, modal P serotype of dividing with VP4 and the G serotype of dividing with VP7 have been comprised, preparation multivalence lactobacillus live vector Rotavirus Vaccine, for inducing the antibody of the A group rotavirus of the generation of human or animal's body and various serotype.
Take Human reoviruslike agent as example, need the different serotypes antigen of single expression to comprise: VP7 (G1, G2, G3, G4, G9, G12 serotype), VP6 (A papova), VP4 (P[4], P[6], P[8] serotype), wherein VP5* and the VP8* protein subunit form of VP4 antigen after decomposing with trypsin expressed.
Take porcine rotavirus as example, need the different serotypes antigen of single expression to comprise: VP7 (G3, G4, G5, G9, G11 serotype), VP6 (A papova), VP4 (P[6], P[7], P[13], P[19], P[23], P[26] serotype), wherein VP5* and the VP8* protein subunit form of VP4 antigen after decomposing with trypsin expressed.
Take sheep rotavirus as example, need the different serotypes antigen of single expression to comprise: VP7 (G6, G8, G10 serotype), VP6 (A papova), VP4 (P[1], P[5], P[11], P[12], P[15] serotype), wherein VP5* and the VP8* protein subunit form of VP4 antigen after decomposing with trypsin expressed.
Vaccine adjuvant of the present invention, comprises coli heat instability mode toxin B (LTB) and cholera toxin subunit b (CTB).Not synantigen of the present invention and vaccine adjuvant, its aminoacid sequence is from GenBank data base.
Antigen of the present invention and vaccine adjuvant are all optimized nucleotide sequence according to the codon usage bias of lactobacillus, are beneficial to express at lactobacillus cell.Its optimization principles is: TTC → TTT; GTA → GTT; TCC → TCT; GAG → GAA; TGC → TGT; AGA → CGT; AGG → CGT
Of the present invention through the antigen after optimizing and the encoding gene of vaccine adjuvant, be to obtain by the nucleic acid Composite service business gene on market is synthetic.
Promoter of the present invention refers to constitutive promoter.This constitutive promoter is the similar sequence of lactobacillus rRNA promoter.Expression cassette by antigen and vaccine adjuvant of the present invention is incorporated on the chromosome of expressive host lactobacilli strain, and removes antibiotics resistance gene, by the restructuring of Cre/lox locus specificity, realizes.
Of the present invention antigen or vaccine adjuvant are finally secreted into outside expressive host lactobacillus cell, or grappling being illustrated on cell wall, is by using different signal peptides and grappling and antigen or vaccine adjuvant amalgamation and expression to realize.
Compared with prior art, the present invention has following beneficial effect:
1) because used expression system and all expression regulation elements all come from food stage lactobacillus cell self, the expression cassette of antigen protein has been incorporated into lactobacillus chromosome, replaced thyA gene, and the antibiotics resistance gene of introducing in genetic manipulation process is finally all removed, so final rotavirus lactobacillus live vector vaccine is real food grade products, after recombination lactic acid bacterial cell is discharged from human body or animal body, owing to can not obtain thymidylic acid or the cytidine of enough concentration from environment, will be dead, thereby can not cause the diffusion of recombinant bacterial strain, horizontal transfer with antibiotics resistance gene, meet the requirement of bio-safety.
2) product is polyvalent vaccine, covers all common serotype antigens, can be more in the past than the vaccine-induced more fully neutrality antibody of unit price lactobacillus of reporting.
3) because expression cassette is present on chromosome rather than plasmid, recombinant lactic acid bacteria character when successive 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 inducer, thereby its production technology is very easy.
4) compare with traditional inactivated vaccine or weak malicious Seedling, lactobacillus live vector vaccine is safer, cost is lower.
Accompanying drawing explanation
Accompanying drawing 1: the expression cassette of antigen protein forms schematic diagram
The specific embodiment
Below in conjunction with instantiation, the present invention will be further described.Protection scope of the present invention is not limited to following example.
Embodiment 1: antigen encoding gene is codon optimized
Because rotavirus gene is at eukaryotic cell expression, its codon usage bias and lactobacillus are different.In order to make rotavirus gene express at lactobacillus cell smoothly, need to be optimized its nucleotide sequence, remove rare codon.Its optimization principles is:
TTC→TTT；GTA→GTT；TCC→TCT；GAG→GAA；TGC→TGT；AGA→CGT；AGG→CGT
1) take the encoding gene of Human reoviruslike agent G2 serotype VP4 albumen is example, and its nucleotide sequence after optimizing is as follows, with two parts of underscore, is sequentially respectively the sequence of VP8* and VP5* according to front and back:
GCA?AGT?TTG?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?CAA?ACC?CGT?TAT?GCC?CCG?GTT?AAC?TGG?CGC?CAT?GGT GAA?ATT?AAT?GAT?TCA?ACC?ACG?GTT?GAA?CCA?GTG?CTG?GAT?GGC?CCG?TAT?CAG?CCA ACT?ACA?TTT?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?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?GGC?TCA?TCT?CAG?AGT?AAC?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?ACT?GCC?GAT?CTG?AAT?AAC ATT?TCT?ATC?GTT?ATC?CAC?AGT?GAA?TTC?TAC?ATC?ATC?CCG?CGT?TCA?CAA?GAA?TCT AAG?TGT?AAC?GAA?TAC?ATC?AAC?AAC?GGT?TTA?CCG?CCA?ATT?CAG?AAC?ACC?CGC?AATGTT?GTG?CCA?CTG?AGC?TTA?TCA?TCT?CGT?TCA?ATT?CAA?TAT?CGT?CGC?GCG?
CAG?GTG AAC?GAA?GAT?ATC?ACT?ATC?AGT?AAG?ACA?AGC?CTG?TGG?AAG?GAA?ATG?CAA?TAC AAC?CGT?GAT?ATC?ATC?ATC?CGC?TTC?AAG?TTC?GGT?AAC?TCA?GTT?ATT?AAA?TTG?GGT GGC?CTG?GGC?TAC?AAG?TGG?TCA?GAA?ATC?TCT?TAC?AAG?GCG?GCC?AAC?TAC?CAA TAC?AGT?TAT?AGC?CGC?GAT?GGT?GAA?CAG?GTG?ACG?GCC?CAT?ACT?ACA?TGT?AGT?GTT AAC?GGC?GTG?AAC?AAC?TTC?TCT?TAC?AAC?GGT?GGC?AGT?TTG?CCG?ACT?GAT?TTT?TCA ATT?TCT?CGT?TAT?GAA?GTT?ATC?AAG?GAA?AAC?AGT?TAT?GTT?TAC?ATT?GAT?TAT?TGG GAT?GAT?TCT?AAA?GCG?TTT?CGT?AAT?ATG?GTT?TAT?GTG?CGC?TCT?CTG?GCT?GCA?AAT TTA?AAC?AGT?GTT?AAA?TGT?GCC?GGT?GGC?AGC?TAT?AAC?TTT?CGT?CTG?CCG?GTG?GGT GAA?TGG?CCA?ATT?ATG?AAT?GGT?GGC?GCT?GTT?AGT?TTG?CAC?TTT?GCA?GGC?GTG?ACG CTG?AGC?ACT?CAA?TTC?ACA?AAC?TTC?GTT?AGT?TTG?AAC?AGC?TTG?CGT?TTT?CGC?TTT AGT?TTA?ACC?GTG?GAT?GAA?CCG?AGT?TTT?AGC?ATT?ATT?CGT?ACT?CGC?ACA?GTT?AAT CTG?TAT?GGT?TTA?CCG?GCG?GCC?AAC?CCA?AAC?AAC?GGT?AAC?GAA?TAC?TAC?GAA ATG?AGC?GGC?CGC?TTT?TCA?TTG?ATT?TCT?CTG?GTT?CCG?ACC?AAC?GAT?GAT?TAC?CAG ACA?CCA?ATC?ATG?AAC?AGT?GTT?ACG?GTG?CGT?CAA?GAT?TTA?GAA?CGC?CAG?TTA?AGC GAT?TTG?CGT?GAA?GAA?TTC?AAC?TCA?TTG?TCT?CAA?GAA?ATT?GCT?ATG?AGT?CAG?CTG ATT?GAT?TTA?GCA?TTA?TTG?CCA?CTG?GAT?ATG?TTC?AGT?ATG?TTC?AGC?GGC?ATC?AAA TCA?ACC?ATC?GAT?TTG?ACG?AAG?TCT?ATG?GCT?ACG?AGT?GTT?ATG?AAG?AAG?TTC?CGC AAG?TCT?AAG?TTA?GCA?ACC?AGT?ATT?AGC?GAA?ATG?ACG?AAT?TCA?TTG?TCT?GAT?GCT GCA?AGT?AGC?GCT?AGT?CGT?AGC?GCA?TCA?GTT?CGC?TCA?AAC?TTA?TCT?GTG?ATT?AGT AAT?TGG?ACT?GAT?GCG?TCT?AAA?AGT?ACT?AGC?AAC?ATT?ACA?GAT?TTG?GTT?AAT?GAT GTG?AGC?ACC?CAA?ACG?AGC?ACT?ATC?AGT?AAA?AAA?TTG?CGT?CTG?AAG?GAA?ATG ATT?ACA?CAG?ACC?GAA?GGT?ATG?AGC?TTT?GAT?GAT?ATT?TCA?GCG?GCC?GTG?TTG?AAG ACT?AAG?ATC?GAT?ATG?TCA?ACA?CAA?ATC?GGC?AAA?AAT?ACC?CTG?CCG?GAT?ATC?GTT ACG?GAA?GCT?TCA?GAA?AAG?TTC?ATC?CCA?AAG?CGT?TCT?TAT?CGC?GTT?CTG?AAG?GAT AAC?GAA?GTG?ATG?GAA?ATC?AAC?ACC?GAA?GGT?AAG?TTT?TTC?GCA?TAC?AAG?GTT GAT?ACG?CTG?AAC?GAA?ATC?CCG?TTC?GAT?ATC?AAC?AAG?TTC?GCG?GAA?TTA?GTT?ACG GAT?AGC?CCA?GTG?ATT?TCA?GCC?ATC?ATC?GAT?TTC?AAG?ACT?TTG?AAG?AAC?CTG?AAC GAT?AAC?TAC?GGC?ATC?ACA?CGC?ATG?GAA?GCT?TTA?AAC?TTG?ATT?AAA?AGC?AAC?CCG AAT?GTG?CTG?CGT?AAC?TTC?ATC?AAC?CAG?AAC?AAC?CCA?ATC?ATT?CGT?AAT?CGC?ATT GAA?CAA?CTG?ATT?TTA?CAG?TGT?AAA?TTA?TAA
2) take the encoding gene of A group Human reoviruslike agent VP6 albumen is example, and its nucleotides sequence after optimizing is classified as:
GAA?GTC?TTA?TAC?TCA?TTA?TCG?AAA?ACC?CTG?AAG?GAT?GCC?CGC?GAC?AAA?ATC?GTTGAA?GGC?ACC?CTG?TAC?TCG?AAC?GTC?TCG?GAT?TTA?ATT?CAA?CAG?TTT?AAT?CAA?ATGATC?GTC?ACT?ATG?AAT?GGC?AAC?GAT?TTC?CAG?ACA?GGT?GGC?ATT?GGT?AAT?TTG?CCGATC?CGT?AAC?TGG?ACC?TTT?GAT?TTC?GGT?CTG?TTA?GGC?ACC?ACG?TTG?CTG?AAT?CTGGAC?GCA?AAC?TAT?GTG?GAA?ACG?GCG?CGC?ACT?ACA?ATC?GAA?TAC?TTC?ATC?GAT?TTCATC?GAC?AAC?GTC?TGT?ATG?GAT?GAA?ATG?GCT?CGC?GAA?AGT?CAA?CGG?AAT?GGTGTT?GCA?CCA?CAG?AGC?GAA?GCC?CTG?CGC?AAA?TTA?GCT?GGT?ATC?AAG?TTC?AAGCGG?ATC?AAC?TTC?GAC?AAC?AGT?AGC?GAA?TAC?ATC?GAA?AAC?TGG?AAC?TTA?CAAAAC?CGT?CGC?CAG?CGG?ACC?GGC?TTT?GTT?TTC?CAT?AAG?CCG?AAT?ATT?TTT?CCA?TACTCA?GCA?TCT?TTC?ACT?TTG?AAT?CGC?TCA?CAA?CCT?ATG?CAC?GAT?AAC?CTG?ATG?GGTACC?ATG?TGG?TTA?AAT?GCC?GGC?TCA?GAA?ATT?CAG?GTT?GCT?GGT?TTT?GAC?TAT?TCTTGC?GCA?CTG?AAT?GCG?CCA?GCC?AAC?ATT?CAA?CAG?TTT?GAA?CAT?ATC?GTG?CAA?TTGCGG?CGT?GCG?CTG?ACC?ACG?GCC?ACC?ATT?ACG?TTA?TTG?CCG?GAT?GCG?GAA?CGT?TTTTCT?TTC?CCG?CGT?GTT?ATC?AAC?TCG?GCG?GAC?GGC?GCC?ACT?ACA?TGG?TTT?TTC?AACCCA?ATC?ATC?TTG?CGT?CCT?AAC?AAC?GTT?GAA?GTG?GAA?TTC?CTG?TTG?AAC?GGC?CAAATC?ATC?AAC?ACC?TAT?CAG?GCG?CGT?TTT?GGT?ACG?ATT?ATC?GCC?CGG?AAC?TTC?GATACG?ATC?CGT?TTA?AGT?TTC?CAA?TTG?ATG?CGC?CCG?CCA?AAT?ATG?ACT?CCG?GCT?GTGAAC?GCA?CTG?TTT?CCT?CAA?GCT?CAG?CCG?TTC?CAG?CAT?CAC?GCA?ACT?GTC?GGT?TTAACA?TTG?CGT?ATT?GAA?AGC?GCG?GTT?TGT?GAA?TCA?GTG?TTA?GCT?GAT?GCA?AAT?GAAACT?TTG?CTG?GCT?AAC?GTC?ACA?GCA?GTT?CGC?CAG?GAA?TAT?GCC?ATT?CCA?GTG?GGTCCT?GTC?TTT?CCT?CCG?GGC?ATG?AAT?TGG?ACC?GAA?CTG?ATC?ACG?AAC?TAC?TCG?CCGAGT?CGG?GAA?GAC?AAC?TTA?CAA?CGG?GTG?TTT?ACA?GTC?GCA?TCT?ATT?CGC?TCA?ATGTTA?ATC?AAG?TAA
3), take HRV P12 serotype VP7 albumen encoding gene as example, its nucleotides sequence after optimizing is classified as:TAT, GGT, ATT, GAA, TAC, ACC, ACG, ATC, CTG, ACG, ATC, TTG, ATC, TCT, ATC, GTT, CTG, TTGAAC, TAC, ATT, CTG, AAA, TCA, ATT, ACT, TCT, ATG, ATG, GAT, TTC, ATC, ATC, TAC, CGC, TTTTTG, CTG, GTT, TTC, GTG, ATC, GTT, TTG, CCG, TTC, ATC, AAG, GCT, CAG, AAC, TAC, GGT, ATCAAC, TTG, CCA, ATT, ACA, GGC, AGT, ATG, GAT, ACC, GCG, TAT, GTT, AAT, AGC, ACC, CAA, CAAGAA, AGT, TTC, ATG, ACG, AGC, ACT, TTG, TGT, TTG, TAC, TAT, CCG, AAC, AGT, GTT, ACT, ACAGAA, ATT, ACG, GAT, CCA, GAT, TGG, ACA, CAC, ACC, CTG, AGC, CAA, CTG, TTT, TTA, ACG, AAAGGT, TGG, CCG, ACT, AAT, AGT, GTG, TAT, TTT, AAA, AGC, TAT, GCG, GAT, ATT, GCC, AGT, TTTAGC, GTT, AAC, CCA, CAG, TTG, TAC, TGT, GAT, TAC, AAC, ATC, GTG, TTG, GTT, CAA, TAT, CAGAAC, TCA, TTG, GCT, CTG, GAT, GTG, TCT, GAA, TTG, GCA, GAT, TTG, ATC, TTG, AAC, GAA, TGGCTG, TGC, AAC, CCG, ATG, GAT, GTT, ACA, TTG, TAC, TAC, TAC, CAA, CAG, ACC, GAT, GAA, GCGAAT, AAA, TGG, ATT, TCA, ATG, GGC, GAT, TCT, TGT, ACA, GTG, AAA, GTT, TGC, CCA, TTG, AACATG, CAA, ACC, CTG, GGT, ATT, GGC, TGC, ACC, ACG, ACT, GAT, GTG, GCC, ACG, TTT, GAAGAA, GTT, GCT, AAT, GCA, GAA, AAA, CTG, GTG, ATT, ACT, GAT, GTT, GTG, GAT, GGT, GTT, AACCAT, AAG, ATC, AAC, ATC, ACA, TTG, AAC, ACG, TGT, ACT, ATC, CAA, AAC, TGC, AAG, AAG, CTGGGC, CCA, CGT GAA AAC GTG GCG ATT ATT CAG GTT GGT GGC AGT GAT ATT ATT GATATT ACG GCC GAT CCG ACA ACC ATT CCA CAA ACT GAA CGT ATC ATG CGC ATC AACTGG AAG AAG TGG TGG CAG GTG TTT TAT ACG GTT GTG GAT TAC ATC AAC CAA ATCGTG CAG GTT ATG TCA AAA CGT TCA CGC TCT TTA AAC TCT GCG GCC TTT TAT TATCGC ATT TAA
4) take the encoding gene of coli heat instability mode toxin B (LTB) is example, and its nucleotides sequence after optimizing is classified as:
ATG?AAT?AAA?GTT?AAA?TTT?TAT?GTT?TTA?TTT?ACG?GCG?TTA?CTA?TCT?TCT?CTA?TGTGCA?CAC?GGA?GCT?CCC?CAG?TCT?ATT?ACA?GAA?CTA?TGT?TCG?GAA?TAT?CGC?AAC?ACACAA?ATA?TAT?ACG?ATA?AAT?GAC?AAA?ATA?CTA?TCATAT?ACG?GAA?TCG?ATG?GCA?GGCAAA?CGT?GAA?ATG?GTT?ATC?ATT?ACA?TTT?AAG?AGC?GGC?GCA?ACA?TTT?CAG?GTC?GAAGTC?CCG?GGC?AGT?CAA?CAT?ATA?GAC?TCT?CAA?AAA?AAA?GCC?ATT?GAA?CGT?ATG?AAGGAC?ACA?TTA?CGT?ATC?GCA?TAT?CTG?ACC?GAA?ACC?AAA?ATT?GAT?AAA?TTA?TGT?GTTTGG?AAT?AAT?AAA?ACC?CCC?AAT?TCA?ATT?GCG?GCA?ATC?AGT?ATG?GAA?AAC?TAA
5) take the encoding gene of cholera toxin subunit b (CTB) is example, and its nucleotides sequence after optimizing is classified as:
CAT?GGA?ACT?CCA?CAA?AAT?ATT?ACT?GAT?CTT?TGT?GCT?GAA?TAT?CAT?AAT?ACT?CAAATT?CAT?ACT?CTT?AAT?GAT?AAG?ATT?TTT?TCT?TAT?ACT?GAA?TCT?CTT?GCT?GGA?AAGAGA?GAA?ATG?GCT?ATT?ATT?ACT?TTT?AAG?AAT?GGA?GCT?ACT?TTT?CAA?GTT?GAA?GTTCCA?GGA?TCT?CAA?CAT?ATT?GAT?TCT?CAA?AAG?AAG?GCT?ATT?GAA?AGA?ATG?AAG?GATACT?CTT?AGA?ATT?GCTTAT?CTT?ACT?GAA?GCT?AAG?GTT?GAA?AAG?TTG?TGT?GTT?TGGAAT?AAT?AAG?ACT?CCA?CAT?GCT?ATT?GCT?GCT?ATT?TCT?ATG?GCT?AAT?TAA
Embodiment 2: the structure of antigen protein or vaccine adjuvant protein expression frame
The expression of antigen protein or vaccine adjuvant albumen and location are to be used in combination to realize by a plurality of expression regulation elements.These expression regulation elements comprise the catenation sequence (seeing accompanying drawing 1) between promoter, signal peptide sequence, antigen encoding gene, grappling subsequence and each expression regulation element.
1), in the present invention, the expression of antigen protein is finally controlled with constitutive promoter.This constitutive promoter derives from lactobacillus rRNA promoter, is a kind of in following sequence, or surpasses a kind of in 85% sequence with the similarity of following sequence:
a)GTC?GGT?CGA?GTT?GTT?GAC?AGG?ATA?AAG?GTC?GCC?TGG?TAT?GGT?CTC?AATATA?GCG
b)GTC?GTG?GCA?GTT?GTT?GAC?AAC?CTG?TGG?GCG?GTT?TGA?TTT?GTT?CTT?GCTATA?GCG
c)ACC?GAG?CCA?GTT?GTA?GAC?AGG?GCT?GTG?CTG?ACC?TGG?TAA?GGT?ATA?TTATAG?CG
d)GAG?CTG?CGA?GTT?GTT?GAC?ATT?GTT?AAT?GGC?CCC?TGA?TAT?ATT?TGG?CGTATA?GCG
e)GGG?GTA?GTT?GTT?GAC?AGC?GTG?GGT?TGG?TGC?TGG?TAA?TTT?TGC?GCT?ATAGCG
f)GAG?TCT?TGC?GCT?ATA?GTT?GTT?GTC?AGA?ATG?GAG?ATA?CTA?TGA?TAT?AATGTT?GCT?ATA?GCG
g)AGG?GAG?TGA?GTT?GTG?ACA?GGG?CTG?TGA?TGG?TGT?GGT?ATT?GTT?TTG?GTATAG?CG
h)ACG?AGT?TGT?TGG?TAT?TGT?GGC?GGT?ATA?GCG
i)AGG?GGT?TGA?GTT?TGA?CAC?TGA?TCC?CGG?CTG?GTG?GTA?AAT?TTT?CGT?TATAGC?G
2), in the present invention, between promoter sequence and signal peptide sequence, with CATATG, the specificity enzyme action site sequence of Cobra venom endonuclease NdeI connects, and introduces thus atg start codon ATG, facilitates genetic manipulation.
3) signal peptide sequence in the present invention is a kind of in following sequence, or surpasses a kind of in 85% sequence with the similarity of following sequence:
a)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
b)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?GAA?CCG?CGT?GTT?TCA
c)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
d)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
e)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?CCA?TTT?GCG?CAA?ACC?CGT
f)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
g)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
h)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
4), in the present invention, between signal peptide sequence and antigen encoding gene sequence, with GTC GAC, the specificity enzyme action site sequence of Cobra venom endonuclease SalI connects, to facilitate genetic manipulation.
5) the grappling subsequence that cell wall surface display of the present invention is used, come from one of LPXT-block cell wall 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
6), in the present invention, between antigen encoding gene sequence and grappling, with GGT GGA, the coded sequence of two glycosides propylhomoserins connects.
7) in the present embodiment,, according to different expression objects, need to select different expression regulation element combinations.As secreting, expressing, select the combination of promoter-signal peptide-antigen, if cell wall surface display is expressed, select promoter-signal peptide-antigen-grappling sub-portfolio.
8) in the present embodiment, gene is synthetic to be completed by the nucleic acid Composite service business on market, is powdered DNA.Synthetic gene is present on the cloned plasmids that nucleic acid Composite service business provides.DNA is dissolved in to 100 microlitre ultra-pure waters, as the template of the PCR step in embodiment 3.
Embodiment 3: Human reoviruslike agent P12 serotype VP7 protein expression frame inserts Lactobacillus fermenti chromosome, and removes antibiotics resistance gene
Lactobacillus described in the present embodiment refers in particular to Lactobacillus fermenti (Lactobacillus fermentium).
Experiment completes in two steps, first a DNA segment that contains Human reoviruslike agent P12 serotype VP7 protein expression frame, lox site, upstream, chloramphenicol resistance gene, lox site, downstream is recombinated and is inserted into the chromosomal thyA gene location of Lactobacillus fermenti by locus specificity, replace thyA gene; And then use Cre enzyme to remove antibiotics resistance gene.
Wherein, insert segment design as follows: from DNA segment-Human reoviruslike agent P12 serotype VP7 protein expression frame-lox site sequence-chloramphenicol resistance gene-lox site sequence of the 1000bp of Lactobacillus fermenti Chromosome t hyA upstream region of gene-from the DNA segment of the 1000bp in Lactobacillus fermenti Chromosome t hyA gene downstream.
Wherein, from the chromosomal DNA segment of Lactobacillus fermenti, take Lactobacillus fermenti genomic DNA as template, by PCR, obtain.The Human reoviruslike agent P12 serotype VP7 expression plasmid described in embodiment 2 of take 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 overlap extension PCR inserts segment.This DNA is inserted to segment to be connected with pNZ plasmid, and transform escherichia coli Top10 competent cell, on the BHI solid medium flat board that contains 5 mcg/ml chloromycetin, screen positive recombinant, and further expanding propagation in the BHI fluid medium that contains 5 mcg/ml chloromycetin, large quantity extracting plasmid DNA sequence verification.Extracted plasmid is converted into Lactobacillus fermenti by electric shock, and the bacterium colony of only not growing on erythromycin flat board at chloromycetin flat board by parallel coated plate screening, further verifies by bacterium colony PCR, to obtain double crossing over recon.
The plasmid that contains Cre recombinase encoding gene is transformed above-mentioned by the double crossing over 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 culture medium that contains respectively 30 mcg/ml erythromycin and 10 mcg/ml chloromycetin, screening is responsive but have the recon of erythromycin resistance to chloromycetin, 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 culture medium containing how, so that the plasmid loss that contains Cre recombinase encoding gene.Culture fluid 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 (9)
1. food stage lactobacillus live vector A group rotavirus vaccine and preparation method thereof.It is characterized in that, in the lactobacillus cell expression of thyA Gene Deletion secretion or cell wall, shown VP7 antigen protein (P serotype) and VP4 antigen protein (G serotype, with VP5* and VP8* protein subunit form single expression), vaccine adjuvant coli heat instability mode toxin B (LTB) and the cholera toxin subunit b (CTB) from the VP6 antigen protein of A papova, common serotype.The expression of antigen protein and vaccine adjuvant albumen is controlled by induction type or constitutive promoter, and protein expression frame is incorporated on the chromosome of expressive host lactobacilli strain, and has removed the external source antibiotics resistance gene of introducing in genetic manipulation process.
2. according to the lactobacilli strain of claim 1, comprise bacillus acidophilus (Lactobacillus acidophilus), lactobacillus casei (Lactobacillus casei), Lactobacillus crispatus (Lactobacillus crispatus), Lactobacillus bulgaricus (Lactobacillus bulgaricus), Deshi Lactobacillus (Lactobacillus delbrueckii), Lactobacillus fermenti (Lactobacillus fermentium), Lactobacillus gasseri (Lactobacillus gasseri), lactobacillus helveticus (Lactobacillus helveticus), Lactobacillus johnsonii (Lactobacillus johnsonii), Lactobacillus paracasei (Lactobacillus paracasei), Lactobacillus plantarum (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 claim 1 lactobacilli strain, the expression cassette of antigen protein and vaccine adjuvant albumen is incorporated into the chromosomal thyA gene location of lactobacillus, when integrating, knock out thyA gene, and use subsequently the restructuring of Cre/lox locus specificity to remove antibiotics resistance gene.
4. according to the antigen protein of claim 1 and vaccine adjuvant albumen, when preparing Group A human rotavirus vaccine, comprising: VP7 (G1, G2, G3, G4, G9, G12 serotype), VP6 (A papova), VP4 (P[4], P[6], P[8] serotype), coli heat instability mode toxin B (LTB) and cholera toxin subunit b (CTB).Wherein VP4 antigen protein is with VP5* and VP8* protein subunit form single expression.
When preparing pig A group rotavirus vaccine, comprising: VP7 (G3, G4, G5, G9, G11 serotype), VP6 (A papova), VP4 (P[6], P[7], P[13], P[19], P[23], P[26] serotype), coli heat instability mode toxin B (LTB) and cholera toxin subunit b (CTB).Wherein VP4 antigen protein is with VP5* and VP8* protein subunit form single expression.
When preparation sheep A group rotavirus vaccine, comprising: VP7 (G6, G8, G10 serotype), VP6 (A papova), VP4 (P[1], P[5], P[11], P[12], P[15] serotype), coli heat instability mode toxin B (LTB) and cholera toxin subunit b (CTB).Wherein VP4 antigen protein is with VP5* and VP8* protein subunit form single expression.
5. according to the antigen protein of claim 1 and vaccine adjuvant albumen, it is characterized in that, the nucleotide sequence of its encoding gene is optimized the use Preference of codon according to lactobacillus.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?GAA?CCG?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?CCA?TTT?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 wall 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.
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