CN101780272A - Application of autovaccine preparation containing TGF beta 1 - Google Patents

Abstract

The invention discloses application of autovaccine preparation containing TGF beta 1. Adjuvant is added in the autovaccine preparation to prepare medicinal preparation for treating continuous pathogen infection of livestock. The vaccine preparation is fusion protein obtained by the way that part or total of the TGF beta 1 or part or total of mutant or total of similar gene sequences is recombined with at least one helper T cell capable of improving immunogenicity or the gene sequence of carrier protein to construct eukaryon or pronucleus expression plasmid and transform host cell for expression, or is directly prepared by eukaryon expression plasmid obtained by construction. The vaccine preparation can be directly used for preparing medicinal preparation for treating the continuous pathogen infection of livestock, induce domestic animals to generate antibody capable of neutralizing self TGF beta 1, and remove immunodepression function caused by too high level of TGF beta 1 in continuous process of infection; simultaneously, the vaccine preparation can depress the generation of inducible regulatory T cell at the persistent infection part of an organism, thus being beneficial to breaking the immune tolerance state of in-vivo pathogen by the organism and promoting the removing function of the organism on the continuously infectious pathogen.

Description

A kind of TGF β 1 application of autovaccine preparation that contains

Technical field

The invention belongs to immunology and animal vaccine manufacturing technology field, be specifically related to treat TGF β 1 application of autovaccine preparation of domestic animal persistence pathogenic infection.

Background technology

The persistence pathogenic infection phenomenon of domestic animal is very general in modern farming.Though infected domestic animal does not often show tangible clinical symptoms, but has the ability that continues to extraneous toxin expelling; Also can be delivered to the next generation by fetus female animal, these pathogen that continue to exist also may be in to morph in the carcass and be caused new popularly, become a kind of important source of infection.Persistent infection also may cause immunologic tolerance or paralysis, the effect when having a strong impact on vaccination once more.This prevention and control and purification work to the infectious disease in the aquaculture has brought great difficulty.

In aquaculture, the disease that persistent infection takes place mainly contains porcine circovirus, reproductive and respiratory syndrome virus, swine fever, parvovirus, mycoplasma hyopneumoniae, Pseudorabies virus, bovine viral diarrhea virus, contagious equine abortion, the secondary haemophilus of pig, Actinobacillus pleuropneumoniae, Mycobacterium tuberculosis at present.Also also have simultaneously some persistent infections that cause in the part by multiple pathogen as diseases such as the chronic endometritis of female animal, mastitiss.For the domestic animal that the persistence pathogenic infection takes place, its possible result has: domestic animal is through one section long period course of infection, but final body is removed intravital pathogen completely, and body enters health status; Though second kind of situation is body persistent infection pathogen, do not cause obvious pathological change, is in Infection Status all the life; The third situation is the final immune system that breaks through or destroy body of the pathogen of persistent infection, makes animal finally die from former or secondary infection.The domestic animal that the persistence pathogenic infection takes place has infectiousness usually, and this infection is often subtle and have a bigger hazardness.

Though people have prepared the infection that various vaccines prevent domestic animal generation pathogen at present, and have replaced great achievement.But but still there is not the persistent infection that effective method is treated domestic animal at present.Though the persistence pathogenic infection that heavy dose of, long Drug therapy helps to treat domestic animal, effect is limited, brings high expense simultaneously.The effective measures of current solution domestic animal persistence pathogenic infection are to take strict elimination system.This measure is promoted in production practices and is got up to have certain degree of difficulty.At first, its symptom of domestic animal that is in the persistent infection state is often also not obvious, determine whether it is that persistent infection need be taked the laboratory diagnosis means, and these operating technologies require height, cost height; Simultaneously, large-scale elimination system causes enormous economic loss, and this makes most of plants to accept, and therefore carries out suitable difficulty.Explore the problem of persistence pathogenic infection in the novel solution domestic animal body, spread unchecked for the current disease of control and have great importance.

Cause the mechanism of pathogen persistent infection that many aspects are arranged, every kind of pathogen persistent infection machine-processed different, but the following aspects is arranged substantially: 1) pathogen infringement immune system, thus the immune system normal function is impaired; 2) pathogen is invaded immunity special pardon position (in central nervous system, testis, eye conjunctiva), causes immune system effectively not play a role; 3) pathogen gene makes a variation and causes epitope to change to escape immune monitoring; 4) cause the host cell gene change of Expression, particularly the expression of main histocompatibility complex descends, and makes host cell can not effectively offer the antigenic peptides of intra-cellular pathogens; 5) host produces some antigenic immunologic tolerance of body because of congenital or posteriority factor cause the low or host of whole immunne response.Wherein the host is to cause the one of the main reasons of some pathogen persistent infections to the immunologic tolerance of pathogen.Break the immune tolerance state of body, remotivate or strengthen body immune system, thoroughly remove pathogen for body and have important function at the reaction of the specific immune response of pathogen to pathogen.

In recent years, people had obtained bigger progress in the immunologic tolerance Study on Mechanism of host to pathogen.Be in the domestic animal of persistence pathogenic infection state and pathogen and normally be in a temporary transient poised state between body immune system and pathogen.The immune system of body also can cause the damage of autologous tissue when launching the pathogen attack.In order to prevent that immunoreation from producing excessive infringement to body self, body can be regulated and control immunoreactive intensity by several different methods: the part of body can be secreted the various kinds of cell factor such as IL-10, TGF β and be waited and suppress immunoreation; Body is induced the CD4 of generation simultaneously ⁺CD25 ⁺Regulatory T cells also can be directly or is waited by secrete cytokines and to suppress the immunoreation of immune effector cell to pathogen.In this course, TGF β 1 has brought into play critical effect, and itself can be inhibited to the panimmunity effector lymphocyte, simultaneously, can also induce CD4 ⁺CD25 ⁺The generation of regulatory T cells and bring into play immunosuppressive action (Song Guo Zheng.Int JClin Exp Med.2008; 1 (3): 192-202; Chen W.J.Exp.Med.198:1875-1886).

TGF β 1 is identical by two structures, and molecular weight is the dimer that the subunit of 12.5KD is formed by connecting by intermolecular disulfide bond.TGF β 1 is a kind of macromolecular complex (molecular weight is 100-250kD) of non-activity at the synthetic initial stage, be referred to as the TGF β 1 of potentiality, producing molecular weight after activating is the dimer of 25kD, be connected by disulfide bond by two identical 12.5kD subunit peptide chains, every strand contains 112 aminoacid.TGF β 1 is the multi-functional cytokine of a class, can the synthetic and secretion by various kinds of cell.Mode with paracrine and autocrine plays a role.The various pathophysiological processes of its wide participation animal.Mainly comprise many aspects such as the secretion of Growth and Differentiation, extracellular matrix of immune regulation and control, cell and deposition, transforming growth factor plays a role by combining with I class and II receptoroid, and signal path has SMAD albumen and the non-dependence approach of SMAD.

TGF β 1 is a kind of intensive immunosuppressive factor in immune system, can act on the panimmunity cell with autocrine or paracrine mode.Can regulate immunologic tolerance by regulating aspects such as lymphocytic propagation, differentiation and survival, lymphocyte, natural killer cell, granulocyte, mastocyte, macrophage are all had important regulating action, and TGF β 1 can directly or by indirect approach come the suppressor T cell reaction.The propagation that suppresses helper T lymphocyte, B cell, natural killer cell (NK) suppresses the generation of cytotoxic T cell (CTL), and it is synthetic to suppress immunoglobulin, the effect of immunoregulatory factor such as antagonism IL-1, IL-2, TNF-α, IFN-γ.TGF β 1 can also suppress CD4 ⁺The maturation of the propagation of T, inhibition dendritic cell reduces the expression of the dendritic cell surface main histocompatibility complex of II class, thereby suppresses the angtigen presentation function of dendritic cell.(Gorelik?L.Nat?Rev?Immunol.2002，2：46-53)。And can also suppress the activation of macrophage and the release of pro-inflammatory cytokine (Tsunawaki, S.Nature, 1988,334 (6179): 260-262).

TGF β 1 under the Pathophysiology situation, can suppress the damage that immunoreation causes autologous tissue excessively by force (Li MO.Annual Review of Immunology, 2006,24:99-146).And can promote impaired tissue repair.Though this mechanism of TGF β 1 suppresses the immunologic injury of body to some extent, but the side effect that brings is to make the removing ability drop of body to pathogen, for condition has been created in the persistent infection of pathogen, because mostly the pathogen of domestic animal persistent infection is to have specific CD8 in the born of the same parents ⁺The T deficiency causes body thoroughly to remove and is hidden in self intracellular pathogen, and this is the one of the main reasons that causes persistent infection.Find that at present TGF β-smad2 signal path causes CD8 ⁺T cell proliferation is main cause (Tinoco R.Immunity.2009,31 (1): 145-57 that cause viral persistent infection; Sanjabi S.Immunity.2009,31 (1): 131-44).

Vaccine is in immunologic process, and possible excitating organism produces the TGF-β 1 of excessive level, and these TGF-β 1 are one of the main reasons (Sudipta Bhowmick.Infect Immun, 2009,77 (4): 1514-1523) of causing immuning failure.Have been found that at present TGF β 1 may be one of major reason that causes persistence parasitic infection (Charles F.The Journalof Immunology, 2008,180,4090-4097).

In recent years have been found that TGF β 1 induces the induction type CD4 of generation ⁺CD25 ⁺Regulatory T cells is brought into play key effect at aspects such as immunologic tolerance and control immunoreation intensity.CD4 ⁺CD25 ⁺Regulatory T cells is that a newfound in recent years class has the inhibiting cell of specific immunity, brings into play immunosuppressive action by many approach, and it can suppress the function of immune effector cell by the mode specificity of cell contact.Isolated CD4 in chronic FV virus infected mice body ⁺CD25 ⁺Regulatory T cells can suppress CD8 by the mode of cells contacting ⁺The cytotoxic effect of T cell, same discovery similar phenomenon in the process of some other virus of mice as HSV, SV40 etc. and people's viral persistent infection such as HCV, HIV, HBV, HTLV, EBV persistent infection.Utilize anti-CD25 monoclonal antibody to remove CD4 ⁺CD25 ⁺Regulatory T cells is enhancement antigen specific C D8+T cell anti-virus immunoreation intensity (Furuichi Y.World J Gastroenterol, 2005,11 (24): 3772-3777) significantly.CD4 ⁺CD25 ⁺Regulatory T cells removes and suppresses CD8 ⁺The T extracellular can also suppress the function of immunocytes such as bone-marrow-derived lymphocyte, NKT cell, NK cell, dendritic cell; Stopping effect T cell can also suppress cytokines such as emiocytosis IFN-γ, TNF and bring into play immune suppression function to the high titre tissue migration of virus.CD4 ⁺CD25 ⁺Regulatory T cells can also be brought into play immunosuppressive action (Kazuhiko Nakamuraa.The Journal ofExperimental Medicine.2001.194 (5): 629-644) by film conjunction type TGF β 1.In the porcine reproductive and respiratory syndrome virus course of infection, the generation of inductivity regulatory T cells may be to cause one of low reason of immunoreation, and the generation of this inductivity regulatory T cells relies on effect (Silva-Campa E.Virology.2009,387 (2): 373-379) of TGF β 1.

The immunoreation level of utilizing inhibition TGF β 1 to improve body is a feasible approach, and existing at present research and utilization TGF-beta 1 antagonists is broken the tumour immunity tolerance, improves the research report of tumor survival phase.Discoveries such as Luc í a Gil-Guerrero can suppress the peptide section P of transforming growth factor ₁₇(KRIWFIPRSSWYERA) can reduce CD4 with external in vivo ⁺CD25 ⁺The activity of regulatory T cells helps strengthening the immune effect of vaccine, and helps body and break for the virus and the immune tolerance state of tumor.(Gil-Guerrero?L.J?Immunol.2008，181(1)：126-135)。

The function that suppresses TGF β 1 can have many approach, mainly contains the chemical compound of monoclonal antibody, inhibitory polypeptide and soluble T GF-β1Shou Ti, synthetic etc.Also there is report to utilize TGF-β 1 to make up autovaccine, but its objective is and be used for breaking the immunologic tolerance of body, promote the immunoreation of body tumor to tumor.But still do not utilize TGF-β 1 autovaccine preparation to carry out the report of the treatment aspect of domestic animal persistence pathogenic infection at present.

Though it is to guarantee the equilibrated key factor of organism immune response that present evidence shows TGF β 1 level of certain level, suitable reduction TGF β 1 level can't cause tangible injury to body.Have experiment show mice for TGF β 1 gene knockout often show fatal autoimmune disease (Kallapur S.Humana Press:Totowa, NJ, 1998:335-368).But when utilizing TGF β 1 inhibitor to handle mice, find that TGF β 1 inhibitor can't cause tangible autoimmune disease.TGF β 1 inhibitor of these uses comprises: neutralizing antibody, soluble T GF β1Shou Ti, receptor kinase agonist drug, antisense agents.Its possible reason is these inhibitor to the signal path inhibitory action of TGF is incomplete.

It is low, easy to use to utilize TGF-β 1 autovaccine preparation to come intravital TGF-β 1 function of inhibition machine to have a cost, advantage such as effect is lasting.Present research has shown that by certain method be the generation that can induce autoantibody in animal body, and the method for its processing generally utilizes oneself protein to connect one section immunoreactive carrier protein of energy excitating organism again or polypeptide is realized.Among the present invention, utilize the B cell epitope of TGF β 1 self, add the external source helper T cell epitope, preparation TGF β 1 autovaccine preparation, and prepare pharmaceutical preparation, it can effectively stimulate body to produce the antibody of anti-self TGF β 1.The antibody that produces can in and TGF β 1, thereby part has suppressed the function of TGF β 1, effectively the immunoreation level of excitating organism.Simultaneously, can also suppress CD4 by said preparation ⁺CD25 ⁺The function of regulatory T cells is broken the immunologic tolerance of body to pathogen, thereby remotivates the immunoreation of body to pathogen, quickens body and removes pathogen.

Summary of the invention

The purpose of this invention is to provide a kind of TGF of containing β 1 application of autovaccine preparation, it is used to prepare the pharmaceutical preparation of treatment domestic animal persistence pathogenic infection.

Domestic animal persistence pathogenic infection is accompanied by the rising of TGF β 1 level in part or the blood, simultaneously induction type CD4 ⁺CD25 ⁺Regulatory T cells quantity and increased functionality, these two factors can be brought into play the effect of immunosuppressant and immunologic tolerance, thereby have suppressed the removing effect of body to the persistent infection pathogen.After using the pharmaceutical preparation of treatment domestic animal persistence pathogenic infection of said preparation preparation, can induce domestic animal to produce the antibody of anti-self TGF β 1, the antibody of these generations TGF β 1 of self that can neutralize, and can suppress CD4 in the part ⁺CD25 ⁺The generation of regulatory T cells and function, thus can impel body to break immunologic tolerance to the persistence pathogen, quicken the pathogen that body is removed persistent infection.This provides a kind of novel method and approach for solving domestic animal persistence pathogenic infection problem.

A kind of TGF of containing β 1 application of autovaccine preparation of the present invention is that described bacterin preparation is used to prepare the pharmaceutical preparation of treatment domestic animal persistence pathogenic infection.In described bacterin preparation, add adjuvant, promptly be prepared into the said medicine preparation.

The infection of described domestic animal persistence pathogen comprises chronic infection, inapparent infection, repeated infection and healthy band poison.Specifically comprise: the infection that the secondary haemophilus of porcine circovirus, reproductive and respiratory syndrome virus, swine fever, parvovirus, mycoplasma hyopneumoniae, Pseudorabies virus, bovine viral diarrhea virus, contagious equine abortion, pig, Actinobacillus pleuropneumoniae, Mycobacterium tuberculosis cause, and multiple pathogen the female animal chronic endometritis, the mastitis that cause.

Described bacterin preparation is by the part of domestic animal self TGF β 1, whole, part or all of mutant or whole similar gene orders and the gene order reorganization that at least one can improve immunogenic helper T cell epitope or carrier protein, make up eucaryon or prokaryotic expression plasmid, transform the host bacterium and express the fusion rotein that obtains; Perhaps the eukaryon expression plasmid that is directly obtained by structure is made.

Described helper T cell epitope comprises the helper T cell epitope in carrier protein, general helper T cell epitope PADRE, tetanus toxoid amino acid fragment TTE, animal infection pathogen source or the t cell epitope that manually designs in conjunction with gene order according to the animal II of main histocompatibility complex class.

Described carrier protein comprises bovine serum albumin, keyhole limpet hemocyanin, hepatitis B surface antigen, hepatitis B virus core antigen, E.coli LT B, ovalbumin, heat shock protein or be directed to label protein fusion rotein on the expression vector.

Described helper T cell epitope comprises also that multiple helper T cell epitope mixes and forms, or the single external source helper T cell epitope form that repeats to be in series.

Mechanism of the present invention is as follows:

Mature T GF β 1 albumen is the dimeric structure that is formed by disulfide bond by two monomeric proteins, the TGF-β 1 proteic crystal three dimensional structure that can obtain aminoacid sequence (seeing aminoacid sequence table 1), the PDB file of mature T GF β 1 easily and resolve from Protein Data Bank (U.S. RCSB Protein Data Bank, PDB ID:1KLA).Can check protein structure with copy editor's software, also can adopt simultaneously three dimensional structure to observe the three dimensional structure that software is observed TGF-β 1 intuitively, these structures comprise proteinic secondary structure, tertiary structure, quarternary structure, can also observe simultaneously the position of whole dimeric surface amino groups acid distribution, disulfide bond, this provides great convenience for B cell epitope of inferring TGF-β 1.This monomeric protein is totally 112 amino acid residues, and wherein 57-68 aminoacid forms the α spiral, and 16-18,43-45,90-92,95-97,77-82,108-112 form 6 βZhe Dies altogether, and 69-72,92-95 form two corners altogether.The selection of B cell epitope can be the whole, partly or mutant of TGF-β 1.Simultaneously, also can be according to the three dimensional structure of TGF-β 1, the software in conjunction with the prediction of B cell epitope optimizes the B cell epitope.Preferred B cell epitope includes but not limited to 1-15,1-30,19-42,46-56,69-75,79-108,83-107 aminoacid sequence.

These preferred sequences are compared by aminoacid sequence, search TGF β 1 family members' similar homologous sequence to it, if possible, avoid having too high homologous amino acid sequence as far as possible, prevent from immunologic process, to produce cross-immune reaction widely with other cytokine partial sequence.

Because TGF β 1 is that eukaryote is expressed, if in prokaryote, carry out expression effect, be necessary this gene order is carried out necessary optimization, making its amino acid whose codon is the high codon of host bacterium frequency of utilization.The person skilled in art can find the codon usage frequency table of various biologies easily and optimize accordingly.

The antibody of how to induce body to produce anti-self TGF β 1 is that the present invention needs the problem that solves emphatically.TGF β 1 is a kind of autologous protein matter for the animal of immunity, because the autoantigen tolerance mechanism effect of body, when directly carrying out immunity with TGF β 1, its peptide section can not effectively be offered to activate helper T lymphocyte by antigen presenting cell, thereby is not enough to induce effective antibody to generate.In order to strengthen immunogenicity from body TGF β 1, be necessary TGF β 1 is carried out necessary modification, these modifications comprise increases at least one t cell epitope.

Solution to this problem is to introduce the external source helper T cell epitope in autoantigen, different and the difference to some extent according to the genetic background of the main histocompatibility complex of immunized animal of these external source helper T cell epitope, the external source helper T cell epitope that this external source is provided can be carrier protein or known helper T cell epitope.Also comprise the aminoacid sequence that increases for enhance immunity originality simultaneously, its aminoacid sequence can be present known helper T cell epitope or carrier protein aminoacid sequence, improve immunogenic helper T cell epitope including, but not limited to general helper T cell epitope (PADRE), tetanus toxoid amino acid fragment (TTE), also can derive from the helper T cell epitope of the pathogen of animal infection, can also be the t cell epitope according to the artificial design of the animal II of main histocompatibility complex class binding motif.Simultaneously, t cell epitope can be that a plurality of t cell epitopes mix and form, and also can be single being in series.Its main purpose of carrier protein provides effective helper T cell epitope, include but not limited to bovine serum albumin, keyhole limpet hemocyanin, hepatitis B surface antigen, hepatitis B virus core antigen, E.coli LT B, ovalbumin, heat shock protein.It also can be the label protein fusion rotein that is directed on the expression vector.

The selection of helper T cell epitope can be with reference to the helper T cell epitope of known pathogen.Because the genetic background difference of domestic animal different cultivars, main histocompatibility complex there are differences, and is necessary to increase the needs that a plurality of different helper T cell epitope are suitable for different genetic backgrounds.In order to strengthen the effect of helper T cell epitope, can adopt a plurality of t cell epitopes or repeat t cell epitope, can adopt suitable aminoacid sequence to connect between the t cell epitope.Those skilled in the art can replace helper T cell epitope that the present invention mentions with different helper T cell epitope easily by prior art.It should be noted that t cell epitope order described here only is used for for example.But the effective t cell epitope that the external source t cell epitope of selecting notes is put to death in the main histocompatibility complex kind of the animal of want immunity, and essential condition is that it can effectively be offered to cell surface by main histocompatibility complex.

Simultaneously, the selection of t cell epitope can also be optimized for the helper T cell epitope of utilizing the persistent infection pathogen.With the pig is example, can select universal t cell epitope, can also select simultaneously the known t cell epitope of common pig persistent infection pathogen, including, but not limited to polypeptide (Stevenson LS.Viral Immunol.2007,20 (3): 389-98) such as the E290 polypeptide (KHKVRNEVMVHWFDD) of corresponding 1446～1460 amino acid residues of swine fever virus NS2-3, porcine circovirus ORF1 81～100; 117～131 of reproductive and respiratory syndrome virus GP5 wait polypeptide (Vashisht K.Vaccine.2008,26 (36): 4747-4753); 20～40 of foot and mouth disease virus VP1 gene are waited polypeptide (Collen T.JImmunol.1991,146 (2): 749-755) etc.As those skilled in the art, can be more convenient pass through the epitope that pertinent literature or immunological technique are determined the persistence pathogenic infection.Simultaneously, the vaccine of these persistent infection pathogen specifics comprises the gene vaccine of attenuated live vaccine, inactivated vaccine, subunit reorganization Seedling, dna vaccination and special carrier etc.Can be used with TGF β 1 autovaccine preparation of the present invention, to reach better effect.

When the design of bacterin preparation, can be with TGF β 1 and carrier protein or helper T cell epitope recombination to construct expression plasmid, expression plasmid can be a prokaryotic expression plasmid, also can be eukaryon expression plasmid.Utilize these expression plasmid transformed host cells that builds, can be at the external fusion rotein that obtains.Add suitable adjuvant and promptly become pharmaceutical preparation of the present invention.Simultaneously, also can directly use by eukaryon expression plasmid as the DNA preparation.

Unless stated otherwise, conventional molecule manipulation technology of the present invention all can be with reference to " molecular cloning experiment guide " third edition (Huang Peitang translates for J. Sa nurse Brooker, D.W. Russell work).

Simultaneously in order to strengthen the effect of TGF β 1 autovaccine preparation, said preparation can also add one or more adjuvants, be prepared into the pharmaceutical preparation of treatment domestic animal persistence pathogenic infection, these adjuvants comprise but do not limit to aluminium hydroxide, white oil, oils and fats, fully or incomplete Freund's adjuvant, cytokine source adjuvant, also can be business-like adjuvant such as MF59, ISA15AVG, ISA206 etc.

The antibody titer that pharmaceutical preparation excited by the preferred TGF-β 1 autovaccine preparation of the present invention is unabiding, but a benefit of bringing like this is to prevent the persistent generation of body TGF-beta 1 specific antibodies, thereby suppresses the normal physiological function of TGF-β 1 for a long time and cause serious adverse.Because after the persistence pathogenic infection is eliminated in the body, the unnecessary continuation of body produces the TGF-beta 1 specific antibodies.Certainly keep suitable anti-TGF-beta 1 specific antibody concentration under specific circumstances if necessary, can regularly adopt preparation of the present invention to come the immune system of excitating organism.

On domestic animal persistence pathogenic infection, took preventive measures in the past, infected the back Drug therapy, concentration or the like, not only the cost cost is big in these measures, and weak effect.The present invention is applied to TGF β 1 autovaccine preparation prepare in the pharmaceutical preparation of treatment domestic animal persistence pathogenic infection first, and use cost is low, and therapeutic effect is good.Also do not have at present relevant research report, this is an important breakthrough technically.

The specific embodiment

The present invention will explain in more detail by following embodiment, but should not be limited to this.

The autovaccine preparation preparation of embodiment 1:TGF-β 1 full amino acid sequence and LTB carrier protein amalgamation and expression comprises the steps:

1.1 the structure of recombiant plasmid pET-TGF-LTB plasmid

1.1.1 download the aminoacid sequence of TGF β 1 and LTB respectively from the genebank website, the aminoacid sequence of TGF β 1 is seen SEQID NO:1, the aminoacid sequence of LTB is seen SEQ ID NO:2, be not subjected to the influence of LTB for the B cell epitope that makes TGF β 1, add flexible joint sequence (GGGGSGGGGSGGGGS) between LTB and TGF β 1 dinucleotide sequence, sequence is seen SEQ ID NO:3.Wherein LTB is positioned at 5 ' end of flexible joint sequence, and TGF β 1 is positioned at 3 ' end of flexible joint sequence.Sequence is LTB-GGGGSGGGGSGGGGS-TGF β 1.Convert nucleotide sequence to according to the preferred codon of escherichia coli, nucleotide sequence is seen SEQ ID NO:4.

Synthesize 1.1.2 nucleotide sequence intergrowth thing company is carried out full gene, and increase EcoR I and HindIII restraining line restriction enzyme site respectively at 5 ' end and 3 ' end.

1.1.3 express synthetic gene order and prokaryotic expression plasmid pET28a (+) (Novagen company commercial product) with EcoR I and HindIII double digestion.And utilizing gel electrophoresis to be purified into the purpose fragment, the T4 dna ligase connects, and transforms DH5 α antibacterial, cultivates the back and extracts plasmid and send biotech firm's evaluation of checking order.The all errorless plasmid of picking nucleotide sequence and reading frame transforms BL21 (DE3) pLysS competent cell (Novagen company commercial product).Picking list bacterium colony cultivate or freezing preservation standby.

1.2 the abduction delivering of recombiant plasmid

The process of inducing is carried out with reference to the pET system workbook of carrier description Novagen company.The bacterium of will recombinating is streak culture, single bacterium colony that picking typical case is fresh, shaken cultivation is spent the night in fresh LB culture medium, next day is by the 1% volume ratio fresh LB culture fluid of transferring, 37 ℃ of shaken cultivation are to exponential phase, add IPTG to final concentration be 1mmol/ml.Continue shaken cultivation 4h.Centrifugal collection thalline, the PBS washing concentrates by 1/20 volume ratio.Ultrasonic disruption behind the high speed centrifugation, is transferred to supernatant in another centrifuge tube.The gained precipitation returns to original volume with the TE buffer.Precipitation and supernatant carry out SDS-PAGE electrophoresis detection, coomassie brilliant blue staining.Set up the recipient bacterium that contains pET28a (+) empty carrier simultaneously and do not add that the inductive E.coliBL21 of IPTG (DE3)/pETLTB-TGF in contrast.The result shows that fusion rotein has obtained expressing preferably, and the expression product major part is arranged in inclusion body.

1.3 the western-blot of recombiant protein analyzes

Earlier recombiant protein is made the SDS-PAGE electrophoresis, electrotransfer is to nitrocellulose filter.Operations such as concrete transfer printing colour developing are all undertaken by " molecular cloning experiment guide ".Spend the night with the TBST sealing that contains 5%BSA then, anti-as one with rabbit anti-TGF-beta 1 polyclonal antiserum, the sheep anti-mouse igg of HRP labelling is two anti-, the colour developing of DBA colour reagent box.The result show expressed fusion protein can with the antiserum good binding, show that expressed albumen has kept the antibodies characteristic of TGF-β 1.

1.4 the purification of recombiant protein

Cultivate the positive colony bacterium, add IPTG and induce expressing fusion protein,, extract inclusion body protein, utilize the His-Tag characteristic through nickel post affinity purification through ultrasonication.

1.5 reorganization LTB-TGF protein drug formulation preparation

Get the good LTB-TGF fusion rotein of purification, adjusting its protein concentration is 1mg/ml.Mix with the equal-volume adjuvant respectively,, promptly be formed for treating the pharmaceutical preparation of domestic animal persistence pathogenic infection through fully emulsified.The concrete operations step can be carried out with reference to the adjuvant description.

Embodiment 2: the autovaccine preparation preparation of general helper T cell epitope (PADRE), tetanus toxin t cell epitope peptide and TGF-β 1 whole protein sequence amalgamation and expression:

The aminoacid sequence of general helper T cell epitope (PADRE) is seen SEQ ID NO:5, the aminoacid sequence of tetanus toxin t cell epitope peptide (TTE) is seen SEQ ID NO:6, the fusion rotein aminoacid sequence that makes up is: PADRE-TTE-GGGGSGGGGSGGGGS-TGF β 1, with aminoacid sequence by e. coli codon optimization and adopt the synthetic method of full gene to obtain the nucleotide sequence of this fusion rotein, and be cloned into expression vector, transform expression strain and obtain fusion rotein.Its specific solution is carried out with reference to embodiment one and " molecular cloning experiment guide ".

Embodiment 3:TGF β 1 autovaccine preparation is to the influence of mice CD4+CD25+ regulatory T cells quantity.

Utilizing the LTB-TGF or the PARDE-TGF fusion rotein secondary immunity mice of preparation, is fusion rotein and complete Freund's adjuvant mixed immunity for the first time wherein, adopts fusion rotein and incomplete Freund's adjuvant mixed immunity mice for the second time.Secondary immunity interval 15 days, CD4 was measured in immunity for the second time in back 15 days ⁺CD25 ⁺Regulatory T cells quantity and distribution.The reagent that is adopted is the FITC rat anti-mouse CD4 monoclonal antibody and the PE rat anti-mouse CD25 monoclonal antibody of U.S. eBioscience company, utilizes BD FACSCaliburTM type cells were tested by flow cytometry CD4 ⁺CD25 ⁺Regulatory T cells quantity and distribution.The result shows that autovaccine preparation can significantly reduce the quantity of regulatory T cells.

Embodiment 4:TGF β 1 autovaccine preparation is to the therapeutic effect of porcine circovirus inapparent infection pig.

2.1 choose about 30 kilograms of body weight, healthy anosis, detect 20 of the pigletss that pig parvoviral, porcine circovirus, reproductive and respiratory syndrome, Pseudorabies virus, encephalitis b virus all be negative.The criterion of porcine circovirus persistent infection pig: outward appearance and behavior do not have the symptom of obviously causing a disease, and body temperature is normal, circovirus antibody is positive, sleeve type PCR detects pathogen and is positive.The pig that is judged to be persistent infection is divided into two groups at random.

2.1.1 circovirus antibody detection method: adopt indirect ELISA method to carry out, test kit is Wuhan Keqian Animal Biological Products Co., Ltd.'s product, and operational approach reference reagent box description is carried out.Sample OD ₆₃₀Value is judged to the positive greater than 0.42; Sample OD ₆₃₀Value is judged to suspicious between 0.38 to 0.42; Sample OD ₆₃₀Value is judged to feminine gender less than 0.38.

2.1.2 the detection method of PCR: adopt the method for sleeve type PCR to detect porcine circovirus DNA.Outside primer is P1 (5 '-CAACTGCTGTCCCAGCTGTAG-3 ') and P2 (5 '-AGGAGGCGTTACCGCAGAAG-3 '); Inner primer is P3 (5 '-TAGGTTAGGGCTGTGGCCTT-3 ') and P4 (5 '-CCGCACCTTCGGATATACTG-3 ').Primer is synthetic by biotech firm.The secondary PCR reaction condition is all by 94 ℃ of 1min, 65 ℃ of 1min, and 72 ℃ of 1min totally 30 circulations, last 72 ℃ are extended 5min.Getting the PCR product observes with the 10g/L agarose gel electrophoresis.

2.2 experiment grouping and processing: 20 pigs that are judged to be persistent infection are divided into two groups at random, and one group is matched group, does not do any processing.Another group is experimental group, the pharmaceutical preparation treatment that TGF-β 1 bacterin preparation of the employing embodiment of the invention 1 is made.

2.3 the result observes: the result shows, the anti-circovirus antibody level of experimental group is apparently higher than matched group, two groups of significant differences (P＜0.05).The antibody horizontal that improves helps body to remove the pig circular ring virus of inapparent infection.PCR result shows in 10 pigs of experimental group have 8 to be shown as porcine circovirus DNA feminine gender, and 10 pigs to have only 1 to be negative in the matched group.The result shows that the pharmaceutical preparation of TGF-β 1 autovaccine preparation preparation can effectively promote the removing speed of body to porcine circovirus.

Sequence table

＜110〉Agricultural University Of Hunan

＜120〉a kind of application that contains TGF β 1 body bacterin preparation

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<170>PatentIn?version?3.3

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Val?Arg?Gln?Leu?Tyr?Ile?Asp?Phe?Arg?Lys?Asp?Leu?Gly?Trp?Lys?Trp

20 25 30

Ile?His?Glu?Pro?Lys?Gly?Tyr?His?Ala?Asn?Phe?Cys?Leu?Gly?Pro?Cys

35 40 45

Pro?Tyr?Ile?Trp?Ser?Leu?Asp?Thr?Gln?Tyr?Ser?Lys?Val?Leu?Ala?Leu

50 55 60

Tyr?Asn?Gln?His?Asn?Pro?Gly?Ala?Ser?Ala?Ala?Pro?Cys?Cys?Val?Pro

65 70 75 80

Gln?Ala?Leu?Glu?Pro?Leu?Pro?Ile?Val?Tyr?Tyr?Val?Gly?Arg?Lys?Pro

85 90 95

Lys?Val?Glu?Gln?Leu?Ser?Asn?Met?Ile?Val?Arg?Ser?Cys?Lys?Cys?Ser

100 105 110

<210>2

<211>103

<212>PRT

<213>Escherichia?coli

<400>2

Ala?Pro?Gln?Thr?Ile?Thr?Glu?Leu?Cys?Ser?Glu?Tyr?Arg?Asn?Thr?Gln

1 5 10 15

Ile?Tyr?Thr?Ile?Asn?Asp?Lys?Ile?Leu?Ser?Tyr?Thr?Glu?Ser?Met?Ala

20 25 30

Gly?Lys?Arg?Glu?Met?Val?Ile?Ile?Thr?Phe?Lys?Ser?Gly?Glu?Thr?Phe

35 40 45

Gln?Val?Glu?Val?Pro?Gly?Ser?Gln?His?Ile?Asp?Ser?Gln?Lys?Lys?Ala

50 55 60

Ile?Glu?Arg?Met?Lys?Asp?Thr?Leu?Arg?Ile?Thr?Tyr?Leu?Thr?Glu?Thr

65 70 75 80

Lys?Ile?Asp?Lys?Leu?Cys?Val?Tyr?Asn?Asn?Lys?Thr?Pro?Asn?Ser?Ile

85 90 95

Ala?Ala?Ile?Ser?Met?Lys?Asn

100

<210>3

<211>15

<212>PRT

<213>Artificial

<220>

＜223〉flexible joint sequence

<400>3

Gly?Gly?Gly?Gly?Ser?Gly?Gly?Gly?Gly?Ser?Gly?Gly?Gly?Gly?Ser

1 5 10 15

<210>4

<211>693

<212>DNA

<213>Artificial

<220>

＜223〉artificial sequence

<400>4

atggctcccc?agactattac?agaactatgt?tcggaatatc?gcaacacaca?aatatatacg 60

ataaatgaca?agatactatc?atatacggaa?tcgatggcag?gcaaaagaga?aatggttatc 120

attacattta?agagcggcga?aacatttcag?gtcgaagtcc?cgggcagtca?acatatagac 180

tcccagaaaa?aagccattga?aaggatgaag?gacacattaa?gaatcacata?tctgaccgag 240

accaaaattg?ataaattatg?tgtatggaat?aataaaaccc?ccaattcaat?tgcggcaatc 300

agtatgaaaa?acggtggtgg?tggtagcggt?ggtggtggta?gtggcggtgg?cggtagcgcc 360

ctggatacca?actattgctt?tagctctacg?gagaaaaact?gctgtgttcg?tcagctgtac 420

attgatttcc?gcaaagatct?gggctggaaa?tggatccatg?aaccgaaagg?ctatcacgca 480

aacttctgcc?tgggtccgtg?tccgtacatt?tggtctctgg?atacgcagta?tagtaaagtg 540

ctggccctgt?acaaccagca?taatccgggt?gcaagcgcag?caccgtgctg?tgttccgcag 600

gcgctggaac?cgctgccgat?tgtgtattac?gttggtcgta?aaccgaaagt?ggaacagctg 660

tctaatatga?tcgttcgcag?ttgcaaatgt?agc 693

<210>5

<211>13

<212>PRT

<213>Artificial

<220>

＜223〉artificial sequence amino acid

<400>5

Ala?Lys?Phe?Val?Ala?Ala?Trp?Thr?Leu?Lys?Ala?Ala?Ala

1 5 10

<210>6

<211>14

<212>PRT

<213>Artificial

<220>

＜223〉artificial sequence amino acid

<400>6

Gln?Tyr?Ile?Lys?Ala?Asn?Ser?Lys?Phe?Ile?Gly?Ile?Thr?Glu

1 5 10

Claims (8)

1. one kind contains TGF β 1 application of autovaccine preparation, it is characterized in that, described bacterin preparation is used to prepare the pharmaceutical preparation of treatment domestic animal persistence pathogenic infection.

2. the application of bacterin preparation according to claim 1 is characterized in that, adds adjuvant in the described bacterin preparation, is prepared into the pharmaceutical preparation that is used for the treatment of domestic animal persistence pathogenic infection.

3. the infection of domestic animal persistence pathogen according to claim 1 is characterized in that: the infection of described domestic animal persistence pathogen comprises chronic infection, inapparent infection, repeated infection and healthy band poison.

4. according to the application of claim 1 or 2 or 3 described bacterin preparations, it is characterized in that, described domestic animal persistence pathogenic infection comprises: the infection that the secondary haemophilus of porcine circovirus, reproductive and respiratory syndrome virus, swine fever, parvovirus, mycoplasma hyopneumoniae, Pseudorabies virus, bovine viral diarrhea virus, contagious equine abortion, pig, Actinobacillus pleuropneumoniae, Mycobacterium tuberculosis cause, and multiple pathogen the female animal chronic endometritis, the mastitis that cause.

5. the application of bacterin preparation according to claim 1 and 2, it is characterized in that, described bacterin preparation is by the part of domestic animal self TGF β 1, whole, part or all of mutant or whole similar gene orders and the gene order reorganization that at least one can improve immunogenic helper T cell epitope or carrier protein, make up eucaryon or prokaryotic expression plasmid, transform the host bacterium and express the fusion rotein that obtains; Perhaps the eukaryon expression plasmid that is directly obtained by structure is made.

6. the application of bacterin preparation according to claim 5 is characterized in that: described helper T cell epitope comprises the helper T cell epitope in carrier protein, general helper T cell epitope PADRE, tetanus toxoid amino acid fragment TTE, animal infection pathogen source or the t cell epitope that manually designs in conjunction with gene order according to the animal II of main histocompatibility complex class.

7. the application of bacterin preparation according to claim 5, it is characterized in that: described carrier protein comprises bovine serum albumin, keyhole limpet hemocyanin, hepatitis B surface antigen, hepatitis B virus core antigen, E.coli LT B, ovalbumin, heat shock protein or be directed to label protein fusion rotein on the expression vector.

8. according to the application of claim 5 or 6 described bacterin preparations, it is characterized in that: described helper T cell epitope comprises also that multiple helper T cell epitope mixes and forms, or the single external source helper T cell epitope form that repeats to be in series.