CN102657865A - Method for conveying proteins or antigens to mammal intestinal tract in targeted way by using transgenic coccidian - Google Patents
Method for conveying proteins or antigens to mammal intestinal tract in targeted way by using transgenic coccidian Download PDFInfo
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Abstract
The invention provides a method for conveying proteins or antigens to a mammal intestinal tract in a targeted way by using a transgenic coccidian. The coccidian is taken as a carrier for conveying proteins or antigens in a targeted way, and foreign proteins or antigens are expressed in the sporangium cavity of the coccidian by performing transgenic operation on the coccidian. The invention provides a novel application of the transgenic coccidian. The foreign proteins are targeted onto the sporangium cavity of the coccidian by using an excretion signal peptide of the coccidian, an oocyst breaks and releases sporangium under the mechanical creeping action of stomach in the stomach, the sporangium breaks and releases daughter spores under the digestion actions of gall and pancreatic enzyme in a small intestine, proteins in the sporangium are released into an intestinal tract, and the denaturation degradation effect of gastric juice on the proteins is avoided, so that proteins in the sporangium are directly contacted with intestinal mucosa, and the aim of activating enteric immune response or treating enteric diseases is fulfilled. The coccidian used in the method has the advantages of high type specificity, high biological safety, capability of releasing foreign proteins into intestinal tracts in a targeted way, suitability for oral application, easiness for operating and wide application prospect.
Description
Technical field
The present invention relates to the application of transgenic coccidiosis, specifically, relate to and a kind ofly utilize the transgenic coccidiosis to transport albumen or antigenic method to mammal intestinal targeting.
Background technology
Mucosa is the main position of most of cause of disease invasion body, with the exogenous antigen mucosal sites of practicing shooting, is a kind of Critical policies of opposing cause of disease invasion thereby excite specific mucosal immune response therefore.Common mucosa transports antigen or protein system and mainly contains two big types: a kind of is the system of synthetic, is that the antigen with purification is wrapped in microsphere, and liposome is in nano-particle or the immunostimulation complex; Another kind is the live vector system, comprises that some cause weak bacterial isolates or virus stain etc.But all there is weak point in these two kinds of systems---the former needs the artificial preparation purifying antigen and wraps up, and its complicated operation needs more a large amount of antigen or albumen, and antigen protein degraded easily under one's belt during the oral administration immunity; Though the latter can express and transport antigen, these cause weak bacterial strain or strain might recover virulence, has certain bio-safety risk.Transport antigen or proteic system so demand developing the targeting mucosa of a kind of biological safety height and easy operating urgently.
Summary of the invention
The objective of the invention is to overcome existing mucosa and transport problems such as the complicated operation, the biological safety that exist in antigen or the protein system are not high, provide a kind of new transgenic coccidiosis that utilizes to transport albumen or antigenic method to mammal intestinal targeting.
In order to realize the object of the invention; Of the present inventionly a kind ofly utilize the transgenic coccidiosis to transport albumen or antigenic method to mammal (comprising the people) intestinal-specific; It is to transport albumen or antigenic carrier with coccidiosis as targeting; Through coccidiosis is carried out transgeneic procedure, make foreign protein or antigen presentation spore blister cavities in coccidiosis.
Aforesaid method, wherein said coccidiosis are Amy otology coccidiosis, are preferably birds Amy otology coccidiosis or mammal Amy otology coccidiosis.
Aforesaid method, wherein said foreign protein or antigen are human cytokines or pathogen antigen.Preferably, said human cytokines is IFN-γ, TNF-α, IL-10, curative monoclonal antibody of human or animal etc.; Pathogen antigen is the CTB subunit of vibrio cholera, the VP4 of human or animal's rotavirus, VP6 antigen, and the gp160 antigen of HIV (HIV) produces the antigen of enterotoxigenic Escherichia coli etc.
In the preceding method, coccidiosis is carried out transgeneic procedure may further comprise the steps:
1) optimize said foreign protein or antigenic genetic fragment according to the codon preference of coccidiosis and carry out the full length DNA of genetic fragment synthetic;
2) genetic fragment with above-mentioned optimization is integrated among the expression vector pMEAAssRA;
The expression vector pMEAAssRA that 3) will contain exogenous segment carries out the zygoblast of transfection coccidiosis after the linearisation, and through the inoculation of chicken cloaca, collects egg capsule in the feces row filter of going forward side by side, and obtains to express said foreign protein or antigenic transgenic coccidiosis.
Wherein, the nucleotide sequence of expression vector pMEAAssRA is shown in SeqID No.1 step 2).
In an optimal technical scheme, the present invention has made up the transgenic chicken coccidiosis of a strain stably express Mucasal Adjuvant Cholera Toxin Subunit B (CTB).CTB is a kind of mucosal adjuvants of classics, can promote the proteic immunne response that merges with it significantly.CTB is expressed in the transgenic coccidiosis oral vaccination mice of spore blister cavities, and mice increases significantly to the antibody response of this transgenic coccidiosis mice than the wild type immunity to coccidiosis.Proved that the CTB that the transgenic coccidiosis is expressed in the spore blister cavities has brought into play important adjuvant effect.
Utilize DE frame carrier that yellow fluorescence protein is expressed in the coccidiosis endochylema, simultaneously CTB is practiced shooting in the spore blister cavities of coccidiosis.Utilize flow cytometer to screen luminous polypide and go down to posterity, passed to for the tenth generation repeatedly, luminous efficiency is about 90%, and the worm strain is basicly stable.Carrying out mouse immuning test, 30 mices are divided into 4 groups, is respectively transgenic coccidiosis worm group alive, transgenic coccidiosis deactivation worm group, wild type coccidiosis worm group alive and PBS matched group.At interval two weeks, immunity twice altogether respectively at gathering serum after the immunity, detects IgG and IgA content in the serum.
One exempt from after, antibody titer is all lower in the serum, two exempt from the back transgenic coccidiosis worm group of living has all produced than the higher antibody response that is directed against somatic antigen with transgenic coccidiosis deactivation polypide group.Show with wild type coccidiosis polypide and compare that the transgenic polypide has excited antibody response better.
The invention provides the new purposes of a kind of transgenic coccidiosis; The coding sequence of secretory signal peptide of utilizing coccidiosis is with the practice shooting spore blister cavities of coccidiosis of foreign protein; Egg capsule is under one's belt because of the mechanical creeping effect of the stomach release Sporangium that breaks; Sporangium broken capsule of Digestion because of bile and pancreatin in small intestinal discharges zygoblast, and the albumen in the Sporangium is discharged into intestinal thereupon, has avoided gastric juice to proteic degeneration Degradation; Thereby the albumen in the Sporangium is directly contacted with intestinal mucosa, reach and excite intestinal immune to reply or treat the purpose of intestinal tract disease.
Transport antigenic system compared to existing two kinds, coccidiosis new is transported albumen or antigenic carrier system has clear superiority to intestinal-specific as a kind of.One of which, coccidiosis have very high biological safety, and its host specificity is strong, and except that chicken, aplasia in people and other animal bodies can not work the mischief with other animals to the people; Its two, coccidiosis is easy to go down to posterity and purification, obtain easily, and when being applied to people or other animals, oral vaccination, easy and simple to handle; Its three, coccidiosis can be anti-crosses the Digestion of gastric acid, skin lesion of the scrotum in enteric cavity can directly be released to intestinal with the antigen in the spore blister cavities, and then brings into play these antigenic effects; Its four, the genetic manipulation of coccidiosis is comparative maturity at present, can exogenous antigen be positioned the different parts of polypide respectively, utilizes the signal peptide of secreting type, can antigen be secreted in the spore blister cavities of coccidiosis.
The invention has the advantages that:
(1) the present invention is expressed in a kind of mucosal adjuvants CTB of classics the spore blister cavities of chicken coccidiosis first, and use in a creative way mice as scale-model investigation the adjuvant effect of CTB to coccidiosis antigen itself.
(2) the invention provides the new purposes of coccidiosis, can exogenous antigen (HRV VP4, VP6, HIV gp160 antigen or produce the antigen of enterotoxigenic Escherichia coli) and some curative albumen (IFN-γ, TNF-α, IL-10 or some specific monoclonal antibodies) targeting be transported in the enteric cavity.
(3) this transports carrier and is suitable for oral use, can the anti-Digestion of crossing gastric acid, and simple to operate, have a extensive future.
(4) the coccidiosis species specificity of the present invention's use is strong, and chicken coccidiosis or mammal coccidiosis be infected person not, can not stride species and propagate, and has very high biological safety.
Description of drawings
Fig. 1 is the ideograph of transfection used carrier pMEAAssCTBA of the present invention: MicI is the promoter of the little linear protein I of coccidiosis among the figure; Actin is the upstream and downstream regulating and controlling sequence of coccidiosis actin; Gra8ss is the signal peptide of Toxoplasma gondii dense granule 8; EYFP is reinforced yellow fluorescence protein gene coding region, and CTB is choleratoxin B subunit (other antigenic protein gene on position is identical with CTB).
Fig. 2 is the 10th a generation luminous polypide and western-blot qualification result figure, and wherein A is depicted as the egg capsule after the 10th generation sporeization, and B is protein expression qualification result figure.
Fig. 3 inoculates mice after 6 hours, gastric content (A) and mucous membrane of small intestine (B) smear for the transgenic coccidiosis.
Fig. 4 is the antibody response of immunity back mice to the transgenic chicken coccidiosis, and wherein A exempts from the reaction of exempting from back IgG with two, and B two exempts from the reaction of back IgA.
The specific embodiment
Following examples are used to explain the present invention, but are not used for limiting scope of the present invention.If do not specialize the conventional means that used technological means is well known to those skilled in the art among the embodiment, the raw materials used commercial goods that is.
Embodiment
Present embodiment is a carrier with transgenic Eimeria tenella (Eimeria tenella), expresses a kind of mucosal adjuvants CTB of classics, and its targeting is transported in the enteric cavity of mice, strengthens the effect of mice to the reaction of chicken immunity to coccidiosis thereby reach.
1, vector construction
Login password uses the usage frequency of each codon in the database website http://www.kazusa.org.jp/codon/ inquiry eimeria tenella protein translation process; Go up the operations such as key entry of completion vibrio cholera CTB gene order and Eimeria tenella codon usage frequency table at OPTIMIZER server (http://genomes.urv.es/OPTIMIZER/) after; Can obtain the improved sequence of CTB, the CTB gene Selection is used the prioritization scheme of right codon fully.Sequence after optimizing is transferred to directly synthetic (the Seq ID No.2) that dna sequencing Synesis Company carries out full length sequence.With restriction enzyme A geI, NotI enzyme action cholera toxin gene and carrier pMEAAssRA (Seq ID No.1); 37 ℃ were reacted 8 hours; Endonuclease bamhi is carried out dna fragmentation reclaim, connect and conversion, then little upgrading grain identifies, will identify that male plasmid carries greatly; Promptly get the coccidiosis transfection carrier that builds, pMEAAssCTBA (Fig. 1).
Wherein, Carrier pMEAAssRA is to be skeleton carrier with the pEASY-Blunt-Simple carrier; Inserting following fragment successively makes up and to obtain: the little linear protein 1 promoter (MIC1 of coccidiosis; 779bp), the yellow fluorescence protein gene (EYFP, 726bp), the promoter of the termination regulating and controlling sequence of coccidiosis actin (ACT3 ' 632bp), coccidiosis actin (ACT5 ' 1128bp), red fluorescent protein gene (RFP, 678bp) and the termination regulating and controlling sequence of coccidiosis actin (ACT3 ' 632bp).The base sequence of carrier pMEAAssRA is shown in Seq ID No.1.
2, consideration convey dyes the screening with the transgenic coccidiosis
Carrier for expression of eukaryon pMEAAssCTBA is carried out enzyme action with SnaBI, reclaim linearizing dna segment (8397bp) then.Get 1 * 10
7The zygoblast of individual Eimeria tenella; The linearization plasmid of 5-10 μ g; The consideration convey of 5 μ LSnaB I restricted enzyme and 100 μ L dyes buffer; Move to behind the mixing in the electric shock cup, the electric shock cup is inserted in the electric shock tank of consideration convey instrument Nucleofector II, select consideration convey to dye program U-033 and press the button the consideration convey mixture is shocked by electricity; In the electric shock cup, add 1000 μ L DMEM culture fluid, the liquid in the electric shock cup is transferred to given young chicken cloaca inoculation in the 1.5 μ L centrifuge tubes.Collect 6～9 days egg capsules in the feces, with the luminous polypide of flow cytometer screening, the inoculation chicken is collected egg capsule again, inoculates chicken after the sporeization, and repeatedly ten generations, visible luminescent efficiency reaches about 90%, and CTB has obtained stable expression in the chicken coccidiosis.The result is as shown in Figure 2.
3, Western blot identifies
Get spore egg capsule 2 * 10
7Individual adding equal-volume bead with full temperature agitator vibration 20min, is got supernatant with liquid nitrogen multigelation 3 to 5 times, and supernatant adding protease inhibitor places ultrasonic degradation on ice, ultrasonic 2 seconds, stops 3 seconds, ultrasonic 5 minutes.4 ℃ of centrifugal 10min of 12000rpm get supernatant, in suspension, add 1 * sample-loading buffer mixing.Boil 10min in the boiling water.Place 2min on ice, 4 ℃ of centrifugal 10min of 12000rpm get supernatant; Last appearance is carried out the SDS-PAGE electrophoresis and is changeed film then, seals 1h in advance with 5% defatted milk powder room temperature; Be directed against the Myc tag antibody with 5% defatted milk powder by 1: 1000 dilution Mus source, add incubated at room 1h on the film.Anti-with 5% defatted milk powder by the anti-Mus two of 1: 1000 dilution HRPO labelling, incubated at room 1h.Take out washed film with 1: 1 blended ECL A liquid and B liquid immersion 1min.Film taken out to be placed on cover (bubble can not be arranged) with preservative film on the plastic plate and put into the tabletting box.In the darkroom, the X-ray sheet is placed on the film, makes its sensitization, place developer solution to develop the X-ray sheet then, photographic fixing in the fixative solution, the result is as shown in Figure 3.By having Sporangium to exist in the visible stomach of figure, zygoblast discharges from Sporangium in the small intestinal, the invasion intestinal mucosa.
4, inoculation mice
30 mices are divided into 4 groups, are respectively transgenic coccidiosis worm groups alive, transgenic coccidiosis deactivation worm group, wild worm group alive and PBS matched group.At interval two weeks, immunity twice altogether respectively at gathering serum after the immunity, detects IgG and IgA in the serum.The result is as shown in Figure 4.With 100 times of serum dilutions, two dilutions in anti-equal 1: 5000 are compared with wild insect mice immunized by figure is visible, and transgenic insect mice immunized has produced higher antibody response.
Though, the present invention has been done detailed description in the preceding text with general explanation and specific embodiments, on basis of the present invention, can to some modifications of do or improvement, this will be apparent to those skilled in the art.Therefore, these modifications or the improvement on the basis of not departing from spirit of the present invention, made all belong to the scope that requirement of the present invention is protected.
Claims (6)
1. utilize the transgenic coccidiosis to transport albumen or antigenic method to mammal intestinal targeting; It is characterized in that; It is to transport albumen or antigenic carrier with coccidiosis as targeting, through coccidiosis is carried out transgeneic procedure, makes foreign protein or the antigen presentation spore blister cavities in coccidiosis.
2. method according to claim 1 is characterized in that, described coccidiosis is an Amy otology coccidiosis.
3. method according to claim 2 is characterized in that, described coccidiosis is birds Amy otology coccidiosis or mammal Amy otology coccidiosis.
4. according to each described method of claim 1-3, it is characterized in that described foreign protein or antigen are human cytokines or pathogen antigen.
5. method according to claim 4 is characterized in that, described human cytokines is human or animal's IFN-γ, TNF-α, IL-10 or a curative monoclonal antibody; Pathogen antigen is the CTB subunit of vibrio cholera, the VP4 of human or animal's rotavirus, VP6 antigen, the antigen of gp160 antigen of HIV or product enterotoxigenic Escherichia coli.
6. according to each described method of claim 1-3, it is characterized in that, saidly coccidiosis is carried out transgeneic procedure may further comprise the steps:
1) optimizes said foreign protein or antigenic genetic fragment according to the codon preference of coccidiosis;
2) genetic fragment with above-mentioned optimization is integrated among the expression vector pMEAAssRA;
The expression vector pMEAAssRA that 3) will contain exogenous segment carries out the zygoblast of transfection coccidiosis after the linearisation, and through the inoculation of chicken cloaca, collects egg capsule in the feces row filter of going forward side by side, and obtains to express said foreign protein or antigenic transgenic coccidiosis;
Wherein, the nucleotide sequence of expression vector pMEAAssRA is shown in SeqID No.1 step 2).
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| CN104673830A (en) * | 2014-01-28 | 2015-06-03 | 中国农业大学 | Method for improving calcium and phosphorus utilization ratio of livestock and poultry |
| CN104830882A (en) * | 2014-02-10 | 2015-08-12 | 中国农业大学 | Fusion gene for expressing Fc fragment of chicken IgY molecule and avian influenza virus antigen and application of same |
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Cited By (3)
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| CN104673830A (en) * | 2014-01-28 | 2015-06-03 | 中国农业大学 | Method for improving calcium and phosphorus utilization ratio of livestock and poultry |
| CN104830882A (en) * | 2014-02-10 | 2015-08-12 | 中国农业大学 | Fusion gene for expressing Fc fragment of chicken IgY molecule and avian influenza virus antigen and application of same |
| CN104830882B (en) * | 2014-02-10 | 2018-02-06 | 中国农业大学 | Express fusion and its application of chicken IgY molecule Fc fragments and avian influenza virus antigen |
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Application publication date: 20120912 |