CN101352157B - Method for constructing chicken model for transgenosis and production exogenous protein - Google Patents
Method for constructing chicken model for transgenosis and production exogenous protein Download PDFInfo
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- CN101352157B CN101352157B CN2008101184331A CN200810118433A CN101352157B CN 101352157 B CN101352157 B CN 101352157B CN 2008101184331 A CN2008101184331 A CN 2008101184331A CN 200810118433 A CN200810118433 A CN 200810118433A CN 101352157 B CN101352157 B CN 101352157B
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Abstract
The invention discloses a method for constructing a chick model which is used for transgene and generating heterologous protein. The method of the invention comprises the following steps: 1) the heterologous protein is introduced to the yolk sac of a breeding egg; 2) the breeding egg in step 1) is incubated still for 46 to 50 hours and incubated continuously until hatching birds, thus obtaining the chick model which is used for transgene and generating the heterologous protein. The method of the invention has the advantages of simple and easily obtained operation condition, low cost, high maneuverability, low introducing dosage of the heterologous protein, good repeatability and high hatching rate. Besides, the method of the invention has little influence on chick embryo development, the hatching rate can reach 73 percent to 83 percent, the durable time of immune tolerance is relatively long, and the chick model which is used for transgene of 60 days still has immune tolerance to specific heterologous protein antigens.
Description
Technical field
The present invention relates to a kind of method of producing the chicken model for transgenosis that foreign protein uses that makes up.
Background technology
The foundation of eighties of last century DNA seventies recombinant technique, allogeneic dna sequence DNA is directed in the eukaryotic, this gene transfer technique provides the ability that changes the higher mammal cell from heredity for the mankind, and the basis that becomes the preparation transgenic animal and carry out gene therapy.Use the DNA recombinant technique, people have obtained successfully to comprise multiple transgenic animal such as mouse, ox, pig, rabbit, chicken, fish, nematode and sea urchin that nineteen ninety, transgenic method also is used in human gene therapy's the research at present.For many higher mammals, particularly have perfect immune vertebrate, there is potential immunostimulating in gene delivery system, and expression of heterologous genes causes that easily the host produces immune response.Generally, no matter be to prepare transgenic animal or carry out gene therapy, its purpose is exactly in order to obtain the expression of exogenous gene product efficiently.Because host itself is these albumen not, nature is identified as non-autoantigen with it and is removed, and causes the transient expression of transgene protein, even causes inflammatory reaction.For fear of the generation of this situation, need the host that specific foreign protein is produced the immunity tolerance, immunological rejection does not take place, promptly form immune tolerance.
Immune tolerance is meant that destructive immunoreactive a kind of physiological status does not take place for immune system and body proper constituent or the antigen that is introduced into, this state can be by antigen being introduced developmental immune system the people for inducing.According to the clonal selective theory that antibody produces, under the normal condition, embryo and outside antigenic stimulus are kept apart, and embryo's lymphatic system only can run into autoantigen, thereby have caused the elimination (immunity is tolerant) of autoimmune response.Immune system has been learned tolerance in growth course, its task is the rearrangement generation structure diversity at random by T cell and B cell antigen receptor gene, the molecule that identification is met unexpectedly is also reacted, thereby be a kind of acquired phenomenon, need antigen induction to produce, even if also be like this to the tolerance of autoantigen.But for most of transgenic animal, particularly as the transgenic animal of bio-reactor, foreign gene is expressed in the full grown adults of immune system usually, therefore need set up a kind of by introduce the method for exogenous antigen inducing immune tolerance in growing early stage embryo.
Can antigenicity substance induce the generation immune tolerance after entering body, depends on mainly and the reason of antigen and body two aspects that generally speaking, the antigen of little molecule solubility, non-polymeric state mostly is toleragen as haemocyanin, polysaccharide and lipopolysaccharides etc.These little molecule soluble antigens are difficult for being absorbed by phagocyte in vivo, might induce body to produce immune tolerance by the mode of direct and lymphocyte effect with optimum concentration.And macromolecular particle material and protein polymer are good immunogene as haemocyte, bacterium or human gamma globulin (HGG) polymer etc.These macromolecular substances are easily absorbed by phagocyte, but the effective stimulus lymphocyte produces immune response after processing is handled.Induce the required antigen dose of tolerance different with antigen type, tolerance cell type, animal genus kind, strain and age.Generally speaking, antigen is through the easiest generation immune tolerance of intravenous injection, and lumbar injection takes second place, and is subcutaneous the most difficult with intramuscular injection, but the result that the different parts intravenous injection causes is also inequality.For example human gamma globulin (HGG) injects through mesenteric vein and can cause tolerance, injects through jugular vein then to cause immune response; IgG or albumin inject portal vein can cause tolerance, injects PeV and then causes immune response.The difficulty or ease that inducing immune tolerance forms are also relevant with the development degree of body immune system, and generally the easiest in embryonic period, embryonic phase, introduction stage takes second place, and the manhood is the most difficult.Experiment in vitro confirms that the prematurity immunocyte is easy to induce the generation immune tolerance, and the maturation immunity cell then is difficult to induce the generation tolerance; Induce the required antigen amount of maturation immunity cell tolerance than inducing high tens of times of the required antigen amount of prematurity immunocyte tolerance.At present a large amount of experimental evidences shows, when the formation of immune tolerance contacts antigen for the first time with immune system the residing developmental stage closely related, immune tolerance can be by inducing embryo or newborn young bird antigen inoculation.
Different with mammal is, the embryonic development of bird is independently to finish in leaving the airtight eggshell of parent, this for embryonic period, embryonic phase antigen inoculation more convenient condition is provided.
Summary of the invention
The purpose of this invention is to provide a kind of method of producing the chicken model for transgenosis that foreign protein uses that makes up.
The method of the chicken model for transgenosis that structure production foreign protein provided by the present invention is used may further comprise the steps:
1) foreign protein is imported in the yolk sac of kind of egg;
2) with above-mentioned steps 1) the kind egg leave standstill hatch 46-50 hour after, continue again to hatch to hatching, obtain producing the chicken model for transgenosis that foreign protein is used.
In the said method, be 5-7 days embryo age of described kind of egg.
Described foreign protein is bovine serum albumin(BSA), bovine casein or human serum albumins, is preferably bovine serum albumin(BSA); The concentration of described bovine serum albumin(BSA) is 0.8-1.2g/L, and the dosage of importing is every kind egg 80-120 μ l.
During concrete operations, can make a call to two holes on the air chamber of kind of egg, one of them hole site is 0.3-0.5cm place, below, air chamber edge, by the fenestra on the air chamber foreign protein is imported in the yolk sac.
The present invention grows early stage at Embryo Gallus domesticus, before its immune system is not improved as yet, by in the yolk sac of kind of egg, importing foreign protein, it is absorbed by developmental embryo with the yolk nutriment, enter into embryo's blood circulation system, interact with the immunocyte of growing during along with blood circulation process thymus gland, induce the immunity silence of embryo immunization system generation, the final chicken model for transgenosis that obtains specific foreign protein immune tolerance foreign protein.
When kind of egg yolk sac is imported foreign protein, shorten the time as far as possible, the operating time of average every kind egg was controlled at 1-1.5 minute.Whole process needs to carry out in superclean bench, can not occur the situation of seepage when sealing the pin hole on the eggshell.The whole operation process can be finished at ambient temperature, for shortening the operating time, can a people prick that hole, a people import foreign protein, a people seals egg, shorten every kind egg time outside incubator as far as possible, to reduce the influence of environmental change to the hatching of chicken embryo, improve its incubation rate greatly, and then improve the immune tolerance rate.
Method of the present invention have operating condition be simple and easy to, low, the favorable repeatability of importing dosage with low cost, workable, foreign protein, tolerance rate and incubation rate time high and that the keep tolerance advantage of waiting so long, and the influence that the inventive method is grown Embryo Gallus domesticus is less, can obtain the incubation rate of 73-83%, successfully solved the problem that transgene product may cause the bion immune response, and the time that immune tolerance is kept is more of a specified duration, and still there is the immune tolerance to specific foreign protein antigen in the chicken model for transgenosis of 60 ages in days.The inventive method for the immune response that solves transgene product and cause provide one simply, solution efficiently, be the efficient exogenous genes products that obtains in Study on Transgenic Animal, setting up provides scientific basis and effective method to the bird model of gene outcome immune tolerance and the using value of raising chicken salpingo biological reactor.
Description of drawings
Fig. 1 is for importing the serum ELISA testing result of the chicken model for transgenosis that obtains behind the bovine serum albumin(BSA) to 3-7 days kind egg embryo of hatching
Embodiment
The present invention is described further below by specific embodiment.
Embodiment 1, the kind egg in homeomorphism not age is imported foreign protein-bovine serum albumin(BSA) (BSA)
Get each 30 pieces on the white leghorn chicken kind egg that embryo was respectively 3,4,5,6 and 7 days age (planting chicken house) available from China Agricultural University, after the sterilization of alcohol swab wiping eggshell surface, determine the residing position of embryo by micro-light source according to egg, on the eggshell of embryo's offside, mark out the edge of air chamber then with pencil, the time can effectively avoid the fetal blood pipe network in punching like this, when avoiding importing foreign protein to the mechanical damage of embryonic blood vessel.A punching mark is done at the 0.3-0.5cm place below the air chamber edge of mark, in order to keep the air pressure balance of air chamber and embryo chamber, do a punching mark at the air chamber top again,, use dissecting needle to prick two apertures (aperture is 1mm) fast in kind of an egg punching mark with alcohol swab wiping punching mark.
Use the 1ml disposable syringe, use No. 1 syringe needle, the syringe needle internal diameter is 0.1mm, drawing 100 μ l concentration is foreign protein-bovine serum albumin solution of 1g/L, vertically plant egg eggshell tangent plane from the aperture place at air chamber edge below 0.3-0.5cm place and penetrate in the egg in syringe needle submerges kind of egg fully, touch syringe piston in foreign protein-bovine serum albumin solution importing yolk sac.Keep former angle and gently extract syringe out, seal two holes with atoleine.
Kind egg after the sealing is put back in the incubator, leave standstill under the condition of turning egg(s) not hatch 48 hours after, under normal condition, continue hatching again until hatching, obtain chicken model for transgenosis.
The statistics incubation rate, the result shows, and the homeomorphism incubation rate of inoculating the chicken model for transgenosis of bovine serum albumin(BSA) age is not respectively 76.67%, 80.00%, 83.33%, 80.00% and 73.33%, and incubation rate is between 73%-83%.
Embodiment 2, utilize enzyme linked immunoassay (ELISA) to detect the immune tolerance result that homeomorphism is not inoculated the chicken model for transgenosis of bovine serum albumin(BSA) age
Bovine serum albumin(BSA) is through Freund (antigen emulsification when Freund's complete adjuvant is used for immunity for the first time, incomplete Freund is used for the antigen emulsification in other ages in days stage) after the emulsification, the 20th day, the 30th day, the 40th day and the 50th day after the chicken model for transgenosis that the not homeomorphism that the foregoing description 1 obtains is inoculated bovine serum albumin(BSA) age is hatched is the bovine serum albumin solution of 1g/L with 100 μ l concentration respectively, carry out 4 hypodermic injections, after the 4th hypodermic injection the 7th day, get the serum of chicken model for transgenosis, carry out ELISA and detect.Simultaneously with embryonic period, embryonic phase undressed chicken birth back carry out four chicken serums after the immunity as positive control according to above-mentioned dosage, to adopt chicken serum that specific pathogen free bacterium (SPF) handles as negative control.
Before the chicken serum sample is respectively organized in detection, need the chicken serum of elder generation, to determine the optimum response concentration of antigen-antibody by serial doubling dilution experimental group, positive controls and negative control group.Choose positive controls OD value about 1.0, and with same dilution factor under the different antigen coated concentration corresponding when maximum of negative control group 0D value difference and the serum diluting multiple antigen concentration and the extension rate that use during as test experience group serum sample.When the 0D of experimental group sample value during greater than 2.1 times of negative control group 0D value, judge that this sample is positive, on the contrary then negative.
All experimental datas adopt the SPSS11.5 statistical software to carry out statistical analysis.(represent by the mode of mean ± SEM) with mean+SD for data.Adopt independent sample t check (two tail), significant difference o'clock is thought in P＜0.05.
Concrete testing result as shown in Figure 1.As can be seen from Figure 1, the average level that to embryo is the anti--bovine serum albumin(BSA) antibody in the chicken model for transgenosis serum of kind egg inoculation bovine serum albumin(BSA) of 3 days or 4 days age is respectively 1.03 ± 0.13 (n=21) and 0.92 ± 0.12 (n=20), with positive control (1.02 ± 0.04, n=6) compare the not remarkable (p=0.984 of difference, p=0.636), show that to import bovine serum albumin(BSA) when embryo is 3 days or 4 days age bad to the effect of the tolerance of inducing chicken, the anti--bovine serum albumin(BSA) antibody that still has higher level in its blood; And to embryo be age that the average level of the anti--bovine serum albumin(BSA) antibody in the chicken model for transgenosis serum of kind egg inoculation bovine serum albumin(BSA) of 5 days, 6 days or 7 days is respectively 0.55 ± 0.08 (n=21), 0.66 ± 0.08 (n=20) and 0.44 ± 0.04 (n=16), significantly be lower than positive control (p=0.003,5 days groups; P=0.020,6 days groups; P＜0.001,7 day group), show that to import bovine serum albumin(BSA) when embryo is 5 days, 6 days or 7 days age better to the effect of the tolerance of inducing chicken.Further analyze relatively these five groups of data, found that, embryo is the average level (p＜0.05) that the average level of anti--bovine serum albumin(BSA) antibody significantly is lower than anti--bovine serum albumin(BSA) antibody in the chicken model for transgenosis serum that embryo is 3 days or 4 days groups age in the chicken serum of 5 days, 6 days or 7 days groups age, only illustrating just can make the level of anti--bovine serum albumin(BSA) antibody in the chicken serum minimum at specific developmental stage inoculation exogenous antigen, be not the time more early good more, the effect that imports foreign protein when embryo is 5-7 days age is best.
The chicken model for transgenosis of experimental group through four immunity after, this moment, chicken reached 60 ages in days, the average level of anti--bovine serum albumin(BSA) antibody significantly is lower than positive control (p=0.003,5 days groups in its body; P=0.020,6 days groups; P＜0.001,7 day group), illustrate that tolerating the time limit can keep the long period.
Claims (4)
1. one kind makes up the method for producing the chicken model for transgenosis that foreign protein uses and may further comprise the steps:
1) foreign protein is imported in the yolk sac of kind of egg; Be 5-7 days embryo age of described kind of egg;
2) with above-mentioned steps 1) the kind egg leave standstill hatch 46-50 hour after, continue again to hatch to hatching, obtain producing the chicken model for transgenosis that foreign protein is used.
2. method according to claim 1 is characterized in that: described foreign protein is bovine serum albumin(BSA), bovine casein or human serum albumins.
3. method according to claim 2 is characterized in that: described foreign protein is a bovine serum albumin(BSA).
4. method according to claim 3 is characterized in that: the concentration of described bovine serum albumin(BSA) is 0.8-1.2g/L, and the dosage of importing is every kind egg 80-100 μ l.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| GB2370750B (en) * | 1999-07-29 | 2003-09-24 | Ajinomoto Kk | Method for treating fertilized chicken eggs and method for hatching the fertilized eggs |
| CN101155509A (en) * | 2005-02-01 | 2008-04-02 | 奥瑞金治疗公司 | Transgenic chickens |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| GB2370750B (en) * | 1999-07-29 | 2003-09-24 | Ajinomoto Kk | Method for treating fertilized chicken eggs and method for hatching the fertilized eggs |
| US6938575B1 (en) * | 1999-07-29 | 2005-09-06 | Ajinomoto Co., Inc. | Method for treating hatching eggs and method for hatching eggs |
| CN101155509A (en) * | 2005-02-01 | 2008-04-02 | 奥瑞金治疗公司 | Transgenic chickens |
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