CN104789514A

CN104789514A - Somatostatin overexpression DNA vaccine and construction method and application thereof

Info

Publication number: CN104789514A
Application number: CN201510175578.5A
Authority: CN
Inventors: 杨利国; 罗璇; 董方晓; 梁爱心
Original assignee: Huazhong Agricultural University
Current assignee: Huazhong Agricultural University
Priority date: 2015-04-14
Filing date: 2015-04-14
Publication date: 2015-07-22
Anticipated expiration: 2035-04-14
Also published as: CN104789514B

Abstract

The invention discloses a somatostatin overexpression DNA vaccine and a construction method and application thereof. The method comprises the following steps: digesting a plasmid pEGS/2SS by two incision enzymes of Xho I and EcoR I to obtain a pEGS/2SS fragment containing kanamycin, wherein two incision enzyme loca of Xho I and EcoR I are formed at the two ends of the pEGS/2SS fragment; then connecting synthesized tpa and the pEGS/2SS fragment for reaction so as to obtain a plasmid pEGS/2SS-tpa. The plasmid pEGS/2SS-tpa is used for transferring escherichia coli competent cell DH5 alpha so as to obtain escherichia coli DH5 alpha (pEGS/2SS-tpa), and the preservation number is CCTCC NO: M2010105. The plasmid pEGS/2SS-tpa is used as a DNA vaccine to immunize mice, growth, reproduction and lactation of the mice can be promoted, and wide application prospect is achieved.

Description

A kind of amicine overexpression DNA vaccination and construction process and application

Technical field

The present invention relates to the construction and application of animal DNA vaccine, particularly relate to a kind of amicine overexpression DNA vaccination and construction process and application.

Technical background

Somatostatin (Somatostatin, SS) gene vaccine, a recombinant plasmid exactly by the gene clone of encoding growth chalone-14 is constructed on carrier for expression of eukaryon.Then this plasmid is imported in animal body, make SS-14 give expression to activated polypeptide in vivo, the antibody being produced anti-Somatostatin by immunne response with in reaching and the object of endogenous growth chalone, thus promotes growth of animal and lactation.Obviously, what play Main Function in this approach is Somatostatin.According to the effect that Somatostatin " suppresses somatotropin (growth hormone; GH) and the release of lactagogue parahormone ", recent year is about the research display of SS genetic immunization in animal reproduction, SS genetic immunization is in its effectiveness in promotion growth of animal and lactation, and obtains comparatively significantly immune effect.

As everyone knows, milk-product, as a kind of nutritious, comprehensive, high-quality liquid foodstuff that digestibility is high, are more and more subject to liking of consumers in general, and are subject to common concern.Containing rich in protein, carbohydrate, amino acid, VITAMIN, mineral substance and enzyme etc. in fresh milk, and the sequence of milk nutritive ingredient is buffalo milk, goat milk, Holstein cow milk, people's milk and mare's milk from high to low according to this.This is because compared with ordinary milk, the comprehensive nutrient of buffalo milk is worth and exceeds nearly 2 times, casein content is high, and cholesterol is low, and somatic number is low, dry matter content (17.5%) is high 0.5 times, doubly, protein content (4%) is high 0.5 times, milk fat content 7.5% for unsaturated fatty acid content height 60-100, calcium contents is high 0.3 times, Zn content is high 12 times, and iron level is high 78 times, vitamin A content high 32 times (Zhang Chunxi 2006).Therefore, normal drinking-water milk can promote more tyrosine calcium absorption, contributes to bone and intelligent growth, reduces cardiovascular disease incidence rate, improve body immunity.But 6.4% of the Chinese milk ultimate production Jin Zhan world, the milk-product level of consumption is 1/5 of world average level, and simultaneously buffalo lactation amount, reproductive efficiency heritability are lower.The existence of above-mentioned present situation causes to launch research to raising milk-quality and milk performance.

It is have example governed that Somatostatin is applied to animal lactation.Chemical antagonist cysteamine (the cysteamine hydrochloride of SS such as Wang Yanling, the holstein cow of CSH) feeding lactation mid-term, finds that the daily yielding of milk cow and feed conversion rate all significantly improve (Wang Yanling etc. 1999).According to reports such as Sun, use external source SS hormone active immunity lactation ewe can significantly improve its lactation amount (Sun et al.1990).Imgap etc. find when immune rat, only have active immunity SS-14 could produce the antibody of anti-SS and improve milk yield (Yiet al.1999).Xiao Shenhua use " swashs and gives birth to No. 1 " (i.e. a kind of SS vaccinia virus recombinant live vector vaccine) immune milk cow, there is the effect promoting lactation, and the milk yield decline process of Lactation of Dairy Cow middle and later periods is slowed down, but milk-quality uninfluenced (Xiao Shen China 2005).Above result of study shows, the milk performance using somatostatin DNA vaccine to improve domestic animal is feasible.

Tissue plasminogen activator (tissue plasminogen activator, tpa) be a special signal peptide being positioned endoplasmic reticulum, the albumen of its coupling can be directly transferred into endoplasmic reticulum, avoid that antigen is processed is transported to kytoplasm, target protein with secreted form in vivo a large amount express and can ensure desirable immunogenic maintenance.Tpa molecule comprises 4 structural domains, first F district, i.e. 47 residues of N-terminal, with the finger structure territory homology of the fibrin plates of adjustment fibronectin; It two is E districts, with Urogastron homology; It three is K1 and K2 two loop domains; It four is P districts, i.e. serine protease district (Pennica et al.1983).Wherein, the function in F district and K2 district is combined with scleroproein, and by removing fast under the adjustment of F district, E district and polysaccharide chain, P district then plays enzymic activity (Lijnen and Collen 1991).Along with the accuracy of Isolation and purification process improves, investigator finds that tpa discharges primarily of endotheliocyte, and tpa and vascular plasminogen activator (vascular plasminogen activator, vpa), blood plasminogen activator (blood plasminogen activator, bpa) there is immune homology, but be different from plasma urokinase-type plasminogen activator (urokinase plasminogen activator, upa) (Rijken et al.1980).Shown by further Biochemical Research, tpa is the main component in fibrinolytic system, physiological regulation fibrinolysis;

But tpa is not only the major function factor in fibrinolytic system, it is an efficient signal peptide that its structure also determines it.Therefore, tpa possesses the total effect of all signal peptides, namely first by signal recognition particle (signal recognition particle, SRP) identify, be attached on ER film and carry out Signal analysis, the rrna being in translation state being combined on RER film, completing after translation until rrna, just be secreted into outside born of the same parents along leading peptide approach cross-film through shearing the maturation protein modified.In addition, as signal peptide, tpa not only can make its coupling protein become secretory protein, and is convenient to positioning protein matter and new polypeptide chain (Wei Xuefang etc. 2006) in cell.At present, the tpa signal peptide of 23bp is used as heterologous leader sequence, be used as method (Yang et al.2001 target protein being brought into emiocytosis approach, Costa et al.2006, Jalah et al.2007, Golden et al.2008, Luo et al.2008, Kaur et al.2009, Seo et al.2009).Subsequently, the application of tpa is not confined to the research of cell levels, but is constantly generalized to other field, especially prevents and treats the new approach of the gene vaccine blaze of Animal diseases for building.Because modificator gene vaccine produces efficient and a large amount of immune responses, depend on the secretion capacity (Watanabe et al.2003, Liang et al.2005) of antigen expressed; And in gene vaccine, the existence of tpa signal peptide effectively can improve the secretion of recombinant protein to medium outside born of the same parents, and induce body to produce very strong immune response (Li et al.1999, Alves et al.2000, Ashok and Rangarajan 2002) to pathogenic agent.The NS1 full-length gene of tpa and dengue fever virus DENV-2 merges by Sinone M.Costa etc., the NS1 albumen of restructuring is secreted into surrounding medium with dimeric forms by the vaccine pcTPANS1 built, higher levels of NS-1 specific antibody is produced compared with control group after attacking poison, and based on IgG1, trend towards Th2 type reaction (Costa et al.2006, Costa et al.2007, Yao Yan rich 2012).Yang etc. are for improving the antibody activity of hepatitis E vaccine, compare and insert these two kinds of modes of 3 C3d complement tumor-necrosis factor glycoproteinss at hepatitis E virus HVE-p179N terminal fusion tpa signal peptide with at HVE-p179C end, find to be improved with the level of antigen in the immune rear antibody produced, the time of merging the hepatitis E vaccine generation antibody of tpa is done sth. in advance 2 weeks (Yang et al.2010).But the application about tpa signal peptide does not terminate in simple copying and continues to use.When trimer protein structural domain is introduced in plasmid vector by Wang etc., find that tpa signal peptide 22 sports L-Ala or glycine by proline(Pro), expression and the secretion situation (Wang et al.2011) of target protein can be improved.In addition, correlative study emerges in an endless stream, such as containing the influenza A vaccine ptPAs/NP of tpa fragment, pulmonary tuberculosis DNA vaccination, human papillomavirus (the papillomavirus of conjunction with electroporation immunity, HPV) vaccine etc. (Delogu et al.2002, Luo et al.2008, Seo et al.2009).In a word, after the goal gene in carrier is with the coupling of tpa signal peptide, not only make destination gene expression secreted form albumen, and humoral immunity level (Wang et al.2004, Costa et al.2006) can be strengthened.

Lactation is a physiological process of breaking up to play a role from mammary tissue, relate generally to mammogenesis, lactation starts and maintenance and the link such as mammary gland degeneration, and each stage participates in regulation and control by hormone.Mammary gland development is from fetal period, body of gland now and conduit system are still undeveloped, along with jenny ontogeny, oestrogenic hormon and progesterone level improve, oestrogenic hormon promotes the growth of breast duct, and progestogen stimulates the growth (Lamote et al.2004) of mammary gland alveolus.And oestrogenic hormon stimulates the synthesis of IGFs in breast stroma cell, thus promote that mammary epithelial cell generates (Tucker 2000).In addition, galactagogin (Prolactin, PRL), thyroliberin (adrenocorticotrophic hormone, ACTH), the hormone such as thyrotropic hormone (thyrotropic hormone, TSH) and external stimulus all can affect breast cell proliferation.During childbirth, lactation is started, and notable feature is now that the secretion of prolactin and glucocorticosteroid peaks.Peak of lactation, is decomposed into master with alienation, and GH level raises and Regular Insulin and thyroid hormone content reduce; After this assimilation synthesizes main (Zhang Shaoying 1990).But the startup of lactation and maintenance stage are mainly by the regulation and control of PRL and GH.Concerning ruminating animal, GH and PRL joint effect lactation amount, GH affects butterfat synthesis, and PRL affects the synthesis (Knight 2001) of milk-protein and lactose.Hydrocortisone can maintain epithelial secretion activity, but does not find that it promotes the effect (Sjaastad et al.2010) that milk generates.

Find according to the study, not only contain SS in blood plasma, the existence of SS in milk, also can be detected.Just in blood plasma, the SS of 70% is SS-28, and only has SS-14 in milk.This is because only express PC-1 proteinase gene in mammiferous mammary tissue, PC-1 proteolytic enzyme can impel growth promotion chalone former synthesis SS-14 (Werner et al.1985, Holst et al.1990).The research of Chen et al. is pointed out, in the mammary gland of rat, detectable unique a kind of neuropeptide is exactly SS, and infers that SS may participate in lactation activity, namely mammary gland growth and play function (Chen et al.1999).In addition, SS can suppress the secretion (Lee and Shin 1996) of PRL under estrogenic synergy.

With regard to ruminating animal, PRL is the required factor of starting lactation.But lactation is once be started, and PRL determines lactation amount limiting factor just (Zhang Shaoying 1990).As far back as 1940, British scientist just confirmed that GH may have certain lactagogue effect (Peel and Bauman 1987).At present, more demonstrate by the practice result that hypophysis or gene recombination Trobest (bST) improve lactation amount the effect that GH maintains lactation.Han Zheng's health etc. injects 12.5-50mg GH to every cow in milk every day, and after found that injection, the milk production of cow in milk generally can improve 15-24%, and its food utilization efficiency can increase 5-20% (Han Zheng's health 1999).In fact, GH non-immediate have an impact to mammary gland, but GH first stimulates liver, make liver produce IGFs, and IGFs can affect mammary gland activity (seedling tree monarch 1999).That is, GH is the differentiation of medium indirect stimulation mammary gland cell by IGFs, strengthens the milking capacity of mammary gland.GH has and coordinates mammary gland and mammary gland and organize competition to nutrition outward, and the nutritive substance intake that mammary gland is organized outward reduces.Research in neuroendocrinology empirical tests, growth hormone releasing hormone (the growth hormone releasing hormone that GH is discharged by hypothalamus and gi tract, GHRH) and the dual regulation and control of SS, namely the secretory volume of GH balanced by the acting in conjunction of these two kinds of hormones of GHRH and SS.Therefore, some researchs promote GHRH secretion by regulation and control or suppress SS level to improve the content of GH in body, thus reach the effect (Han Zheng's health 1999) improving lactation amount.A lot of research shows, lactation amount can be improved 20-30% by the method using GH to inject cow in milk, can't affect the healthy state (seedling tree monarch 1999) of composition of milk or ox through inspection this method.The GH that GH and DNA that the researchists such as Machlin, Baird extract respectively by injection is re-combined into, in lactating cow body, makes the milk performance of lactating cow improve 10-25% (Machlin 1973, Baird and Durkin 1986).Cornell Univ USA's restructuring Methionyl bovine somatotropin (MBS) in 1981 carries out a short-time test to milk cow, result shows at increase lactation amount in this, the effect suitable (Peeland Bauman 1987) of the GH that MBS and hypophysis derive.Trobest (the bovine growth hormone that Chadio etc. produce with gene recombination, bST) lactational goat is processed, find except raising lactation amount, also improve the content of butterfat and lactose, but the content of milk-protein little (Chadio et al.2000).In addition, Castigliego etc., to being in lactational milk cow effect bST, detect that bST and the IGF-1 level in process milk Ox blood plasma all significantly rises (Castigliego et al.2009).Nowadays recombinant bovine GH has been applied in dairy industry production by the U.S., and domestic also have similar research to obtain good achievement.Such as east, land woods etc. cultivate intestinal bacteria, gene recombination technology is utilized to obtain the crude extract of bST, process milk cow one month with this, every bovine lactation amount increases 1.65kg/d (about 10.75%), but the index such as milk fat content, newborn density has no difference (Lu Donglin etc. 1990).Although test method and Testing index different, must can promote mammiferous Milking ability by GH.

From above bibliographical information, tpa signal peptide and Somatostatin SS gene vaccine cooperate with animal lactation and breeding acts on both at home and abroad without relevant report, therefore the present invention recombinates to original somatostatin DNA vaccine pEGS/2SS, construct the novel overexpression somatostatin DNA vaccine containing tpa signal peptide fragment, be intended to improve the effect of this vaccine to animal immune originality, lactation and breeding, and inquire into working conditions and immune programme for children preferred embodiment, for the application of New somatostatin gene vaccine in actual production process provides theoretical foundation.

Summary of the invention

The object of the invention is to the defect overcoming existing somatostatin gene immunological technique, provide a kind of eukaryon expression plasmid pEGS/2SS-tpa of overexpression Somatostatin, this plasmid is that tpa fragment is inserted plasmid pEGS/2SS Xho I and EcoR I acquisition.

Another object of the present invention there are provided a kind of bacterial strain including plasmid pEGS/2SS-tpa; This bacterial strain delivers to China typical culture collection center preservation on March 12nd, 2015, Classification And Nomenclature: intestinal bacteria Escherichia coli DH5 α (pEGS/2SS-tpa), deposit number: CCTCC NO:M2015105, this bacterial strain can obtain pEGS/2SS-tpa plasmid by the extracting of SDS alkaline lysis.

The eukaryon expression plasmid pEGS/2SS-tpa that last object of the present invention is to provide a kind of overexpression Somatostatin or the intestinal bacteria comprising it are preparing the application in amicine DNA vaccination, this DNA vaccination is applied to mouse, significantly improves the function of mouse growth, breeding and lactation.

The present invention implements by following technical solution:

An eukaryon expression plasmid pEGS/2SS-tpa for overexpression Somatostatin, prepares by the following method

First plasmid pEGS/2SS Xho I and EcoR I two restriction endonucleases are digested, obtain the pEGS/2SS fragment having Xho I and EcoR I two restriction enzyme sites containing the two ends of kantlex, then synthetic tpa fragment is passed through Xho I and EcoR I two endonuclease digestion, obtain the tpa fragment of Xho I and EcoR I two restriction enzyme sites, by this gene fragment and the ligation of pEGS/2SS fragment, obtain plasmid pEGS/2SS-tpa.

By in plasmid pEGS/2SS-tpa transformation of E. coli competent cell DH5a, obtain with intestinal bacteria be carrier ultra-high efficiency start express somatostatin DNA vaccine.This bacterial strain delivers to China typical culture collection center preservation on March 12nd, 2015, Classification And Nomenclature: intestinal bacteria Escherichia coli DH5 α (pEGS/2SS-tpa), deposit number: CCTCC NO:M2015105, preservation address: Wuhan, China Wuhan University; This bacterial strain can obtain pEGS/2SS-tpa plasmid by the extracting of SDS alkaline lysis.

Intestinal bacteria Escherichia coli DH5 α (pEGS/2SS-tpa) is the mixed culture of the Somatostatin eukaryon expression plasmid pEGS/2SS-tpa containing tissue plasminogen activator (tpa), can obtain pEGS/2SS-tpa plasmid by the extracting of SDS alkaline lysis.Substratum: 1L LB solid medium forms: 10g sodium-chlor, 5g yeast extract, 10g Tryptones, 1.5g agar powder.Culture condition: pH:6.8-7.8, temperature: 37 DEG C is Kan ⁺resistance; In LB solid medium, detect this culture with plate streak whether survive.

The eukaryon expression plasmid pEGS/2SS-tpa of overexpression Somatostatin is preparing the application in amicine DNA vaccination, using this plasmid as vaccine direct immunization animal or immune animal after mixing with DNA vaccination adjuvant.

Successfully be applied in the present invention and improved growth of animal, breeding and milk performance, as improved the application of function of growth, breeding and lactation in mouse.

More detailed technical scheme is shown in " embodiment ".

Compared with prior art, the present invention has the following advantages:

1, a kind of eukaryon expression plasmid pEGS/2SS-tpa of efficient startup amicine is obtained;

2, obtain a kind of can in the growth of mouse, breeding and milk performance the somatostatin DNA vaccine pEGS/2SS-tpa of unusual effect.Reproductive performance has certain superiority judged by the Cluth size index of female mouse, its pEGS/2SS-tpa vaccine experiments group, than high many 1 of pEGS/2SS Somatostatin positive controls, reaches 1.4 ≈ 2 than feminine gender group pEGFP-N1; By the Body Mass Index of newborn mouse of weaning, pEGS/2SS-tpa group can be obtained higher by 10.7% than pEGS/2SS Somatostatin positive controls body weight, higher than negative control group pEGFP-N1 by 21.8%; And efficient start vaccine high dose group (200 μ g/ only), add exempt from 1 time weightening finish, reproductive performance best results.

Accompanying drawing explanation

Fig. 1 is that eukaryon expression plasmid pEGS/2SS-tpa builds collection of illustrative plates.

Fig. 2 is the electroresis appraisal figure of synthesis fragment tpa.

M is 50bp DNA Ladder Marker; 1-3 swimming lane is the result that the signal peptide tpa after annealing to be measured detects through 1% agarose gel electrophoresis.

Fig. 3 is that eucaryon plasmid carrier pEGS/2SS enzyme cuts qualification collection of illustrative plates.

M represents DL 2,000Marker; 1 swimming lane is the result of eucaryon plasmid carrier pEGS/2SS after restriction enzyme EcoR I and Xho I double digestion.

Fig. 4 is that eucaryon plasmid carrier pEGS/2SS-tpa enzyme cuts qualification collection of illustrative plates.

M representation DNA ladder; 1 swimming lane is plasmid pEGS/2SS-tpa electrophoresis result, and 2 swimming lanes are the result of plasmid pEGS/2SS-tpa after restriction enzyme Xho I and EcoR I double digestion.

Fig. 5 is expression and the distribution situation of different plasmids Plasmids transfection MCF-7 cell GFP green fluorescence.

Wherein in Fig. 5, A is the expression observing GFP green fluorescence after pEGFP-N1, pEGS/2SS and pEGS/2SS-tpa tri-kinds of plasmid transfection MCF7 cell 24h.Bright and fluorescence is that scale represents 200 μm in light field and fluorescence observations after the match respectively.

In Fig. 5, B is the expression observing GFP green fluorescence after pEGFP-N1, pEGS/2SS and pEGS/2SS-tpa tri-kinds of plasmid transfection MCF7 cell 48h.Bright and fluoresce is in light field and fluorescence observations after the match respectively; Scale represents 200 μm.

In Fig. 5, C is the distribution situation of observing GFP green fluorescence after pEGFP-N1, pEGS/2SS and pEGS/2SS-tpa tri-kinds of plasmid transfection MCF7 cell 24h; Scale represents 200 μm.

Fig. 6 is the overexpression effect comparison chart of SS after quantitative real-time PCR eucaryon plasmid transfection MCF-7 cell.

48h after pEGFP-N1, pEGS/2SS or pEGS/2SS-tpa difference transfection MCF-7 cell, detects the expression of Somatostatin SS in cell.The expression amount of SS carries out stdn with the expression amount of GAPDH, and expressing multiple is calculate in the expression of pEGFP-N1 transfection group relative to SS, finally represents with average standard error, and each data have 3 biological samples for repeating.**P<0.01

Fig. 7 is protein expression result after eucaryon plasmid pEGS/2SS-tpa transfection MCF-7 cell.

Western Blot is utilized to detect the expression of Somatostatin SS-14 albumen after MCF-7 cell transfecting pEGFP-N1, pEGS/2SS and pEGS/2SS-tpa; The expression amount simultaneously choosing GAPDH albumen carries out stdn correction, expresses multiple and calculates relative to pEGFP-N1.

Fig. 8 is that Trizol method extraction kunming mice organizes RNA, the agarose gel electrophoresis detected result of RNA concentration.

M is DNA ladder, and swimming lane 1-10 is the heart of kunming mice, liver, spleen, lung, kidney, muscle, small intestine, stomach, brain and pancreas tissue RNA successively.

Fig. 9 is Somatostatin SS expression amount detected result during kunming mice is respectively organized.

Swimming lane 1-9 is the result organizing RNA amplification Somatostatin of heart, liver, spleen, lung, kidney, muscle, small intestine, stomach, brain successively.

In Fig. 9 A be 5d after immune pEGS/2SS-tpa kunming mice body in SS distribution situation;

In Fig. 9 B be 5d after immune pEGS/2SS Mice Body in SS distribution situation.

Figure 10 is the secretion level result of tethelin GH after vaccine immunity kunming mice.

Compared with zero load injection group pEGFPN1, after immunity pEGS/2SS and pEGS/2SS-tpa, the secretory volume of mouse GH promotes all to some extent, but after intramuscular injection pEGS/2SS, mouse GH improves not remarkable, and after injecting pEGS/2SS-tpa, mouse GH content is significantly higher than the mouse (P<0.05) of injection pEGS/2SS.Can increase the release of GH after somatostatin DNA vaccine pEGS/2SS immunity is described, after inserting tpa signal peptide, this effect is more obvious.

Figure 11 is the secretion level result of prolactin PRL after vaccine immunity kunming mice.

Compared with unloaded immune group, the mouse PRL content of pEGS/2SS and pEGS/2SS-tpa immune group is improved; And mouse PRL content remarkable rising (P<0.05) compared with the mouse of injection pEGS/2SS after intramuscular injection pEGS/2SS-tpa, difference extremely significantly (P<0.01) compared with control group mice.Secretion by increasing mouse PRL after somatostatin gene immunity is described, and this promoter action is especially obvious after insertion tpa signal peptide.

Figure 12 is the secretion level result of Somatostatin SS after vaccine immunity kunming mice.

Compared with zero load injection group, after immune pEGS/2SS, the secretory volume of mouse SS declines to some extent, but it is not remarkable to decline; After immunity pEGS/2SS-tpa, mouse SS content is on the contrary higher than the mouse of control group, although difference is also not remarkable.After somatostatin DNA vaccine pEGS/2SS and pEGS/2SS-tpa immunity are described, remarkably influenced can not be caused on the SS in mouse blood.

Embodiment

Embodiment 1:

The structure of eukaryon expression plasmid pEGS/2SS-tpa

1, tissue plasminogen activator's (tpa) gene clone and sequential analysis

According at GenBank (ID:5327) plasminogen activator, tissue [Homo sapiens (human)] gene order, synthesis plasminogen activator, the restriction enzyme site at its two ends of cDNA sequence of tissue (tpa) is Xho I and EcoR I, adopt Primer5.0 primer-design software, tpa sequences Design is as follows: (be tpa sequence in square frame, two ends are restriction enzyme site)

tpa-F：

5-TCGAG G-3

tpa-R：

3-C cTTAA-3, Sangon Biotech (Shanghai) Co., Ltd. obtains tpa gene fragment by base synthesis h.

After sequent synthesis, upstream and downstream fragment is diluted to every OD and adds 140 μ L ddH ₂o, is made into 10 μMs of stock solutions, then two strand annealing is formed double-strand.Annealing system is that 10 μ L tpa-F mix with 10 μ L tpa-R, and annealing conditions is 95 DEG C of 30s, 72 DEG C of 2min, 37 DEG C of 2min, 25 DEG C of 2min.After annealing, agarose gel electrophoresis detects, and observes electrophoretic band and conforms to object stripe size, store for future use in 4 DEG C.(Fig. 2)

2, the enzyme of carrier pEGS/2SS is cut and recovery

With restriction enzyme Xho I and EcoR I double digestion pEGS/2SS plasmid (Cao Shaoxian, Yang Liguo, Zhang Wenwei, Deng. the construction and expression [J] of somatostatin DNA vaccine plasmid pEGS/2SS. Chinese animal doctor's journal, 2005,25 (5): 499-502.), 16bp is excised, expose the sticky end at two ends, it is as follows that enzyme cuts system:

Enzyme reclaims gel electrophoresis through 1% agarose after cutting and isolates plasmid band, uses sepharose DNA to reclaim test kit and carries out the recovery of plasmid large fragment, reclaim rear electrophoresis and detect, observe electrophoretic band and conform to object stripe size.(accompanying drawing 3)

3, goal gene connects

Measure the concentration of the pEGS/2SS plasmid enzyme restriction fragment of recovery and the tpa fragment of dilution 10 times respectively, the connection volume ratio calculating carrier and object fragment is 6:1, pEGS/2SS carrier and goal gene tpa fragment is connected according to Invitrogen T4 DNA Ligase specification sheets, 16 DEG C are spent the night, and linked system is as follows:

4, recombinant plasmid transformed competence colibacillus bacterium

1) competence bacterium DH5 α (TIANGEN Biotech's production)

2) competent escherichia coli cell DH5 α is taken out ice bath from-80 DEG C to thaw, add 10 μ L connection products and enter in 50 μ L competent cells to mix, and setting does not add the competent cell of connection product as blank, ice bath 30min; Go to 42 DEG C of heat shock 90s, then rapid ice bath 5min; Add 400 μ L LB liquid nutrient mediums, shaking table is adjusted to 200r/min, 37 DEG C of shaking culture 1h; Whether mixing is rear gently draws 100 μ L, uses spreader to be evenly applied on the LB solid plate containing Kan, after 37 DEG C of cultivation 12h, observe and have conversion bacterium colony to grow.

5, the selection systems of pEGS/2SS-tpa plasmid

Picking list bacterium colony to vibrate enlarged culturing 8h in containing the LB liquid nutrient medium of Kan, and plasmid is little to be carried to use test kit to carry out, and cuts should cut out tpa (75bp) through Xho I and EcoR I enzyme.It is as follows that enzyme cuts system:

Get 5 μ L digestion products and carry out 1% agarose gel electrophoresis detection, observe electrophoretic band to conform to object stripe size, deliver to Sangon Biotech's order-checking, analyze sequencing result, plasmid pEGS/2SS-tpa sequencing result 100% comparison success.Identify that correct pEGS/2SS-tpa plasmid-20 DEG C saves backup, by plasmid pEGS/2SS-tpa transformation of E. coli competent cell DH5a, obtain with intestinal bacteria be carrier ultra-high efficiency start express somatostatin DNA vaccine.This bacterial strain delivers to China typical culture collection center preservation on March 12nd, 2015, Classification And Nomenclature: intestinal bacteria Escherichia coli DH5 α (pEGS/2SS-tpa), deposit number: CCTCC NO:M2015105, this bacterial strain can obtain pEGS/2SS-tpa plasmid by the extracting of SDS alkaline lysis.Containing the bacterium liquid conservation of pEGS/2SS-tpa plasmid ,-80 DEG C of preservations.(Fig. 4)

Embodiment 2:

The detection of Somatostatin overexpression plasmid pEGS/2SS-tpa plasmid protein expression in vitro

1, statin recombinant plasmid pEGS/2SS-tpa transfectional cell

With plasmid extraction kit (purchased from TIANGEN Biotech (Beijing) Co., Ltd.) extracting intestinal bacteria Escherichia coli DH5 α (pEGS/2SS-tpa) plasmid, by Lipofectamine when MCF-7 cell (human breast cancer cell) individual layer grows to 60%-70% ^tM2000 lipofectamine boxes (purchased from Invitrogen company) specification sheets operates, and adds 0.2 μ g plasmid and 1.5 μ L Lipofectamine according to every hole ^tM2000 calculate, and dissolve mixing with serum free medium OPTI-MEM respectively, room temperature leaves standstill 5min.Then the plasmid configured and liposome solutions equal-volume are mixed, room temperature leaves standstill 20min.Period absorbs the substratum in Tissue Culture Plate, and PBS cleans 2-3 time, adds OPTI-MEM and cultivates based on 37 DEG C, 5%CO ₂process is hatched in incubator.Take out 24 orifice plates after 20min, absorb OPTI-MEM substratum, every hole adds 100 μ L OPTI-MEM substratum, and slowly drips 100 μ L plasmid-liposome complex.Cell process arranges and is respectively: pEGFP-N1 transfection group, pEGS/2SS transfection group, pEGS/2SS-tpa transfection group and blank group, often organize and arrange 3 parallel repetitions respectively.37 DEG C, 5%CO ₂after cultivating 5-6h in incubator, change DMEM complete culture solution (i.e. DMEM+15%FBS) into and continue to cultivate.

2, statin recombinant plasmid pEGS/2SS-tpa transfectional cell egfp expression

Cell growth state and green fluorescence respectively in fluorescence microscopy Microscopic observation different treatment group after transfection 24h and 48h, judged the transfection efficiency of MCF-7 cell by the contrast white field of cell and green fluorescence intensity, indirectly determine carrier target protein optimum expression time.

Difference transfection pEGFP-N1, pEGS/2SS and pEGS/2SS-tpa when MCF-7 cell 80% converges, after transfection 24h and 48h, under fluorescence inverted microscope, the green fluorescence in blue-light excited rear field of view.Can see that test group all has green fluorescence to present, and the green florescent signal intensity after transfection 48h is apparently higher than the strength of signal after transfection 24h.Therefore, following test cell line all adopts the cell (Fig. 5) of 48h after transfection.

Green fluorescence distribution in uniformly continous in MCF-7 cell of pEGFP-N1 and pEGS/2SS transfection group cell, the green fluorescence of pEGS/2SS-tpa transfection group cell is that loose spot distribution is in cell.Illustrate that inserting signal peptide tpa fragment changes the expression and distribution of target protein in cell, makes target protein be transferred in endoplasmic reticulum and then be secreted into extracellular.(Fig. 5 C)

3, the detection of overexpression Somatostatin plasmid pEGS/2SS-tpa vivoexpression

3.1 the expression of Western Blot testing goal albumen

The extraction of cell protein: get cell pyrolysis liquid RIPA, puts upside down mixing for several times (if having precipitation can put 37 DEG C of dissolvings), packing after thawing, with protein inhibitor phenylmethylsulfonyl fluoride (PMSF, 100mM) mix in proportion, make final concentration be 1mM, now with the current.MCF-7 cell after transfection abandons nutrient solution, and the PBS of 4 DEG C of precoolings cleans 2-3 time.Add appropriate lysate, the abundant cracking of piping and druming cell.The centrifugal 3-5min of 10,000rpm, gets supernatant, packing, and-80 DEG C save backup.Above all operations all operates on ice.

The mensuration of protein concentration: according to BCA determination of protein concentration test kit confirmed standard curve and sample protein concentration.Concrete steps are as follows:

(1) get 0.8mL protein standard preparation liquid to join in protein standard (20mg BSA), fully dissolve, be made into the protein standard liquid of 25mg/mL, use immediately after preparation;

(2) get the protein standard liquid of appropriate 25mg/mL, be diluted to final concentration 0.5mg/mL;

(3) number per sample, adds 1 times of volume BCA reagent B (i.e. A:B=50:1) by 50 times of volume BCA reagent A and joins appropriate BCA working fluid, fully mix;

(4) standard substance are pressed 0,1,2,4,8,12,16,20 μ L are added in 96 hole enzyme plates, and complement to 20 μ L with standard dilutions (i.e. PBS), namely concentration is respectively 0, and 0.025,0.05,0.1,0.2,0.3,0.4,0.5mg/mL, as standard orifice;

(5) add in the sample well of equal-volume sample to 96 hole enzyme plate, be diluted to 20 μ L;

(6) each hole adds 200 μ L BCA working fluids, 37 DEG C of temperature bath 30min; Light absorption value under microplate reader mensuration 562nm (between 540-595nm) wavelength;

(7) production standard curve, according to typical curve calculation sample protein concentration.

SDS-PAGE electrophoresis: according to the molecular size range 55KDa of target protein, the separation gel of configuration 15%, the concentrated glue of 5% carries out SDS-PAGE gel electrophoresis.Be preventing from cementing leakage during preparative separation glue, available a small amount of lower concentration agarose gel back cover, the separation gel that reinjects, to blank, is then divided equally from glue liquid surface with hydraulic pressure, is solidified about 30min; Concentrated glue is filled it up with after insertion comb, solidifies 1-2h.Glue plate is put into electrophoresis chamber, is about 400mL with electrophoretic buffer 1 × Tris-Gly.Before loading, albumen mixes rear boiling water bath 5min with Loading Buffer, makes protein denaturation.The albumen applied sample amount 50 μ g in every hole, ice bath electrophoresis, is first about 20min to concentrated glue and separation gel interface with constant voltage 80V electrophoresis, then changes constant voltage 120V into and continue electrophoresis and be about 50min, makes sample go to bottom glue, stops electrophoresis, take out sheet glass.

Transferring film: measure the length and width of getting glue with a yardstick, the filter paper of the corresponding size of clip 6, is immersed in transferring film damping fluid.Weigh the approximate location of object fusion protein S/2SS-GFP (55KDa) and internal reference Protein G APDH (37KDa) according to Marker band, the pvdf membrane of the corresponding size of clip, soaks 3-5min bubble removing in methyl alcohol.Distilled water soaks graphite cake, keeps flat and opens, from the bottom to top lay negative plate, 3 metafiltration paper, pvdf membrane, 3 metafiltration paper and positive plates successively, often spreads one deck test tube and catches up with bubble, cover tightly battery lead plate.Ice bath electricity turns, and 250mA electric current transferring film is about 45min.After transferring film terminates, get glue and put into dyestuff and dye, check whether that transferring film is complete.

Close: use TBST to join 1%BSA and close, room temperature horizontal shaker 1h.

Primary antibodie is hatched: TBST washes film 3 times, each 10min; Somatostatin primary antibodie (1:300 dilutes, Santa Cruz) and internal reference GAPDH antibody (1:1000 dilutes, Boster) is diluted, sealer, 4 DEG C of overnight incubation with 0.1%BSA.

Two anti-hatch: TBST washes film 3 times, each 10min; Goat anti-rabbit igg-HRP (1:1000 dilutes, Boster) incubated at room 1h is diluted with 0.1%BSA.

Colour developing: TBST washes film 3 times, each 10min; Finally add nitrite ion colour developing 3-5min, develop and scan, time shutter 600s; By AlphaEaseFC computed in software optical density(OD), by with the comparing of internal reference Protein G APDH, extrapolate the relative expression quantity of albumen.

Cell protein is carried after different plasmid transfection MCF-7 cell 48h, Western Blot detects relative expression's situation of Somatostatin SS-14, result display is compared with blank (monk), pEGS/2SS transfection group and pEGS/2SS-tpa transfection group all have band at 55KDa place, and in the same size with target protein; Infer according to color, after MCF-7 cell transfecting pEGS/2SS-tpa plasmid, the expression amount of SS is a little more than the expression amount of SS after transfection pEGS/2SS plasmid.(Fig. 6)

Embodiment 3:

Somatostatin overexpression DNA vaccination of the present invention on Kunming mouse on its growth and the impact of immunne response

1, vaccine preparation:

By through identify correct pEGFP-N1, pEGS/2SS and pEGS/2SS-tpa bacterium liquid be added to 250mL containing Kan LB liquid nutrient medium in enlarged culturing, 200r/min, 37 DEG C, 12h.Use plasmid to remove the large extraction reagent kit large quantity extracting plasmid of intracellular toxin, survey plasmid concentration, enzyme is cut and sequence verification.As required, plasmid normal saline dilution is to different concns, and after packing ,-20 DEG C store for future use.

2, experimental animal immunity test:

100 female 3 week age Kunming mouse be divided at random 10 groups (weigh and ensure that the original body mass of each experimental group is without significant difference), 10/group, room temp remains on about 28 degree always.In phase trial period Animal House keep cleaning always, health, remove fecaluria in house in time, keep house inner drying.3 factors (vaccine, immunizing dose, immune time), 3 level (pEGFP-N1, pEGS/2SS, pEGS/2SS-tpa are designed with " orthogonal design assistant II 3.1 software "; Basic, normal, high; 2 times, 3 times, 4 times) orthogonal test.Test is grouped as follows:

Table 2 mouse immuning test divides into groups

According to grouping, immunity is carried out to mouse, take mouse quadriceps muscle of thigh multi-point injection, 2w booster immunization in interval after first immunisation, within after immunity continuous one week, observe the mind & body situation of mouse.Mouse is feeding standard routinely, changes weekly bedding and padding 2 times, free choice feeding and drinking-water.

3. mouse weights

To mouse weights before first immunisation, just exempt from rear 2w, 4w, 6w, 8w and mouse is weighed, and record weight data, until off-test.

Before just exempting from, just exempt from rear 2w, 4w, 6w, 8w test mice is weighed, record body weight Changing Pattern, find that mouse is the fastest from the growth in 3 weeks age to 5 week age (namely after initial immunity 2 weeks interior), increase weight afterwards and slow down gradually, these consistent with the weightening finish rule of Kunming mouse growth and development stage (Jing Zhizhong etc. 1996).Respectively to vaccine, dosage with add and exempt from number of times and do one-way analysis of variance, showing different vaccine immunity has impact to female mouse weightening finish, and various dose group and different to add the weight differences of exempting from time array all not obvious.Wherein as shown in table 3 to the one-way analysis of variance of different vaccine, obviously can find out that the first starting weight of pEGS/2SS immune group, pEGS/2SS-tpa immune group and control group mice is basically identical, female mouse of just exempting from rear 4w (namely before second time booster immunization) pEGS/2SS immune group and pEGS/2SS-tpa immune group increases weight, and than control group, all there were significant differences, but female mouse of pEGS/2SS-tpa immune group increases weight without significant difference compared with pEGS/2SS immune group, although the weightening finish of pEGS/2SS-tpa immune group is a little more than pEGS/2SS immune group; Just exempt from all female mouse speeds of growth of rear 4-8 to slow down, though female mouse weightening finish numerical value of visible pEGS/2SS-tpa immune group is without the significance difference opposite sex, but numerically still a little more than pEGS/2SS immune group and control group;

By only just exempting from the rear 2w of the final immunity of Shi Qizhi, female mouse total augment weight in vegetative period difference to some extent of different vaccine group, namely female mouse weightening finish of pEGS/2SS-tpa immune group and pEGS/2SS immune group is all significantly higher than control group, but between pEGS/2SS-tpa immune group and pEGS/2SS immune group, difference is not remarkable.Weigh the growth traits of mouse with the body weight gains of mouse growth phase, illustrate that vaccine pEGS/2SS-tpa and pEGS/2SS all can improve the growth velocity of female mouse, and vaccine pEGS/2SS-tpa is slightly better than vaccine pEGS/2SS to the growth-promoting effect of female mouse.

The impact that the different vaccine immunity of table 3 increases weight on female mouse

Note: in same row, any two digital rearmounted letters are identical is considered as difference remarkable P>0.05, on the contrary be then considered as difference remarkable P<0.05.

4. immunity is on the impact of MOUSE REPRODUCTION proterties

4.1 mouse litter sizes and newborn mouse weightening finish

After last immune 2w, a public mouse thrown in by when 11 week age (i.e. mouse) every cage, mates, observes female mouse vaginal suppository next day, check whether and successfully breed.After this note the pregnancy status of female mouse in 21d, record female mouse pregnancy cycle, litter size and newborn mouse birth weight and weaning weight (i.e. newborn mouse 3 week age) in detail.

Breed when 11 week age of female mouse, Gestation period 20-23d, records female mouse nest litter size, and between different vaccine treatment group, female mouse litter size difference is not significantly (table 4).But we still can find out embody superiority in the number of mouse, its pEGS/2SS-tpa vaccine experiments group, than high many 1 of pEGS/2SS Somatostatin positive controls, reaches 1.4 ≈ 2 than feminine gender group pEGFP-N1.Illustrate that pEGS/2SS-tpa vaccine still has certain superiority on the reproductive performance of female mouse.

The farrowing situation of the female mouse of the different immunization ways of table 4

Note: in same row, any two digital rearmounted letters are identical is considered as P>0.05, otherwise is then considered as P<0.05.

Nest weightening finish between record newborn mouse lactation, lactation, 21d, weighed the milk character of female mouse with nest weightening finish between newborn mouse lactation.Make a general survey of the newborn mouse nest weightening finish of different treatment group, the nest of pEGFP-N1 injection group and blank group increases weight no significant difference, and between all the other treatment group groups, newborn mouse nest increases weight, and there were significant differences.Then respectively to vaccine, immunizing dose with add and exempt from number of times 3 factors and carry out single factor analysis: different vaccine shows the one-way analysis of variance (table 4 (a)) of newborn mouse weaning weight, after immunity pEGS/2SS and pEGS/2SS-tpa, weaning weight significantly increases, and the weaning weight after immune pEGS/2SS-tpa is significantly higher than pEGS/2SS immune group, really can promote the milk performance of female mouse after immune pEGS/2SS and pEGS/2SS-tpa is described, wherein the short lactogenic result of pEGS/2SS-tpa is better; Various dose shows the one-way analysis of variance (table 4 (b)) of newborn mouse weaning weight, the weaning weight of high dosage immunity is higher than middle dose immunization but difference is not remarkable, all there were significant differences with low dosage for high dosage, middle dosage, illustrates that high dosage (200 μ g) immunity significantly can promote lactation; Difference adds to be exempted from number of times and shows the one-way analysis of variance (table 4 (c)) of newborn mouse weaning weight, adds and exempts from 1 time with to add the weaning weight difference exempting from 2 times remarkable, but is all significantly higher than to add and exempts from 3 times, illustrates that to add the short lactogenic result of exempting from 1 time better.In a word, different vaccine, immune time and immunizing dose all make a significant impact newborn mouse weaning weight, consider newborn mouse weaning weight mainly by the impact of female mouse milking capacity; Adopt high dosage (200 μ g) immunity 2 pEGS/2SS-tpa best to the facilitation effect of female mouse lactation.

The different vaccine immunity of table 4 (a) is on the impact of newborn mouse weaning weight

Table 4 (b) immunizing dose is on the impact of newborn mouse weaning weight

Table 4 (c) immune time is on the impact of newborn mouse weaning weight

5, RT-PCR testing goal gene transcribe detection

5.1 total tissue RNA are extracted

Often organize random selecting 1 mouse after immunity 5d and gather heart, liver, spleen, lung, kidney, injection site muscle, small intestine, stomach, brain, pancreas, extract by Trizol method and organize RNA, detailed step is as follows:

(1) liquid nitrogen grinding: tissue block directly puts into mortar, adds a small amount of liquid nitrogen, grinds rapidly, deliquescing to be organized, then adds a small amount of liquid nitrogen, then grinds, three times like this;

(2) homogenate: tissue sample adds Trizol by 50-100mg/mL Trizol, about needs 1-2min with the abundant homogenate of electric homogenizer;

(3), after tissue adds Trizol, room temperature places 5min, makes its abundant cracking;

The centrifugal 5min of (4) 12,000rpm, abandons precipitation;

(5) add chloroform by 200 μ L chloroform/mL Trizol, after vibration mixing, room temperature places 15min;

(6) 4 DEG C of 12,000g centrifugal 15min;

(7) draw upper strata aqueous phase, in another centrifuge tube, add Virahol mixing by 0.5ml Virahol/mL Trizol, room temperature places 5-10min;

(8) 4 DEG C of 12,000g centrifugal 10min, abandon supernatant, RNA is sunken at the bottom of pipe;

(9) add 75% ethanol by 1mL 75% ethanol/mL Trizol, gentle vibration centrifuge tube, suspend precipitation;

(10) 4 DEG C of 8,000g centrifugal 5min, abandons supernatant as far as possible;

(11) room temperature is dried or vacuum-drying 5-10min;

(12) with 50 μ L DEPC H ₂o dissolves RNA sample, 55-60 DEG C, 5-10min;

(13) whether survey OD value quantitative RNA concentration, A260/A280 value is answered between 1.6-1.8, and make agarose gel electrophoresis and check and degrade.

Often organize random selecting 1 mouse after initial immunity 5d and gather each tissue, extract organize RNA by Trizol method, the agarose gel electrophoresis of RNA detects figure as shown in the figure, and 28S, 18S and 5S tri-bands are all high-visible, and the RNA of its mesencephalic tissue has degraded trend; Concentration at 300-1000ng/ μ L not etc., OD ₂₆₀/ OD ₂₈₀value, all between 1.8 ~ 2.0, illustrates that the total tissue RNA purity extracted is better, can be used for follow-up test.(Fig. 8)

5.2 reverse transcriptions (method is with reference to reverse transcription method step in embodiment 2)

The amplification of 5.3 goal gene SS

After the cDNA of reverse transcription being done 5 times of dilutions, the endogenous SS of pcr amplification (design of primers is in table 5).

Somatostatin design of primers in table 5 cell

PCR amplification system is as follows:

Reaction conditions is 95 DEG C of denaturation 3min, 95 DEG C of sex change 30s, 60 DEG C of annealing, 30,72 DEG C of extension 15s, after 35 circulations of increasing, and 72 DEG C of 5min, 15 DEG C of 2min.Whether amplified production detects through agarose gel electrophoresis, checking Somatostatin normal transcription in Mice Body.

The RNA that respectively organizes extracted obtains cDNA through reverse transcription, again respectively with each tissue cDNA for template amplification Somatostatin, find except the central nervous system (as brain etc.) of endogenous growth chalone proper distribution and each tissue of Digestive tract (as stomach, intestines etc.), in the tissues such as heart, lung, kidney and injection site muscle, also occur faint band.Illustrate that the Somatostatin fragment in plasmid can be transcribed at each histoorgan of mouse after injection somatostatin DNA vaccine.(Fig. 9)

6. mouse blood and tissue sampling

6.1 mouse blood collections

Respectively at before immunity and after off-test, gather mouse blood with docking blood-collecting method, be collected in the 1.5mL EP pipe of 20 μ L EDTA antithrombotics/pipes, the centrifugal 10min of 3,000r/min, carefully draws upper plasma, packing about 200 μ L/ manages, and is placed in-20 DEG C and saves backup.

6.2 mouse tissue collections

After off-test, vertebra puts to death mouse, often organize the heart of anatomical acquisition some animals, liver, spleen, lung, kidney, injection site muscle, small intestine, stomach, brain, ovary (ovary is weighed), be placed in 4% paraformaldehyde stationary liquid, be collected in centrifuge tube after each histoorgan collection of another part mouse ,-80 DEG C of preservations.

7. associated hormone and antibody test

The detection of 7.1 tethelin, prolactin and Somatostatin

Detect the content of hormone GH, PRL and SS in the mice plasma of different treatment with double antibody one step Sandwich ELISA, concrete steps are as follows:

(1) from the aluminium foil bag after equilibrium at room temperature 20min, take out required lath, the sealing of residue lath valve bag puts back to 4 DEG C;

(2) arrange standard sample wells and sample aperture, standard sample wells respectively adds the standard substance 50 μ L of different concns;

(3) sample aperture first adds sample to be tested 10 μ L, then adds Sample dilution 40 μ L; Blank well does not add;

(4) except blank well, in standard sample wells and sample aperture, every hole adds the detection antibody 100 μ L that horseradish peroxidase (HRP) marks, and seals reacting hole, 37 DEG C of thermostat container incubation 60min with shrouding film;

(5) discard liquid, thieving paper pats dry, washings is filled it up with in every hole, leaves standstill 1min, gets rid of washings, thieving paper pats dry, so repeat to wash plate 5 times;

(6) every hole adds substrate A, each 50 μ L of B, and 37 DEG C of lucifuges hatch 15min;

(7) every hole adds stop buffer 50 μ L, in 15min, measures the OD value in each hole at 450nm wavelength place;

(8) drawing standard curve, according to the concentration (calculated value is multiplied by 5 for ultimate density value) of GH, PRL and SS in typical curve calculation sample.

7.1.1 the secretion level of tethelin

The typical curve formula that double-antibody sandwich elisa detects GH is: Y=-0.30+8.94X-14.86X ²+ 18.49X ³-6.08X ⁴, wherein Y is GH concentration (ng/mL), X is 450nm place OD value (see annex).By the OD recorded ₄₅₀value substitutes in this typical curve formula, calculates the GH concentration in mice plasma.T check analysis is done between any two to different treatment group and control group, variant.Therefore, respectively to vaccine, immunizing dose with add and exempt from number of times 3 factors and carry out single factor analysis, find that different immunizing dose and the concentration of immune time to GH have no significant effect, but in mouse blood, GH content difference is remarkable after different vaccine immunity, compared with zero load injection group, after immunity pEGS/2SS and pEGS/2SS-tpa, the secretory volume of mouse GH promotes all to some extent, but after intramuscular injection pEGS/2SS, mouse GH improves not remarkable, and after injecting pEGS/2SS-tpa, mouse GH content is significantly higher than the mouse (P<0.05) of injection pEGS/2SS.Can increase the release of GH after somatostatin DNA vaccine pEGS/2SS immunity is described, after inserting tpa signal peptide, this effect is more obvious, although do not show in the growth traits of mouse.(Figure 10)

7.1.2 the secretion level of prolactin

The typical curve formula that double-antibody sandwich elisa detects PRL is: Y=-1.20+22.96X-30.46X ²+ 70.88X ³-32.56X ⁴, wherein Y is PRL concentration (ng/mL), X is 450nm place OD value (see annex).By the OD recorded ₄₅₀value substitutes in this typical curve formula, and the PRL concentration in the mice plasma drawn, does the T check analysis between two between group, and the PRL concentration shown between different groups is variant.Therefore, to vaccine, immunizing dose with add and exempt from number of times 3 factors and carry out single factor analysis respectively, find different immunizing dose and immune time not remarkable on the impact of PRL concentration, but in mouse blood, the content of PRL has significant difference after immune different vaccine, compared with unloaded immune group, the mouse PRL content of pEGS/2SS and pEGS/2SS-tpa immune group is improved; And mouse PRL content remarkable rising (P<0.05) compared with the mouse of injection pEGS/2SS after intramuscular injection pEGS/2SS-tpa, difference extremely significantly (P<0.01) compared with control group mice.Secretion by increasing mouse PRL after somatostatin gene immunity is described, and this promoter action is especially obvious after insertion tpa signal peptide, this result is corresponding with female mouse milk character result, and fully can explain the phenomenon that immune stepmother mouse milking capacity improves.(Figure 11)

7.1.3 the secretion level of Somatostatin

The typical curve formula that double-antibody sandwich elisa detects SS is: Y=-1.31+32.39X-56.62X ²+ 86.87X ³-32.52X ⁴, wherein Y is SS concentration (pg/mL), X is 450nm place OD value (see annex).The 450nm place OD value of sample is updated in above-mentioned typical curve formula, calculates the concentration of SS in mice plasma.T check analysis is done between two to different treatment group and control group, to find between different groups SS content no significant difference in mouse blood.Further to vaccine, immunizing dose with add and exempt from number of times 3 factors and carry out single factor analysis separately, find that different vaccine, different immunizing dose and different immune time all do not make a significant impact the concentration of SS.Different vaccine immunity affects result to SS content in mouse blood, and compared with zero load injection group, after immune pEGS/2SS, the secretory volume of mouse SS declines to some extent, but it is not remarkable to decline; After immunity pEGS/2SS-tpa, mouse SS content is on the contrary higher than the mouse of control group, although difference is also not remarkable.After somatostatin DNA vaccine pEGS/2SS and pEGS/2SS-tpa immunity are described, remarkably influenced can not be caused on the SS in mouse blood.(Figure 12)

The detection of 7.2 somatostatin antibodies

Detect the generation situation of SS antibody in immunized mice body with indirect ELISA method, concrete steps are as follows:

(1) the 96 every hole of hole enzyme plate is wrapped by standard growth chalone antigen (Sigma-Aldrich, S9129) 100ng/100 μ L, 4 DEG C of overnight incubation;

(2) abandon reaction solution, PBST washs 5 times, 300 μ L/ holes, each 1min;

(3) add confining liquid (i.e. 1%BSA solution) 200 μ L/ hole, hatch 1h for 37 DEG C;

(4) abandon reaction solution, PBST washs 5 times, 300 μ L/ holes, each 1min;

(5) add test plasma (1:200 dilution) 100 μ L/ hole, negative control hole, non-specific adsorption hole (namely PBST replaces blood plasma) and zeroing hole are set simultaneously, hatch 1h for 37 DEG C;

(6) abandon reaction solution, PBST washs 5 times, 300 μ L/ holes, each 1min;

(7) sheep anti-mouse igg-HRP (Boster, BA1050,1:2000 dilute) 100 μ L/ holes are added, 37 DEG C of reaction 1h;

(8) abandon reaction solution, PBST washs 5 times, 300 μ L/ holes, each 1min;

(9) tmb substrate nitrite ion 150 μ L/ hole is added, lucifuge reaction 25min;

(10) 2mol/L H is added ₂sO ₄stop buffer 50 μ L/ hole termination reaction, the inherent 450nm wavelength place of 15min measures the OD value in each hole.

According to concentration and the OD of SS antibody standard substance ₄₅₀value drawing standard curve, by sample survey OD ₄₅₀value substitutes into typical curve, then is multiplied by the SS antibody concentration that 200 are sample.The SS antibody concentration of test group carries out stdn correction with the SS antibody concentration of negative blood sample, to get rid of the impact of non-specific adsorption on result.

Calculate the average OD of negative blood sample to be measured ₄₅₀value (X _n) and standard deviation (S _n), draw yin and yang attribute threshold value X _n+ 2S _n.If the OD of testing sample ₄₅₀value>=X _n+ 2S _n, then the positive is judged to; Otherwise, then feminine gender is judged to.Record the positive mice number often organized, and add up the antibody positive rate of different tests grouping.

7.2.1 somatostatin antibody positive rate

According to the OD of positive blood sample ₄₅₀value>=X _n+ 2S _n, judge that often group produces the mouse number of positive antibody, calculates the antibody positive rate of each test group.As shown in Table 6, pEGS/2SS immune group and pEGS/2SS-tpa immune group all have the mouse producing SS antibody, and the antibody positive rate of pEGS/2SS-tpa immune group is higher than pEGS/2SS immune group; Under different immunizing dose process, the antibody positive rate of high dose group (200 μ g) is higher than middle low dose group; Difference is added and exempts from number of times analysis, add the antibody positive rate exempting from 1 time slightly higher.Therefore, with regard to antibody positive rate, adopt the immune effect of high dosage (200 μ g) immunity 2 pEGS/2SS-tpa best.

The antibody positive rate of the different vaccine group mouse of table 6 (a)

The antibody positive rate of the different immunizing dose group of table 6 (b)

Table 6 (c) difference adds the antibody positive rate exempting from time array

Claims

1. a genetic engineering bacterium, is characterized in that: intestinal bacteria escherichia colidH5 α (pEGS/2SS-tpa), deposit number: CCTCC NO: M2015105.

2. the plasmid that comprises of bacterial strain described in claim 1.

3. plasmid described in bacterial strain described in claim 1 or claim 2 promotes the application in growth of animal vaccine in preparation.

4. plasmid described in bacterial strain described in claim 1 or claim 2 promotes the application in animal lactation vaccine in preparation.

5. plasmid described in bacterial strain described in claim 1 or claim 2 promotes the application in animal reproduction vaccine in preparation.

6. plasmid described in bacterial strain described in claim 1 or claim 2 promotes the application in growth of animal, breeding and lactation vaccine simultaneously in preparation.

7. claim 3 or 4 or the application described in 5 or 6, described animal is mouse.

8. promote a method for mouse growth and lactation simultaneously, comprise plasmid 200 μ g immune mouse according to claim 22 times.