CN104789514B - A kind of amicine overexpression DNA vaccination and construction method and application - Google Patents

Abstract

The invention discloses a kind of amicine overexpression DNA vaccination and construction method and applications, plasmid pEGS/2SS is digested with I two restriction endonucleases of Xho I and EcoR, obtaining the both ends containing kanamycins has the pEGS/2SS segments of I two restriction enzyme sites of Xho I and EcoR, then by synthetic tpa and pEGS/2SS segment coupled reactions, plasmid pEGS/2SS tpa are obtained.Escherichia coli will be obtained in plasmid pEGS/2SS tpa conversion competent escherichia coli cells DH5aEscherichia coliDH5 α (pEGS/2SS tpa), deposit number：CCTCC NO:M2015105.Mouse is immunized using plasmid pEGS/2SS tpa as DNA vaccination, mouse growth, breeding and lactation can be promoted, had broad application prospects.

Description

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

Technical field

Structure and application the present invention relates to animal DNA vaccine overexpress DNA epidemic diseases more particularly to a kind of amicine Seedling and construction method and application.

Technical background

Growth hormone release inhibiting hormone (Somatostatin, SS) gene vaccine is exactly by by the gene cloning of encoding growth chalone -14 The recombinant plasmid constructed on carrier for expression of eukaryon.Then the plasmid is imported in animal body, makes SS-14 in vivo Active polypeptide is given expression to, the antibody of anti-growth hormone release inhibiting hormone is generated by immune response to reach the mesh for neutralizing endogenous growth chalone , so as to promote growth of animal and lactation.Obviously, play main function in this approach is growth hormone release inhibiting hormone.According to growth The effect of chalone " inhibiting somatotropin (growth hormone, GH) and the release of lactagogue parahormone ", recent year close Just have in terms of growth of animal and lactation is promoted in the studies have shown that SS genetic immunizations in terms of animal reproduction in SS genetic immunizations Effect, and obtain more apparent immune effect.

It is well known that dairy products are as a kind of high-quality liquid food full of nutrition, comprehensive, digestibility is high, it is more next More liked by consumers in general, and by common concern.Contain abundant protein, carbohydrate, amino acid, dimension life in fresh milk Element, minerals and enzyme etc., and the sequence of milk nutritional ingredient is according to this from high to low buffalo milk, goat milk, Holstein cow milk, people's milk and Mare's milk.This is because compared with ordinary milk, the comprehensive nutrient value of buffalo milk is higher by nearly 2 times, and casein content is high, and courage is solid Alcohol is low, and somatic number is low, and dry matter content (17.5%) is 0.5 times high, and unsaturated fatty acid content is 60-100 times high, and protein contains Amount (4%) is 0.5 times high, and butterfat percnetage 7.5%, calcium content is 0.3 times high, and Zn content is 12 times high, and iron content is 78 times high, and vitamin A contains It measures high 32 times (Zhang Chunxi 2006).Therefore, often drinking-water milk can promote more tyrosine calcium uptakes, contribute to bone and intelligence Development reduces cardiovascular disease incidence rate, improves the immunity of the human body.However the 6.4% of the milk total output Jin Zhan worlds of China, breast The product level of consumption is the 1/5 of world average level, while buffalo lactation amount, reproductive efficiency genetic force are relatively low.Above-mentioned present situation Presence cause must to improve milk-quality and milk performance expansion research.

It is to have the example governed that growth hormone release inhibiting hormone, which is applied to animal lactation,.The chemical antagonist cysteamine of the SS such as Wang Yanling (cysteamine hydrochloride, CSH) feeds the holstein cow of lactation mid-term, finds daily yielding and the feeding of milk cow Material conversion ratio all significantly improves (Wang Yanling etc. 1999).According to reports such as Sun, external source SS hormone active immunity lactation ewes are used Its lactation amount (Sun et al.1990) can be significantly improved.Imgap has found when in immune rat, only active immunity SS-14 The antibody of anti-SS could be generated and improve the output of milk (Yi et al.1999).Xiao Shenhua " swashing No. 1 raw " (i.e. a kind of SS weights Group vaccinia virus live vector vaccine) milk cow is immunized, play the role of promoting lactation, and make under the output of milk of Lactation of Dairy Cow middle and later periods Drop process slows down, but milk-quality is uninfluenced (Xiao Shen China 2005).More than result of study shows using somatostatin DNA vaccine Milk performance to improve domestic animal is feasible.

Tissue plasminogen activator (tissue plasminogen activator, tpa) is a special positioning In the signal peptide of endoplasmic reticulum, the albumen that can be coupled is directly transferred into endoplasmic reticulum, and antigen is avoided to be processed and is transported to cytoplasm, mesh Albumen with secreted form, a large amount expression can ensure the maintenance of ideal immunogenicity in vivo.Tpa molecules include 4 structures Domain, one is 47 residues in F areas, i.e. N-terminal, same with adjusting the finger structure domain of fibrin plates of fibronectin Source；The second is E areas, homologous with epidermal growth factor；The third is two loop domains of K1 and K2；The fourth is P areas, i.e. serine Protease area (Pennica et al.1983).Wherein, the function in F areas and K2 areas is combined with fibrin, passes through F areas, E areas It is quickly removed under the adjusting of polysaccharide chain, and P areas then play enzymatic activity (Lijnen and Collen 1991).With extraction and The accuracy of purification process improves, and researcher has found that tpa is mainly discharged, and tpa swashs with blood vessel plasminogen by endothelial cell Being (vascular plasminogen activator, vpa), blood activator of plasminogen (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).Show that tpa is by further Biochemical Research Main component in fibrinolytic system, physiological regulation fibrinolysis；

However, tpa is not only the major function factor in fibrinolytic system, it is one that structure, which also determines it, Efficient signal peptide.Therefore, tpa has the shared effect of all signal peptides, i.e., first by signal recognition particle (signal Recognition particle, SRP) identification, it is attached on ER films and carries out signal identification, by the ribosomes in translation state It is incorporated on RER films, after ribosomes completes translation, the maturation protein of clipped modification will be secreted along peptide approach cross-film is led To extracellular.In addition, as signal peptide, tpa can not only make its coupling protein become secretory protein, but also convenient for fixed in cell Position protein and new polypeptide chain (Wei Xuefang etc. 2006).At present, the tpa signal peptides of 23bp have been used as heterologous leader sequence, by with Destination protein is brought into method (Yang et al.2001, Costa et al.2006, the Jalah et of cell secretory pathway by work al.2007,Golden et al.2008,Luo et al.2008,Kaur et al.2009,Seo et al.2009).With Afterwards, the application of tpa is not limited solely to the research of cellular level, but is constantly generalized to other field, is especially prevented for structure New approach is opened in terms of the gene vaccine of Animal diseases.Because modificator gene vaccine generates efficiently and a large amount of immune anti- Should, depending on expressing the secretion capacity (Wa tanabe et al.2003, Liang et al.2005) of antigen；And in gene epidemic disease The presence of Miao Zhong, tpa signal peptide can effectively improve secretion of the recombinant protein to extracellular medium, and body is induced to generate pathogen Very strong immune response (Li et al.1999, Alves et al.2000, Ashok and Rangarajan 2002). Sinone M.Costa etc. merge tpa with the NS1 full-length genes of dengue fever virus DENV-2, the vaccine of structure The NS1 albumen of recombination is secreted into surrounding medium by pcTPANS1 with dimeric forms, and higher is generated compared with the control group after attacking poison Horizontal NS-1 specific antibodies, and based on IgG1, it is intended to Th2 types reaction (Costa et al.2006, Costa et Al.2007, Yao Yan are rich 2012).Yang etc. is the antibody activity for improving Hepatitis E vaccine, is compared in Hepatitis E virus HVE-p179 N-terminals merge tpa signal peptides and are inserted into 3 C3d complements repetitive sequence both sides in HVE-p179 C-terminals Formula, finds in the antibody generated after being immunized and the level of antigen is improved, and the Hepatitis E vaccine for merging tpa generates antibody Time is done sth. in advance 2 weeks (Yang et al.2010).It is continued to use however, the application about tpa signal peptides does not terminate in simple duplication. Wang has found that tpa signal peptides 22 sport the third ammonia by proline when trimer protein structural domain is introduced into plasmid vector Acid or glycine can improve the expression of destination protein and secretion situation (Wang et al.2011).In addition to this, correlative study It emerges in an endless stream, such as the Flu-A vaccine ptPAs/NP of the segment containing tpa, pulmonary tuberculosis DNA vaccination, the people that conjunction with electroporation is immunized Papillomavirus (papillomavirus, HPV) vaccine etc. (Delogu et al.2002, Luo et al.2008, Seo et al.2009).In short, after the target gene in carrier is coupled with tpa signal peptides, destination gene expression is not only made to secrete shape Formula albumen, and humoral immunity level (Wang et al.2004, Costa et al.2006) can be enhanced.

Lactation is one and breaks up from breast tissue to the physiology course to play a role, relates generally to mammogenesis, lactation hair The dynamic and links such as maintenance and mammary gland, and each stage is to participate in by hormone.Mammary gland development is Since fetal period, body of gland and conduit system at this time is still undeveloped, with jenny ontogeny, estrogen and pregnant Hormonal readiness improves, and estrogen promotes the growth of breast duct, and progestational hormone stimulates development (the Lamote et of mammary gland alveolus al.2004).And the synthesis of IGFs in estrogen stimulation breast stroma cell, so as to which galactophore epithelial cell be promoted to generate (Tucker 2000).In addition, galactagogin (Prolactin, PRL), corticotropin (adrenocorticotrophic hormone, ACTH), thyrotropic hormone (thyrotropic hormone, TSH) etc. swash Element and environmental stimuli can influence breast cell proliferation.Lactation is started during childbirth, is noteworthy characterized by prolactin and sugar at this time The secretion of cortin peaks.Based on peak of lactation decomposed with alienation, the raising of GH levels and insulin and thyroid gland swash Cellulose content reduces；Hereafter based on assimilation synthesis (Zhang Shaoying 1990).But the startup of lactation and maintenance stage mainly by PRL and The regulation and control of GH.For ruminant, GH and PRL joint effect lactation amounts, GH influences butterfat synthesis, and PRL influences lactoprotein With the synthesis (Knight 2001) of lactose.Cortisol can maintain the secretion activity of epithelial cell, but not find that it promotes milk life Into effect (Sjaastad et al.2010).

It is found by researches that not only containing SS in blood plasma, the presence of SS is also can detect in milk.Only 70% in blood plasma SS is SS-28, and only has SS-14 in milk.This is because PC-1 protease-baseds are only expressed in the breast tissue of mammal Cause, PC-1 protease can promote growth promotion chalone original to synthesize SS-14 (Werner et al.1985, Holst et al.1990). The research of Chen et al. points out that a kind of detectable unique neuropeptide is exactly SS, and infer that SS can in the mammary gland of rat It can participate in lactation activity, i.e., the development of mammary gland and function (Chen et al.1999).In addition, SS can be in the association of estrogen Inhibit the secretion (Lee and Shin 1996) of PRL under same-action.

For ruminant, PRL is the required factor for starting lactation.However, lactation, once being started, PRL is just No longer it has been the limiting factor (Zhang Shaoying 1990) for determining lactation amount height.Early in 1940, British scientist just confirmed that GH can There can be certain lactagogue effect (Peel and Bauman 1987).At present, with hypophysis or genetic recombination bovine growth hormone (bST) practice result for improving lactation amount more demonstrates the effect that GH maintains lactation.Han Zheng's health etc. is daily to every cow in milk 12.5-50mg GH are injected, as a result, it has been found that the milk production of cow in milk can generally improve 15-24% after injection, and its feed conversion Efficiency can increase 5-20% (Han Zheng's health 1999).In fact, GH and it is indirect mammary gland is had an impact, but GH first stimulates liver, Liver is made to generate IGFs, and IGFs can influence mammary gland activity (seedling tree monarch 1999).That is, GH by IGFs between medium It connects stimulation mammary glandular cell to break up, enhances the milking capacity of mammary gland.GH, which has, to be coordinated to organize the competition to nutrition outside mammary gland and mammary gland, Reduce the nutriment intake organized outside mammary gland.Research in terms of Neuroendocrinology it has been experienced that GH by hypothalamus It is dual with the growth hormone releasing hormone (growth hormone releasing hormone, GHRH) and SS of gastrointestinal tract release Regulation and control, the i.e. secretory volume of GH are balanced by the collective effect of both hormones of GHRH and SS.Therefore, some researchs are by regulating and controlling to promote It is secreted into GHRH or SS levels is inhibited to improve the content of internal GH, so as to achieve the effect that improve lactation amount (Han Zheng's health 1999).Much research shows that, lactation amount can be improved 20-30% using the method for GH injection cows in milk, through examining the method It can't influence composition of milk or the health status (seedling tree monarch 1999) of ox.The researchers such as Machlin, Baird pass through respectively It injects in the GH to lactating cow body that the GH extracted and DNA are re-combined into, the milk performance of lactating cow is made all to improve 10- 25% (Machlin 1973, Baird and Durkin 1986).Cornell Univ USA in 1981 uses recombination methionyl ox Growth hormone (MBS) carries out a short term tests to milk cow, the results showed that is increasing lactation amount in terms of this, MBS spreads out with hypophysis The effect of raw GH is quite (Peel and Bauman 1987).The bovine growth hormone that Chadio etc. is produced with genetic recombination (bovine growth hormone, bST) handle lactation period goat, find in addition to lactation amount is improved, also improve butterfat with The content of lactose, but the changes of contents of lactoprotein is less (Chadio et al.2000).In addition, Castigliego etc. is to being in The milk cow effect bST of lactation period, detects that bST the and IGF-1 levels in processing milk cow blood plasma significantly rise (Castigliego et al.2009).Nowadays recombinant bovine GH is applied in dairy industry production by the U.S., and the country also has Similar research obtains preferable achievement.Such as the culture Escherichia coli such as land east woods, obtain the thick of bST using gene recombination technology Extract handles milk cow one month with this, and every bovine lactation amount increases 1.65kg/d (about 10.75%), but butterfat percnetage, newborn density Etc. indexs have no difference (Lu Donglin etc. 1990).Although test method is different with Testing index, must for GH can promote The Milking ability of mammal.

From the point of view of document above report, tpa signal peptides and growth hormone release inhibiting hormone SS gene vaccines cooperate with animal lactation and Breeding effect is at home and abroad without relevant report, therefore the present invention weighs original somatostatin DNA vaccine pEGS/2SS Group constructs the novel overexpression somatostatin DNA vaccine of the peptide fragment of signal containing tpa, it is intended that improve the vaccine to animal immune Originality, lactation and the effect of breeding, and use condition and immune programme optimal case are inquired into, it is New somatostatin gene vaccine Application in the actual production process provides theoretical foundation.

Invention content

The defects of it is an object of the invention to overcome existing somatostatin gene immunological technique, provides a kind of overexpression life The eukaryon expression plasmid pEGS/2SS-tpa of long chalone, the plasmid are that tpa segments are inserted into plasmid pEGS/2SS with I Hes of Xho EcoR I is obtained.

It is another object of the present invention to provide a kind of bacterial strains for including plasmid pEGS/2SS-tpa；The bacterial strain is It is sent on March 12nd, 2015 to China typical culture collection center preservation, Classification And Nomenclature：Escherichia coli Escherichia Coli DH5 α (pEGS/2SS-tpa), deposit number：CCTCC NO:M2015105, the bacterial strain are extracted by SDS alkaline lysis It can obtain pEGS/2SS-tpa plasmids.

Final object of the present invention is to provide a kind of eukaryon expression plasmid pEGS/2SS- for overexpressing growth hormone release inhibiting hormone Tpa or comprising the application in amicine DNA vaccination is prepared its Escherichia coli, is applied to mouse by the DNA vaccination, shows Work improves mouse growth, breeding and the function of lactation.

The present invention is implemented by following technical solution：

A kind of eukaryon expression plasmid pEGS/2SS-tpa for overexpressing growth hormone release inhibiting hormone, is prepared by the following method to obtain

Plasmid pEGS/2SS with I two restriction endonucleases of Xho I and EcoR is digested first, is obtained containing kanamycins There are the pEGS/2SS segments of I two restriction enzyme sites of Xho I and EcoR at both ends, and synthetic tpa segments then are passed through Xho I and EcoR, I two endonuclease digestions obtain the tpa segments of I two restriction enzyme sites of Xho I and EcoR, by the gene piece Section and pEGS/2SS segment coupled reactions, obtain plasmid pEGS/2SS-tpa.

By in plasmid pEGS/2SS-tpa conversion competent escherichia coli cells DH5a, obtain using Escherichia coli as carrier Ultra high efficiency starts expression somatostatin DNA vaccine.The bacterial strain is sent on March 12nd, 2015 into China typical culture collection Heart preservation, Classification And Nomenclature：Escherichia coli Escherichia coli DH5 α (pEGS/2SS-tpa), deposit number：CCTCC NO:M2015105, preservation address：Wuhan, China Wuhan University；The bacterial strain can obtain pEGS/ by the extracting of SDS alkaline lysis 2SS-tpa plasmids.

Escherichia coli Escherichia coli DH5 α (pEGS/2SS-tpa) is contain tissue plasminogen activator (tpa) mixed culture of growth hormone release inhibiting hormone eukaryon expression plasmid pEGS/2SS-tpa can be obtained by the extracting of SDS alkaline lysis To pEGS/2SS-tpa plasmids.Culture medium：1L LB solid mediums form：10g sodium chloride, 5g yeast extracts, 10g pancreas eggs White peptone, 1.5g agar powders.Condition of culture：pH：6.8-7.8, temperature：37 DEG C, be Kan⁺Resistance；With flat in LB solid mediums Plate method of scoring detects whether the culture survives.

A kind of eukaryon expression plasmid pEGS/2SS-tpa for overexpressing growth hormone release inhibiting hormone is in amicine DNA vaccination is prepared Application, after being mixed using the plasmid as vaccine direct immunization animal or with DNA vaccination adjuvant be immunized animal.

It has been applied successfully to improve growth of animal, breeding and milk performance in the present invention, such as improved in mouse The application of the function of growth, breeding and lactation.

More detailed technical solution is shown in《Specific embodiment》.

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

1st, a kind of efficient eukaryon expression plasmid pEGS/2SS-tpa for starting amicine has been obtained；

2nd, obtained it is a kind of can in the growth, breeding and milk performance of mouse remarkable result growth hormone release inhibiting hormone DNA epidemic diseases Seedling pEGS/2SS-tpa.There is certain superiority to judge by the Cluth size index of female rat on reproductive performance, pEGS/2SS- Tpa vaccine experiments group is more 1 higher than pEGS/2SS growth hormone release inhibiting hormone positive controls, than feminine gender group pEGFP-N1 up to 1.4 ≈ 2 Only；By the Body Mass Index for newborn mouse of weaning, pEGS/2SS-tpa groups can be obtained than pEGS/2SS growth hormone release inhibiting hormone positive controls Weight is high by 10.7%, higher than negative control group pEGFP-N1 by 21.8%；And efficiently start vaccine high dose group (200 μ g/ only), Add and exempt from weightening, the reproductive performance best results of 1 time.

Description of the drawings

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

Fig. 2 is the electroresis appraisal figure for synthesizing segment tpa.

M is 50bp DNA Ladder Marker；1-3 swimming lanes are the signal peptide tpa after annealing to be measured through 1% agarose The result of detected through gel electrophoresis.

Fig. 3 identifies collection of illustrative plates for eucaryon plasmid carrier pEGS/2SS digestions.

M represents DL 2,000Marker；1 swimming lane is eucaryon plasmid carrier pEGS/2SS through I Hes of restriction enzyme EcoR Result after I double digestions of Xho.

Fig. 4 identifies collection of illustrative plates for eucaryon plasmid carrier pEGS/2SS-tpa digestions.

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

Fig. 5 is expression and the distribution situation that different plasmids Plasmids transfect MCF-7 cell GFP green fluorescences.

In wherein Fig. 5 A for tri- kinds of plasmid transfection MCF7 cells of pEGFP-N1, pEGS/2SS and pEGS/2SS-tpa for 24 hours after Observe the expression of GFP green fluorescences.Bright and fluorescence be respectively in the observation off field of light field and fluorescence as a result, Scale represents 200 μm.

B is observes in Fig. 5 after tri- kinds of plasmid transfection MCF7 cells 48h of pEGFP-N1, pEGS/2SS and pEGS/2SS-tpa The expression of GFP green fluorescences.Bright and fluoresce is the observation result in light field and fluorescence off field respectively；Scale generation 200 μm of table.

C is observed afterwards for 24 hours for tri- kinds of plasmid transfection MCF7 cells of pEGFP-N1, pEGS/2SS and pEGS/2SS-tpa in Fig. 5 The distribution situation of GFP green fluorescences；Scale represents 200 μm.

Fig. 6 is the overexpression effect comparison chart that quantitative real-time PCR eucaryon plasmid transfects SS after MCF-7 cells.

PEGFP-N1, pEGS/2SS or pEGS/2SS-tpa transfect 48h after MCF-7 cells respectively, detect and are grown in cell The expression of chalone SS.The expression quantity of SS is standardized with the expression quantity of GAPDH, and expression multiple is existed relative to SS The expression of pEGFP-N1 transfection groups is finally represented, each data have 3 biological samples to attach most importance to come what is calculated with mean value standard error It is multiple.**P<0.01

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

Western Blot detection lifes are utilized after MCF-7 cell transfectings pEGFP-N1, pEGS/2SS and pEGS/2SS-tpa The expression of long chalone SS-14 albumen；The expression quantity for choosing GAPDH albumen simultaneously is standardized correction, and expression multiple is It is calculated relative to pEGFP-N1.

Fig. 8 is the agarose gel electrophoresis testing result that Trizol methods extract kunming mice tissue RNA, RNA concentration.

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

Fig. 9 is growth hormone release inhibiting hormone SS expression quantity testing results in kunming mice respectively tissue.

Swimming lane 1-9 is the tissue RNA amplification growth hormone release inhibiting hormone of heart, liver, spleen, lung, kidney, muscle, small intestine, stomach, brain successively As a result.

SS distribution situations in the kunming mice body of 5d after A is immune pEGS/2SS-tpa in Fig. 9；

SS distribution situations in the Mice Body of 5d after B is immune pEGS/2SS in Fig. 9.

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

Compared with unloaded injection group pEGFPN1, the secretory volume of mouse GH is equal after immune pEGS/2SS and pEGS/2SS-tpa It is promoted, but mouse GH raisings are not notable after intramuscular injection pEGS/2SS, and inject mouse GH after pEGS/2SS-tpa Content is significantly higher than the mouse (P of injection pEGS/2SS<0.05).Illustrate that somatostatin DNA vaccine pEGS/2SS can be with after being immunized Increase the release of GH, this effect is more obvious after being inserted into tpa signal peptides.

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

Compared with unloaded immune group, the mouse PRL contents of pEGS/2SS and pEGS/2SS-tpa immune groups are improved；And And mouse PRL contents significantly raising (P compared with injecting the mouse of pEGS/2SS after intramuscular injection pEGS/2SS-tpa<0.05), The difference extremely significantly (P compared with control group mice<0.01).Illustrate that mouse PRL can be increased after being immunized by somatostatin gene Secretion, and this facilitation is especially apparent after tpa signal peptides are inserted into.

Figure 12 is the secretion level result of growth hormone release inhibiting hormone SS after vaccine immunity kunming mice.

Compared with unloaded injection group, the secretory volume of mouse SS is declined after immune pEGS/2SS, but is declined not notable；Exempt from Mouse SS contents are higher than the mouse of control group instead after epidemic disease pEGS/2SS-tpa, although difference is not also notable.Illustrate growth hormone release inhibiting hormone After gene vaccine pEGS/2SS and pEGS/2SS-tpa are immunized, the SS in mouse blood will not be significantly affected.

Specific embodiment

Embodiment 1：

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

1st, tissue plasminogen activator's (tpa) gene cloning and sequence analysis

According in GenBank (ID:5327)plasminogen activator,tissue[Homo sapiens (human)] gene order, the digestion at its both ends of cDNA sequence of synthesis plasminogen activator, tissue (tpa) Site is Xho I and EcoR I, and using Primer5.0 primer-design softwares, tpa sequence designs are as follows：(it is tpa sequences in box Row, both ends are restriction enzyme site)

tpa-F：

5-TCGAG G-3

tpa-R：

3-C CTTAA-3, Sangon Biotech (Shanghai) Co., Ltd. synthesize h by base and obtain tpa gene pieces Section.

After sequent synthesis, upstream and downstream segment is diluted to every OD and adds 140 μ L ddH₂O is made into 10 μM of storage liquid, then will Two single-stranded annealing form double-strand.Annealing system is mixed with 10 μ L tpa-R for 10 μ L tpa-F, annealing conditions be 95 DEG C of 30s, 72℃2min、37℃2min、25℃2min.Agarose gel electrophoresis detects after annealing, observes electrophoretic band and purpose band is big It is small to be consistent, it is stored for future use in 4 DEG C.(Fig. 2)

2nd, the digestion and recycling of carrier pEGS/2SS

With restriction enzyme Xho I and I double digestion pEGS/2SS plasmids of EcoR, (Cao Shaoxian, Yang Liguo, Zhang Wenwei wait The structure of somatostatin DNA vaccine plasmid pEGS/2SS and expression [J] China animal doctor's journal, 2005,25 (5):499- 502.) 16bp, is cut off, exposes the cohesive end at both ends, digestion system is as follows：

Plasmid band is isolated through 1% agarose recycling gel electrophoresis after digestion, uses Ago-Gel DNA QIAquick Gel Extraction Kits Plasmid large fragment recycling, recycling rear electrophoresis detection are carried out, observation electrophoretic band is consistent with purpose band size.(attached drawing 3)

3rd, target gene connects

The concentration of the pEGS/2SS plasmid enzyme restrictions segment of recycling and the tpa segments of 10 times of dilution is measured respectively, calculates load The connection volume ratio of body and target fragment is 6:1, pEGS/2SS is connected according to Invitrogen T4DNA Ligase specifications and is carried Body and target gene tpa segments, 16 DEG C overnight, and linked system is as follows：

4th, recombinant plasmid transformed competence colibacillus bacterium

1) competent bacteria DH5 α (TIANGEN Biotech's production)

2) competent escherichia coli cell DH5 α are taken out ice bath to thaw from -80 DEG C, 10 μ L connection products is added to enter 50 μ L Mixing in competent cell, and set and do not add the competent cell of connection product as blank control, ice bath 30min；Go to 42 DEG C heat shock 90s, then rapid ice bath 5min；400 μ L LB fluid nutrient mediums are added in, shaking table is adjusted to 200r/min, 37 DEG C of oscillation trainings Support 1h；100 μ L are gently drawn after mixing, are uniformly applied to using spreader on the LB solid plates containing Kan, after 37 DEG C of culture 12h, See whether that conversion bacterium colony is grown.

5th, the screening and identification of pEGS/2SS-tpa plasmids

Picking single bacterium colony vibrates expansion culture 8h in the LB fluid nutrient mediums containing Kan, and it is small to carry out plasmid using kit It carries, tpa (75bp) should be cut out through Xho I and I digestions of EcoR.Digestion system is as follows：

5 μ L digestion products is taken to carry out 1% agarose gel electrophoresis detection, observe electrophoretic band and purpose band size phase Symbol is sent to Sangon Biotech's sequencing, analyzes sequencing result, plasmid pEGS/2SS-tpa sequencing results 100% compares successfully.It identifies that correct -20 DEG C of pEGS/2SS-tpa plasmids save backup, plasmid pEGS/2SS-tpa is converted In competent escherichia coli cell DH5a, obtain the ultra high efficiency using Escherichia coli as carrier and start expression somatostatin DNA vaccine. The bacterial strain is sent on March 12nd, 2015 to China typical culture collection center preservation, Classification And Nomenclature：Escherichia coli Escherichia coli DH5 α (pEGS/2SS-tpa), deposit number：CCTCC NO:M2015105, the bacterial strain pass through SDS Alkaline lysis extracting can obtain pEGS/2SS-tpa plasmids.The bacterium solution conservation of the plasmid containing pEGS/2SS-tpa, -80 DEG C of preservations. (Fig. 4)

Embodiment 2：

The detection of growth hormone release inhibiting hormone overexpression plasmid pEGS/2SS-tpa plasmids protein expression in vitro

1st, inhibin recombinant plasmid pEGS/2SS-tpa transfectional cells

With plasmid extraction kit (being purchased from TIANGEN Biotech (Beijing) Co., Ltd.) extracting Escherichia coli Escherichia coli DH5 α (pEGS/2SS-tpa) plasmid, treat MCF-7 cells (human breast cancer cell) individual layer grow to Lipofectamine is pressed during 60%-70%^TM2000 lipofectamine boxes (being purchased from Invitrogen companies) specification carries out Operation, adds 0.2 μ g plasmids and 1.5 μ L Lipofectamine according to every hole^TM2000 calculate, and use serum free medium respectively OPTI-MEM dissolves mixing, is stored at room temperature 5min.Then the plasmid being configured and liposome solutions are mixed in equal volume, room temperature is quiet Put 20min.Period absorb tissue culture plate in culture medium, PBS clean 2-3 times, add OPTI-MEM culture based on 37 DEG C, 5% CO₂Incubation is handled in incubator.24 orifice plates are taken out after 20min, absorb OPTI-MEM culture mediums, add 100 μ L OPTI-MEM per hole Culture medium, and 100 μ L plasmid-liposome complex are slowly added dropwise.Cell processing, which is set, is respectively：PEGFP-N1 transfection groups, PEGS/2SS transfection groups, pEGS/2SS-tpa transfection groups and blank control group, every group sets 3 parallel repetitions respectively.37℃、 5%CO₂After cultivating 5-6h in incubator, change DMEM complete culture solutions (i.e. DMEM+15%FBS) into and continue to cultivate.

2nd, inhibin recombinant plasmid pEGS/2SS-tpa transfectional cell egfp expressions

Transfection for 24 hours with after 48h respectively in the cell growth state and green of fluorescence microscopy Microscopic observation different disposal group Fluorescence judges the transfection efficiency of MCF-7 cells by comparing the white field of cell and green fluorescence intensity, indirectly determining carrier purpose egg White optimum expression time.

Transfect pEGFP-N1, pEGS/2SS and pEGS/2SS-tpa respectively when MCF-7 cells 80% converge, transfection is for 24 hours After 48h, under fluorescence inverted microscope, the green fluorescence after blue light excitation in field of view.It can be seen that test group have it is green Color fluorescence is presented, and transfects the signal strength that the green florescent signal intensity after 48h is apparently higher than after transfection for 24 hours.Therefore, below Test cell line is using the cell (Fig. 5) of 48h after transfection.

The green fluorescence of pEGFP-N1 and pEGS/2SS transfection group cells is distributed in MCF-7 cells in uniformly continuous, The green fluorescence of pEGS/2SS-tpa transfection group cells is distributed in cell in scatterplot shape.Illustrate that being inserted into signal peptide tpa segments changes Expression and distribution of the destination protein in cell makes destination protein be transferred in endoplasmic reticulum and then is secreted into extracellular.(figure 5C)

3rd, the detection of growth hormone release inhibiting hormone plasmid pEGS/2SS-tpa vivoexpressions is overexpressed

The expression of 3.1Western Blot testing goal albumen

The extraction of cell protein：Take cell pyrolysis liquid RIPA, after thawing overturn mixing for several times (if having precipitation can put 37 DEG C it is molten Solution), packing with protein inhibitor phenylmethylsulfonyl fluoride (PMSF, 100mM) mixing in proportion, makes final concentration of 1mM, now with existing With.MCF-7 cells after transfection abandon culture solution, and the PBS of 4 DEG C of precoolings is cleaned 2-3 times.Add appropriate lysate, piping and druming cell is abundant Cracking.10,000rpm centrifugation 3-5min, take supernatant, dispense, -80 DEG C save backup.All of above operation operates on ice.

The measure of protein concentration：Standard curve and sample protein concentration are determined according to BCA determination of protein concentration kits.Tool Body step is as follows：(1) 0.8mL protein standards is taken to prepare liquid to be added in protein standard (20mg BSA), fully dissolves, is made into The protein standard liquid of 25mg/mL, uses immediately after preparation；

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

(3) according to sample number, add 1 times of volume BCA reagent B (i.e. A by 50 times of volume BCA reagent As:B=50:1) with appropriate BCA working solutions, abundant mixing；

(4) standard items are added to by 0,1,2,4,8,12,16,20 μ L in 96 hole elisa Plates, with standard dilutions (i.e. PBS 20 μ L) are complemented to, i.e. concentration is respectively 0,0.025,0.05,0.1,0.2,0.3,0.4,0.5mg/mL, as gauge orifice；

(5) plus in isometric sample to the sample well of 96 hole elisa Plates, 20 μ L are diluted to；

(6) each hole adds 200 μ L BCA working solutions, 37 DEG C of warm bath 30min；Microplate reader measures 562nm (between 540-595nm ) light absorption value under wavelength；

(7) standard curve is made, sample protein concentration is calculated according to standard curve.

SDS-PAGE electrophoresis：According to the molecular size range 55KDa of destination protein, 15% separation gel, 5% concentration is configured Glue carries out PAGE gel electrophoresis.It is preventing from cementing leakage during preparative separation glue, a small amount of low concentration agarose gel back cover can be used, then note Enter separation gel to blank, then divided equally with hydraulic pressure from glue liquid surface, solidify about 30min；Concentration glue is filled it up with after comb is inserted into, and is solidified 1-2h.Plastic plate is put into electrophoresis tank, adds 1 × Tris-Gly of electrophoretic buffer about 400mL.Albumen and Loading before loading Boiling water bath 5min after Buffer mixing, makes albuminous degeneration.50 μ g of albumen applied sample amount per hole, ice bath electrophoresis, first with constant pressure 80V electricity About 20min swim to glue and separation gel interface is concentrated, then change constant pressure 120V into and continue electrophoresis about 50min, sample is made to go to glue bottom, Stop electrophoresis, take out glass plate.

Transferring film：Measure the length and width for taking glue with a yardstick, clip filter paper 6 of corresponding size is opened, is immersed in transferring film buffer solution.According to Marker bands weigh the approximate location of purpose fusion protein S/2SS-GFP (55KDa) and internal reference Protein G APDH (37KDa), cut Pvdf membrane of corresponding size is taken, impregnates 3-5min bubble removings in methyl alcohol.Distill water-soaked graphite cake, keep flat opening, by down toward On placed negative plate, 3 layers of filter paper, pvdf membrane, 3 layers of filter paper and positive plate successively, often spread one layer and catch up with bubble with test tube, cover tightly electrode Plate.Ice bath electricity turns, 250mA electric currents transferring film about 45min.After transferring film, glue is taken to be put into dyestuff and is dyed, check whether that transferring film is complete Entirely.

Closing：It is closed using TBST with 1%BSA, room temperature horizontal shaker 1h.

Primary antibody is incubated：TBST washes film 3 times, each 10min；With 0.1%BSA dilution Somatostatin primary antibodies (1:300 is dilute Release, Santa Cruz) and internal reference GAPDH antibody (1:1000 dilutions, Boster), sealer, 4 DEG C of overnight incubations.

Secondary antibody is incubated：TBST washes film 3 times, each 10min；With 0.1%BSA dilution goat anti-rabbit iggs-HRP (1:1000 is dilute Release, Boster) incubation at room temperature 1h.

Colour developing：TBST washes film 3 times, each 10min；Developing solution colour developing 3-5min is eventually adding, develops and scans, during exposure Between 600s；Optical density is calculated with AlphaEaseFC softwares, by the comparison with internal reference Protein G APDH, extrapolates the opposite of albumen Expression quantity.

Cell protein is carried after different plasmid transfection MCF-7 cells 48h, Western Blot detection growth hormone release inhibiting hormones SS-14's Relative expression's situation, as a result display is compared with blank control (monk), pEGS/2SS transfection groups and pEGS/2SS-tpa transfection groups There is band at 55KDa, and in the same size with destination protein；Speculate according to color, MCF-7 cell transfectings pEGS/ The expression quantity of SS is slightly above the expression quantity of SS after transfection pEGS/2SS plasmids after 2SS-tpa plasmids.(Fig. 6)

Embodiment 3：

Influence of the growth hormone release inhibiting hormone overexpression DNA vaccination of the present invention on Kunming mouse to growth and immune response

1st, prepared by vaccine：

Identified correct pEGFP-N1, pEGS/2SS and pEGS/2SS-tpa bacterium solution is added in LBs of the 250mL containing Kan Expand culture, 200r/min, 37 DEG C, 12h in fluid nutrient medium.The big extraction reagent kit of endotoxin is gone largely to extract matter using plasmid Grain surveys plasmid concentration, digestion and sequence verification.As needed, plasmid normal saline dilution is to various concentration, -20 after packing It DEG C stores for future use.

2nd, experimental animal immunity test：

100 3 week old Kunming mouses of female are randomly divided into 10 groups, and (original body mass for ensureing each experimental group of weighing is without significance difference It is different), 10/group, indoor temperature is always held at 28 degree or so.Animal house is always maintained at cleaning, health in phase experimental period, in time Fecaluria in house is removed, keeps dry in house.With " 3.1 softwares of orthogonal design assistant II " design 3 factors (vaccine, immunizing dose, Immune time), 3 level (pEGFP-N1, pEGS/2SS, pEGS/2SS-tpa；It is basic, normal, high；2 times, 3 times, 4 times) orthogonal examination It tests.Experiment is grouped as follows：

2 mouse immuning test of table is grouped

Mouse is immunized according to grouping, takes mouse quadriceps muscle of thigh multi-point injection, being spaced 2w after first immunisation strengthens It is immune, the mind ＆ body situation of continuous one week observation mouse after being immunized.Mouse routinely feeding standard, changes weekly bedding and padding 2 times, It is freely eaten and drinks water.

3. mouse is weighed

It weighs before first immunisation to mouse, just exempts from rear 2w, 4w, 6w, 8w and weigh to mouse, and record weight number According to until off-test.

Before just exempting from, just exempt from rear 2w, 4w, 6w, 8w and weigh to test mice, record changes of weight rule, find mouse Growth from 3 week old to 5 week old (i.e. after initial immunity in 2 weeks) is most fast, and weightening later gradually slows down, this and Kunming mouse growth are sent out The weightening rule for educating the phase is consistent (scape will loyalty etc. 1996).To vaccine, dosage and add respectively and exempt from number and do one-way analysis of variance, show Show that different vaccine immunities have an impact female rat weightening, and different dosage groups and weight differences that are different plus exempting from time array are unknown It is aobvious.It is wherein as shown in table 3 to the one-way analysis of variance of different vaccines, it can be clearly seen that pEGS/2SS immune groups, pEGS/ The first starting weight of 2SS-tpa immune groups and control group mice is basically identical, just exempts from rear 4w (i.e. before second of booster immunization) pEGS/ There were significant differences, but pEGS/2SS-tpa compared with control group for the weightening of the female rat of 2SS immune groups and pEGS/2SS-tpa immune groups The female rat of immune group increases weight without significant difference compared with pEGS/2SS immune groups, although the weightening of pEGS/2SS-tpa immune groups is omited Higher than pEGS/2SS immune groups；Just exempt from the rear 4-8 weeks female rat speed of growth to slow down, it is seen that the female rat of pEGS/2SS-tpa immune groups increases Though tuple value is numerically still slightly above pEGS/2SS immune groups and control group without the significance difference opposite sex；

By just exempt from Shi Qizhi it is finally immune after 2w stop, the female rat growth period total augment weights of different vaccine groups difference, i.e., The weightening of the female rat of pEGS/2SS-tpa immune groups and pEGS/2SS immune groups is all remarkably higher than control group, but pEGS/2SS-tpa exempts from Difference is not notable between epidemic disease group and pEGS/2SS immune groups.The growth traits of mouse is weighed with the body weight gains of mouse growth phase, is said Bright vaccine pEGS/2SS-tpa and pEGS/2SS can improve the growth rate of female mice, and vaccine pEGS/2SS-tpa is to female mice Growth-promoting effect is slightly better than vaccine pEGS/2SS.

The influence that the different vaccine immunities of table 3 increase weight to female rat

Note：The postposition letter of any two number is identical in same row is considered as the not notable P of difference>0.05, it is on the contrary then be considered as The not notable P of difference<0.05.

4. the immune influence to MOUSE REPRODUCTION character

4.1 mouse litter sizes and newborn mouse weightening

When 11 week old of mouse (i.e.) launches a public mouse per cage after the immune 2w of last time, mates, next day observation female rat the moon Road bolt is checked whether and is successfully bred.Hereafter the pregnancy status of female rat is paid attention in 21d, records female rat pregnancy cycle, litter size in detail And newborn mouse birth weight and weaning weight (i.e. 3 week old of newborn mouse).

It breeds in 11 week old of female rat, gestational period 20-23d records female rat nest litter size, female between different vaccine processing groups Mouse litter size difference is not notable (table 4).But we still can be seen that embodies superiority, pEGS/ in the number of mouse 2SS-tpa vaccine experiments group is more 1 higher than pEGS/2SS growth hormone release inhibiting hormone positive controls, and pEGFP-N1 up to 1.4 is organized than feminine gender ≈ 2.Illustrate that pEGS/2SS-tpa vaccines still have certain superiority on the reproductive performance of female rat.

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

Note：The postposition letter of any two number is identical in same row is considered as P>0.05, it is on the contrary then be considered as P<0.05.

Nest increases weight during recording newborn mouse lactation, nursing period 21d, and the lactation of female rat is weighed with nest weightening during newborn mouse lactation Shape.The nest weightening no significant difference of the newborn mouse nest weightening of different disposal group, pEGFP-N1 injections group and blank control group is made a general survey of, There were significant differences for the weightening of newborn mouse nest between remaining processing group group.Then to vaccine, immunizing dose and add respectively and exempt from 3 factors of number and carry out Single factor analysis：Different vaccines show the one-way analysis of variance (table 4 (a)) of newborn mouse weaning weight, be immunized pEGS/2SS and Weaning weight dramatically increases after pEGS/2SS-tpa, and the weaning weight after immune pEGS/2SS-tpa is significantly higher than pEGS/2SS and is immunized Group illustrates that the milk performance of female rat, wherein pEGS/2SS-tpa can be promoted after immune pEGS/2SS and pEGS/2SS-tpa really Rush lactogenic result it is more preferable；Various dose shows that high dose is immunized to the one-way analysis of variance (table 4 (b)) of newborn mouse weaning weight Weaning weight be immunized higher than middle dosage but difference is not notable, there were significant differences with low dosage for high dose, middle dosage, illustrates high agent Amount (200 μ g) is immune can to remarkably promote lactation；Difference, which adds, exempts from one-way analysis of variance (table 4 (c)) of the number to newborn mouse weaning weight It has been shown that, add exempt from 1 time with plus exempt from 2 times weaning weight difference it is not notable, but be all remarkably higher than plus exempt from 3 times, illustrate the rush for exempting from 1 time is added to secrete Newborn effect is preferable.In short, different vaccines, immune time and immunizing dose make a significant impact newborn mouse weaning weight, it is contemplated that Newborn mouse weaning weight is mainly influenced by female rat milking capacity；2 pEGS/2SS-tpa are immunized to female mice using high dose (200 μ g) The facilitation effect of lactation is best.

Influence of table 4 (a) the difference vaccine immunity to newborn mouse weaning weight

Influence of table 4 (b) immunizing dose to newborn mouse weaning weight

Influence of table 4 (c) immune time to newborn mouse weaning weight

Note：The postposition letter of any two number is identical in same row is considered as the not notable P of difference>0.05, it is on the contrary then be considered as The not notable P of difference<0.05.

5th, the transcription detection of RT-PCR testing goals gene

5.1 total tissue RNAs are extracted

After immune 5d every group randomly select 1 mouse acquisition heart, liver, spleen, lung, kidney, injection site muscle, small intestine, stomach, Brain, pancreas extract tissue RNA with Trizol methods, and detailed step is as follows：

(1) liquid nitrogen grinding：Tissue block is directly placed into mortar, adds in a small amount of liquid nitrogen, rapid to grind, and treats that tissue softens, then Add a small amount of liquid nitrogen, then grind, so three times；

(2) it is homogenized：Tissue sample adds in Trizol by 50-100mg/mL Trizol, is fully homogenized about with electric homogenizer Need 1-2min；

(3) after tissue adds Trizol, 5min is placed at room temperature for, it is made fully to crack；

(4) 12,000rpm centrifuge 5min, abandon precipitation；

(5) chloroform is added in by 200 μ L chloroforms/mL Trizol, 15min is placed at room temperature for after vibrating mixing；

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

(7) upper strata aqueous phase is drawn, until in another centrifuge tube, isopropanol mixing is added in by 0.5ml isopropanols/mL Trizol, It is placed at room temperature for 5-10min；

(8) 4 DEG C of 12,000g centrifuge 10min, abandon supernatant, RNA is sunken to tube bottom；

(9) 75% ethyl alcohol is added in by 75% ethyl alcohol of 1mL/mL Trizol, mildly vibrates centrifuge tube, suspend precipitation；

(10) 4 DEG C of 8,000g centrifuge 5min, abandon supernatant as possible；

(11) 5-10min is dried or be dried in vacuo to room temperature；

(12) with 50 μ L DEPC H₂O dissolving RNA samples, 55-60 DEG C, 5-10min；

(13) OD values are surveyed and quantifies RNA concentration, between A260/A280 values answer 1.6-1.8, and make agarose gel electrophoresis inspection Whether degrade.

1 each tissue of mouse acquisition is randomly selected after initial immunity 5d for every group, and tissue RNA is extracted with Trizol methods, RNA's For agarose gel electrophoresis detection figure as shown in the figure, tri- band of 28S, 18S and 5S is high-visible, the RNA of mesencephalic tissue has drop Solution trend；Concentration is differed in 300-1000ng/ μ L, OD₂₆₀/OD₂₈₀Value illustrates that the tissue of extraction is total between 1.8~2.0 RNA purity is preferable, available 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 target gene SS

After the cDNA of reverse transcription is made 5 times of dilutions, the endogenous SS of PCR amplification (design of primers is shown in Table 5).

Growth hormone release inhibiting hormone design of primers in 5 cell of table

PCR amplification system is as follows：

Reaction condition is 95 DEG C of pre-degeneration 3min, 95 DEG C of denaturation 30s, 60 DEG C of annealing, 30,72 DEG C of extension 15s, expands 35 After cycle, 72 DEG C of 5min, 15 DEG C of 2min.Amplified production is detected through agarose gel electrophoresis, and whether verification growth hormone release inhibiting hormone is small Normal transcription in mouse body.

Each tissue RNA of extraction obtains cDNA, then respectively using each tissue cDNA as template amplification growth hormone release inhibiting hormone through reverse transcription, It was found that each group of the central nervous system (such as brain) and digestive system (such as stomach, intestines) in addition to endogenous growth chalone normal distribution Other than knitting, also there is faint band in the tissues such as heart, lung, kidney and injection site muscle.Illustrate in injection growth hormone release inhibiting hormone After gene vaccine, the growth hormone release inhibiting hormone segment in plasmid can be transcribed in each histoorgan of mouse.(Fig. 9)

6. mouse blood and tissue sampling

6.1 mouse bloods acquire

Before immune and after the test, with docking blood collection method acquisition mouse blood, 20 μ L EDTA have been collected in it and have resisted In the 1.5mL EP pipes of solidifying agent/pipe, 3,000r/min centrifugation 10min carefully draw upper plasma, and packing about 200 μ L/ pipes are put It is saved backup in -20 DEG C.

6.2 mouse tissues acquire

Vertebra puts to death mouse, the heart of every group of anatomical acquisition some animals, liver, spleen, lung, kidney, injection part after the test Position muscle, small intestine, stomach, brain, ovary (ovary is weighed), are placed in 4% paraformaldehyde fixer, each group of another part mouse It is collected in centrifuge tube after knitting organ acquisition, -80 DEG C of preservations.

7. associated hormone and antibody test

The detection of 7.1 growth hormone, prolactin and growth hormone release inhibiting hormone

The content of hormone GH, PRL and SS in the mice plasma of different disposal are detected with one step Sandwich ELISA of double antibody, It is as follows：

(1) lath needed for being taken out from the aluminium foil bag after equilibrium at room temperature 20min, remaining lath valve bag sealing put back to 4 ℃；

(2) standard sample wells and sample aperture, standard sample wells is set respectively to add the 50 μ L of standard items of various concentration；

(3) sample aperture first adds 10 μ L of sample to be tested, then adds 40 μ L of Sample dilution；Blank well is not added with；

(4) in addition to blank well, the detection that horseradish peroxidase (HRP) marks is added in standard sample wells and sample aperture per hole 100 μ L of antibody seal reacting hole with sealing plate film, and 37 DEG C of insulating boxs incubate 60min；

(5) liquid is discarded, is patted dry on blotting paper, cleaning solution is filled it up with per hole, stands 1min, gets rid of cleaning solution, on blotting paper It pats dry, so repeats board-washing 5 times；

(6) substrate A, each 50 μ L of B are added in per hole, 37 DEG C are protected from light incubation 15min；

(7) it is added in terminate liquid 50 μ L, 15min per hole, the OD values in each hole is measured at 450nm wavelength；

(8) standard curve is drawn, (calculated value is multiplied by 5 according to the concentration of GH, PRL and SS in standard curve calculating sample Ultimate density value).

7.1.1 the secretion level of growth hormone

Double-antibody sandwich elisa detection GH calibration curve formula be：Y=-0.30+8.94X-14.86X²+18.49X³- 6.08X⁴, wherein Y is GH concentration (ng/mL), and X is OD values at 450nm (see annex).The OD that will be measured₄₅₀It is bent that value substitutes into this standard In line formula, the GH concentration in mice plasma is calculated.T check analyses are done between any two to different disposal group and control group, It is variant.Therefore, to vaccine, immunizing dose and add respectively and exempt from 3 factors of number and carry out single factor analysis, find different immunizing agents Amount and immune time have no significant effect the concentration of GH, but GH content differences are notable in mouse blood after different vaccine immunities, Compared with unloaded injection group, the secretory volume of mouse GH is promoted after immune pEGS/2SS and pEGS/2SS-tpa, but flesh Mouse GH is improved not notable after meat injection pEGS/2SS, and mouse GH contents are significantly higher than injection after injecting pEGS/2SS-tpa Mouse (the P of pEGS/2SS<0.05).Illustrate that somatostatin DNA vaccine pEGS/2SS can increase the release of GH after being immunized, insert This effect is more obvious after entering tpa signal peptides, although not showed in the growth traits of mouse.(Figure 10)

7.1.2 the secretion level of prolactin

Double-antibody sandwich elisa detection PRL calibration curve formula be：Y=-1.20+22.96X-30.46X²+ 70.88X³-32.56X⁴, wherein Y is PRL concentration (ng/mL), and X is OD values at 450nm (see annex).The OD that will be measured₄₅₀It is worth generation Enter in this calibration curve formula, the PRL concentration in the mice plasma obtained does the T check analyses between group two-by-two, shows different groups Between PRL concentration it is variant.Therefore, to vaccine, immunizing dose and add and exempt from 3 factors of number and carry out single factor analysis respectively, send out The existing influence of different immunizing doses and immune time to PRL concentration is not notable, but PRL in mouse blood is immunized after different vaccines The significant difference of content, compared with unloaded immune group, the mouse PRL contents of pEGS/2SS and pEGS/2SS-tpa immune groups are equal It is improved；And mouse PRL contents significantly raising compared with injecting the mouse of pEGS/2SS after intramuscular injection pEGS/2SS-tpa (P<0.05), the difference extremely significantly (P compared with control group mice<0.01).Illustrate to increase after being immunized by somatostatin gene Add the secretion of mouse PRL, and this facilitation is especially apparent after tpa signal peptides are inserted into, this result and female rat lactation Character result is corresponding, and can fully explain the phenomenon that immune rear female rat milking capacity improves.(Figure 11)

7.1.3 the secretion level of growth hormone release inhibiting hormone

Double-antibody sandwich elisa detection SS calibration curve formula be：Y=-1.31+32.39X-56.62X²+86.87X³- 32.52X⁴, wherein Y is SS concentration (pg/mL), and X is OD values at 450nm (see annex).OD values at the 450nm of sample are updated to In above-mentioned standard curve equation, the concentration of SS in mice plasma is calculated.Are done by T two-by-two and is examined for different disposal group and control group and is divided Analysis, SS contents no significant difference in mouse blood between finding different groups.Further to vaccine, immunizing dose and add exempt from number 3 Factor individually carries out single factor analysis, finds different vaccines, different immunizing doses and different immune times not to the concentration of SS It makes a significant impact.Influence of the different vaccine immunities to SS contents in mouse blood is immunized as a result, compared with unloaded injection group The secretory volume of mouse SS is declined after pEGS/2SS, but is declined not notable；Mouse SS content backs after immune pEGS/2SS-tpa And higher than the mouse of control group, although difference is not also notable.Illustrate somatostatin DNA vaccine pEGS/2SS and pEGS/2SS- After tpa is immunized, the SS in mouse blood will not be significantly affected.(Figure 12)

The detection of 7.2 somatostatin antibodies

With the generation situation of SS antibody in indirect ELISA method detection immunized mice body, it is as follows：

(1) 96 hole elisa Plates coating standard growth chalone antigen (Sigma-Aldrich, S9129) 100ng/100 μ per hole L, 4 DEG C of overnight incubations；

(2) reaction solution is abandoned, PBST is washed 5 times, 300 μ L/ holes, each 1min；

(3) add 200 μ L/ holes of confining liquid (i.e. 1%BSA solution), 37 DEG C of incubation 1h；

(4) reaction solution is abandoned, PBST is washed 5 times, 300 μ L/ holes, each 1min；

(5) add test plasma (1:200 dilutions) 100 μ L/ holes, while negative control hole, non-specific adsorption hole are set (i.e. PBST replaces blood plasma) and zeroing hole, 37 DEG C of incubation 1h；

(6) reaction solution is abandoned, PBST is washed 5 times, 300 μ L/ holes, each 1min；

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

(8) reaction solution is abandoned, PBST is washed 5 times, 300 μ L/ holes, each 1min；

(9) add 150 μ L/ holes of tmb substrate developing solution, be protected from light 25min；

(10) add 2mol/L H₂SO₄50 μ L/ holes of terminate liquid terminate reaction, and each hole is measured at 450nm wavelength in 15min OD values.

According to the concentration and OD of SS antibody standard substances₄₅₀Value draws standard curve, and sample is surveyed OD₄₅₀It is worth substitution standard Curve, multiplied by the SS antibody concentrations for 200 being sample.The SS antibody concentrations of test group with the SS antibody concentrations of negative blood sample into Row standardization correction, to exclude influence of the non-specific adsorption to result.

Calculate the average OD of negative blood sample to be measured₄₅₀It is worth (X_N) and standard deviation (S_N), obtain yin and yang attribute critical value X_N+2S_N。 If the OD of sample to be tested₄₅₀Value >=X_N+2S_N, then it is judged to the positive；Conversely, then it is judged to feminine gender.Every group of positive mice number is recorded, And count the antibody positive rate of different tests grouping.

7.2.1 somatostatin antibody positive rate

According to the OD of positive blood sample₄₅₀Value >=X_N+2S_N, judge the mouse number of every group of generation positive antibody, calculate each experiment The antibody positive rate of group.As shown in Table 6, pEGS/2SS immune groups and pEGS/2SS-tpa immune groups, which have, generates the small of SS antibody Mouse, and the antibody positive rate of pEGS/2SS-tpa immune groups is higher than pEGS/2SS immune groups；Under different immunizing dose processing, high agent The antibody positive rate of amount group (200 μ g) is higher than middle low dose group；To difference plus exempt from number and analyze, add and exempt from the antibody sun of 1 time Property rate is slightly higher.Therefore, for antibody positive rate, using the immune of immune 2 pEGS/2SS-tpa of high dose (200 μ g) Best results.

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

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

Table 6 (c) is different plus exempts from the antibody positive rate of time array

Claims (4)

1. a kind of genetic engineering bacterium, it is characterised in that：Escherichia coli Escherichia coli DH5 α (pEGS/2SS-tpa), Deposit number：CCTCC NO:M2015105.

2. the plasmid pEGS/2SS-tpa that bacterial strain described in claim 1 includes.

3. plasmid described in bacterial strain described in claim 1 or claim 2 is preparing while is promoting mouse growth, breeding and lactation epidemic disease Application in seedling.

4. method that is a kind of while promoting mouse growth and lactation, including 200 μ of plasmid g/ times described in claim 2 is immune Mouse 2 times.