CN101407781A - Inhibin DNA vaccine capable of improving animal fertility, and preparation and use thereof - Google Patents

Abstract

The invention belongs to the preparation technical field of animal vaccines, and particularly relates to a pig cholera salmonella inhibin DNA vaccine which does not contain the residue of antibiotics genes and can improve the propagating fecundity of the animals as well as the preparation and applications thereof. A pig inhibin Alpha (1-32) gene and a hepatitis b surface antigen fusion gene are inserted into a plasmid vector pVAX1 which is lack of a kanamycin resistance gene for obtaining the eukaryotic expression plasmid pXAIS of the inhibin; and the eukaryotic expression plasmid pXAIS of the inhibin is transfected into the pig cholera salmonella C500 for constructing the pig cholera salmonella inhibin DNA vaccine. Compared with the prior art, the vaccine does not contain the residue of antibiotics genes and has the advantages of high moving efficiency, simple immune method and good immune effect. The vector bacteria self can be used as immune adjuvant which can simultaneously activate mucosal immunity and general immunity, and can obtain the immunity to the vector bacteria, and the like.

Description

A kind of inhibin DNA vaccine and preparation and application that improves animal reproduction power

Technical field

The present invention relates to the structure and the application of animal inhibin DNA vaccine, particularly relate to Salmonella choleraesuls inhibin DNA vaccine and the preparation method and the application of the residual raising animal reproduction power of a kind of antibiotic-free gene.

Technical background

Statin (inhibin-INH) is by the sugar-protein hormone of ovarian secretion, can suppress the secretion of hypophysis follitropin (FSH), has vital role for the adjusting of follicular development.Many experimental results show that, use INH active or passive immunization animal, all can promote follicular development, induce many ovulations, improve litter size, and can induce twin (the Medan MS of monotocous animal, Deng .The effect of active immunization againstinhibin on gonadotropin secretions and follicular dynamics during the estrous cycle in cows.J Reprod Dev.2006,52 (1): 107-13; .Effect of active immunization against inhibin onhormonal concentrations and semen characteristics in Shiba bucks.Theriogenology.2006 such as Medan MS, 65 (4): 691-702; .Passive immuno-neutralization of endogenous inhibin increasesovulation rate in miniature Shiba goats.J Reprod Dev.2004 such as Medan MS, 50 (6): 705-10).Well-known immunogen preparing is very difficult: for natural immunogen, still have the problem of separation and purification difficulty at present; The statin immunogen of synthetic or reorganization exists workload big, the difficult problem that cost is high.

For solving these difficulties, the applicant was from beginning in 1998, begun the research of inhibin DNA vaccine, 7 kinds of INH eukaryon expression plasmids have successively been made up, and the immune effect and the security (Jiang Xunping of various vaccines have been compared, Deng. Reproductive Endocrinology is to the influence of mouse propagation. Chinese animal doctor's journal, 2002,22 (4): 368-9; Cogongrass reaches dried, etc. statin α (1-32) genetic immunization rat follicular development and reproductive hormone is influenced .. Scientia Agricultura Sinica, 2003,36 (12): 1554-9; Zhang Dekun, etc. Reproductive Endocrinology is induced the twin research of single tire sheep. China Agricultural University's journal, 2004,9 (4): the 40-5 reference).

But, because in the former statin fusion expression plasmid (pCIS) that makes up: 1. contain resistant genes such as kanamycin gene or penbritin gene; 2. the plasmid DNA after cloning must be extracted, purifying just can be used for immunity, and the cost of purification is very high; 3. immunization route is confined to intramuscular injection, and it is not too convenient to use, and can only be used for experimental study, and can not be used for product development.At the common problem that exists in Gonadostatin (INH) genetic immunization and the research of other gene vaccines, this project utilizes aspartic acid beta galactose dehydrogenase gene (asd)-attenuation salmonella carrier-host's balanced lethal system (hereinafter to be referred as balanced lethal system) to replace the resistant gene that has now in the statin eukaryon expression plasmid, makes up the eukaryon expression plasmid that does not contain resistant gene and obtains a kind of novel inhibin DNA vaccine.The present invention reduces production costs for the security and the effect that improve the Gonadostatin genetic immunization, simplifies service routine etc., and is all significant.

Summary of the invention

Task of the present invention is to overcome the defective of prior art, develops a kind of residual Salmonella choleraesuls inhibin DNA vaccine of antibiotic-free gene that improves animal reproduction power.Purpose of the present invention also comprises the eukaryon expression plasmid that obtains a kind of non-resistant gene and above-mentioned dna vaccination is applied to improve its reproductivity, wherein comprise litter size, the raising of key technical index such as lactation power and newborn mouse surviving rate on the animal (mouse).

The present invention is achieved in that

The applicant is by preparation, obtained Salmonella choleraesuls (Salmonellaenterica sv.Choleraesuis) C500/pXAIS that a strain contains statin eukaryon expression plasmid pXAIS, this bacterial strain is delivered Chinese typical culture collection center (CCTCC) preservation in the Wuhan University of Wuhan City, Hubei Province on October 30th, 2008, and its preserving number is CCTCC NO:M208195.

The applicant utilizes the bacterial strain direct production of above-mentioned preservation to go out the Salmonella choleraesuls inhibin DNA vaccine.

In technical scheme of the present invention, the expression plasmid pXAIS that expresses pig statin fusion gene IS makes up like this: at first plasmid pVAX1 is digested with the pVUII restriction endonuclease, the nucleotide sequence that obtains aspartic acid beta galactose dehydrogenase gene that increases from plasmid PYA3493 is inserted in the pVAX1 plasmid, the eukaryon expression plasmid pVAX-asd that is not contained kantlex, enzyme among the statin fusion expression plasmid PCIS is cut back to close the fusion gene that obtains hepatitis B surface antigen and statin c (1-32), this fusion gene is inserted among the eukaryon expression plasmid pVAX-asd, plain eukaryon expression plasmid pXAIS is inhibited again.

The applicant sets up a kind of preparation method who improves the Salmonella choleraesuls inhibin DNA vaccine of animal reproduction power, and its step is as follows:

(1) plasmid pVAX1 is digested with the pVUII restriction endonuclease, the nucleotide sequence that obtains aspartic acid beta galactose dehydrogenase gene that increases from plasmid PYA3493 is inserted in the pVAX1 plasmid, is not contained the eukaryon expression plasmid pVAX-asd of kantlex;

(2) enzyme among the statin fusion expression plasmid PCIS is cut back to close the gene that obtains hepatitis B surface antigen and statin α (1-32), this fusion gene is inserted among the eukaryon expression plasmid pVAX-asd, plain eukaryon expression plasmid pXAIS is inhibited;

(3) transform Salmonella choleraesuls C500 with statin eukaryon expression plasmid pXAIS, obtaining preserving number is CCTCC NO:M208195 Salmonella choleraesuls (Salmonella enterica sv.Choleraesuis) C500/pXAIS;

(4) Salmonella choleraesuls of enlarged culturing step (3) obtain the Salmonella choleraesuls inhibin DNA vaccine.

More detailed technical scheme is seen " embodiment ".

Salmonella choleraesuls inhibin DNA vaccine of the present invention does not contain kanamycin gene, has better security than existing inhibin DNA vaccine on using, and its preparation technology is simple, does not need through preparation processes such as purifying.

Description of drawings

Fig. 1: be the amplification collection of illustrative plates of the gene fragment of the present invention's aspartic acid beta galactose dehydrogenase gene (asd) of cloning, among the figure: the DNA marker of M:250bp; 1-4: the purpose fragment that to be template with PYA3493 obtain with the primers amplification of asd; 5: the result that to be template with water obtain with the primers amplification of asd.

Fig. 2: be that eukaryon expression plasmid pVAX-asd enzyme is cut the evaluation collection of illustrative plates, among the figure: the DNA marker of M:250bp; 1: eukaryon expression plasmid pVAX-asd carries out the result that enzyme is cut by pVUII.

Fig. 3: the structure collection of illustrative plates that is statin eukaryon expression plasmid pXAIS of the present invention.

Fig. 4: be the collection of illustrative plates after statin fusion gene IS reclaims: lane1-5: carry out result after the double digestion recovery with NheI and HindIII from statin fusion expression plasmid PCIS; The DNA marker of M:250bp.

Fig. 5: be the DNA marker that statin eukaryon expression plasmid pXAIS enzyme is cut evaluation collection of illustrative plates: M:250bp; The result that 1:pXAIS obtains by NheI and HindIII double digestion.

Fig. 6: add microbiotic to being the influence of the inhibin DNA vaccine of carrier with Salmonella choleraesuls C500: the figure left side is for being the inhibin DNA vaccine of carrier what add that the antibiotic LB solid medium of kantlex cultivates with Salmonella choleraesuls C500; The figure right side is for being the inhibin DNA vaccine of carrier what do not add that any antibiotic LB solid medium cultivates with Salmonella choleraesuls C500.

Fig. 7: be statin eukaryon expression plasmid pXAIS carries out transcriptional level by RT-PCR after the PK15 transit cell dyes 48 hours detected result: swimming lane 1: the PK15 cell of plasmid-free transfection; Swimming lane 2: RT-PCR result after the eukaryon expression plasmid pVAX-asd transfection; Swimming lane 3-5: the RT-PCR result after the statin eukaryon expression plasmid pXAIS transfection.

Fig. 8: be the protein expression indirect immunofluorescence detected result of statin eukaryon expression plasmid pXAIS after the PK15 transit cell dyes 48 hours: Fig. 8 A: statin eukaryon expression plasmid pXAIS expression in the PK15 cell detects fluorescence, shows that this fusion rotein has immunocompetence; Fig. 8 B: eukaryon expression plasmid pVAX-asd expression in the PK15 cell detects no fluorescence; Fig. 8 C: the plasmid-free cells transfected detects and is no fluorescence (blank).

Fig. 9: the anti-statin IgG antibody titers of different time detects after the Kunming mouse immunity: the IgG antibody horizontal of immunity back two all test group is apparently higher than other control groups, and antibody horizontal does not have significant difference behind booster immunization.

Figure 10: the anti-statin IgG somatotype antibody horizontal that is different time sections after the Kunming mouse immunity detects: the IgG1 of immunity back two all test group and the antibody horizontal of IgG2a all are higher than other immune group, and the OD value of IgG2a is higher than IgG1, and at the OD value indifference almost of 4,6 weeks, two kinds of somatotypes.

Embodiment

The structure of embodiment 1 eukaryon expression plasmid pVAX-asd

1, (asd) gene clone of aspartic acid beta galactose dehydrogenase gene and sequential analysis

Adopt the Primer5.0 primer-design software, the Salmonellas asd gene order according to GenBank (AF015781) login has designed primer to (forward primer: P1 ATGCAGCTGGCACATCTCTTTGCAGG; Reverse primer: P2 TTACAGCTGCTACGCCAACTGGCGCA), with PYA3493 plasmid (Kang HY etc., Immune responses to recombinant pneumococcal PspA antigen delivered by liveattenuated Salmonella enterica serovar typhimurium vaccine.Infect Immun, 2002,70 (4): 1739-49.) be template, by the asd gene fragment of PCR method clone Salmonella typhimurium.The asd dna homolog of Salmonella typhimurium and Salmonella choleraesuls is 100%, and its cDNA open reading frame is 1107bp, 368 amino acid of encoding.With the PYA3493 plasmid is template, to the row amplification to the greatest extent of asd gene fragment; Reaction cumulative volume 20 μ L wherein contain:

MgCl ₂(available from Toyobo company) 2 μ L

Each 0.5 μ L of primer (it is synthetic that worker's biotechnology company limited is given birth in 10pmol/ μ L Shanghai)

DNTP Mixture (each 10mM is available from Toyobo company) 1 μ L

Taq enzyme (40U/ μ L is available from Toyobo company) 0.5 μ L

Template 0.5 μ L

10 * Taq damping fluid (available from Toyobo company), 1 μ L

Distilled water makes reaction cumulative volume 20 μ L.

Carry out PCR reaction by following condition: 94 ℃ of sex change 4min, then 94 ℃ of sex change 1min, 60 ℃ of annealing 45sec, 72 ℃ extend 1min, 35 circulations are extended 7min for back 72 ℃ again.

At last, with PCR product 1.2% agarose gel electrophoresis, as molecular weight standard, observe electrophoresis result with DL2000.(see figure 1)

2, enzyme is cut and is connected

With PCR product purification test kit (available from TIANGEN Biotech (Beijing) Co., Ltd.), reference reagent box working instructions reclaim pcr amplification product and purifying.PCR product behind the purifying and plasmid vector pVAX carry out enzyme with the pVUII restriction endonuclease respectively and cut.

Enzyme is cut system: 10 * H damping fluid (available from Toyobo company), 5 μ L

Asd (or pVAX) 20 μ L

PVUII (available from Toyobo company) is 2 μ L

Distilled water is to cumulative volume 50 μ L,

37 ℃, reaction 3-5h, enzyme cut back the asd purpose fragment of low melting point agarose gel electrophoresis Separation and Recovery 1107bp and the pVAX fragment of 2640bp.

Carry out ligation by following system then, connect purpose fragment and carrier, linked system is:

10 * ligase damping fluid (available from Toyobo company), 2 μ L

asd?6μL

pVAX?2μL

T4DNA ligase enzyme (available from Toyobo company) 1 μ L

Distilled water is to cumulative volume 20 μ L, and 16 ℃ are spent the night.Connect product-20 ℃ preservation, standby.

3, preparation of competence bacterium and recombinant plasmid transformed competence colibacillus bacterium

1) adopts conventional CaCl ₂Method (J. Sa nurse Brooker, D.W. La Saier, molecular cloning experiment guide [M]. the third edition, Beijing: Science Press, 2002) preparation fresh competence bacteria Escherichia coli 6097 (Nakayama K etc., Construction of an Asd+expression-cloning vector:stable maintenance and high level expression of cloned genes in aSalmonella vaccine strain.Bio/Technology 1988.6:693-697).Concrete steps are as follows:

(1) (1L LB solid medium is formed: 10g sodium-chlor, 5g yeast extract, 10g Tryptones from the LB flat board that contains diaminopimelic acid DAP (the DAP final concentration is 50 μ g/mL), the 15g agar powder, pH 7.0) the single bacterium colony of Escherichiacoli of the new activatory of picking disappearance asd gene, (1L LB liquid nutrient medium is formed: 10g sodium-chlor, 5g yeast extract, 10g Tryptones to be inoculated in (the DAP final concentration is 50 μ g/mL) in the 10mL LB liquid nutrient medium, pH 7.0), about 37 ℃ of following shaking culture 12h, be OD until the logarithmic growth later stage ₆₀₀＞0.5 o'clock.This bacteria suspension is inoculated in the 100mL LB liquid nutrient medium 37 ℃ of shaking culture 2.5h to OD with 1: 100 (volume ratio) ₆₀₀About=0.5.

(2) nutrient solution changes in the centrifuge tube, places 10min on ice, then in 4 ℃ of centrifugal 10min of following 3000g.

(3) remove supernatant, use the CaCl of the 0.05mol/L of precooling ₂Solution 10mL is suspension cell gently, place 30min on ice after, 4 ℃ of centrifugal 10min of following 3000g.

(4) supernatant discarded adds the CaCl that the 4mL precooling contains the 0.05mol/L of 15% glycerine ₂Solution, suspension cell is placed 10min on ice gently, the competent cell suspension.

(5) competent cell is distributed into the aliquot of 100 μ L, can be used at once transforming, perhaps it is stored under-70 ℃, standby.

2) with the heat-shocked method (J. Sa nurse Brooker, D.W. La Saier, molecular cloning experiment guide [M]. the third edition, Beijing: Science Press, 2002) will connect product transformed competence colibacillus bacterium DH5 α, step is as follows:

(1) from-70 ℃ of refrigerators, gets 100 μ L competent cell suspensions, place on ice and melt.

(2) add to connect product D NA solution, shake up gently, place 30min on ice after.

Thermal shock 90s in (3) 42 ℃ of water-baths places cooled on ice 3min rapidly behind the thermal shock.

(4) Xiang Guanzhong adds 400 μ L LB liquid nutrient mediums, and 37 ℃ of shaking culture 1h behind the mixing make the bacterium state that restore normal growth, and the ammonia benzyl antibiotics resistance gene of expressible dna coding.

(5) get 100 μ l after above-mentioned bacterium liquid is shaken up and coat on the screening flat board that does not contain DAP (the DAP final concentration is 50 μ g/mL), face up and place half an hour, treat that bacterium liquid is absorbed the back by substratum fully and is inverted culture dish, cultivate 16-24h for 37 ℃.

4, the screening of positive colony, evaluation and order-checking

Picking positive colony from the flat board of above-mentioned (5), being inoculated in the LB liquid nutrient medium that does not contain DAP cultivates, with plasmid small volume of reagent box (available from TIANGEN Biotech (Beijing) Co., Ltd.) extracting plasmid (reference reagent box working instructions), the pVUII enzyme is cut, the enzyme system of cutting is: 10 * H damping fluid (available from Toyobo company), 1.6 μ L, the recombinant plasmid 9 μ L that extract, pVUII is 0.8 μ L, adds ddH ₂O is to cumulative volume 16 μ L, and 37 ℃, reaction 2-3h observes enzyme and cuts the result, and electrophoretic band conforms to the purpose stripe size, serves extra large Ying Jun Bioisystech Co., Ltd and checks order.Obtain eukaryon expression plasmid pVAX-asd.(see figure 2)

The structure (see figure 3) of embodiment 2 statin eukaryon expression plasmid pXAIS

1, eukaryon expression plasmid pVAX-asd extraction and purification

The single bacterium colony of the new activatory eukaryon expression plasmid of picking pVAX-asd from the LB flat board, be seeded in the 10mL LB liquid nutrient medium, 37 ℃, 240rpm overnight incubation, 1: 250 by volume dilution proportion is in proper volume LB, continue to cultivate 12h, centrifugal collection thalline, extracting and purifying eukaryon expression plasmid pVAX-asd.

2, statin fusion gene produced in fragments

Statin fusion expression plasmid PCIS (the Han L etc. that adopt the applicant to make up in the past, Development and evaluation of anovel DNA vaccine expressing inhibin alpha (1-32) fragment for improving the fertility in ratsand sheep.Animal reproduction science, 2008,109 (1-4): 251-65) can obtain statin and the hepatitis B surface antigen (Davis etc. of 858bp by NheI and HindIII double digestion, DNA-based immunization for Hepatitis B inducescontinuous secretion 269 of antigen and high levels of circulating antibody, Hum.Mol.Genet.1993 (2): 1847-1851) fusion gene fragment IS (see figure 4).

3, the connection of statin eukaryon expression plasmid

Statin fusion gene fragment IS and eukaryon expression plasmid pVAX-asd that purifying, recovery are obtained carry out NheI I and HindIII double digestion by following system respectively.Reaction system is: 10 * H damping fluid (available from Toyobo company), 5 μ L, IS 15 μ L (or eukaryon expression plasmid pVAX-asd 20 μ L), NheI I and HindIII are respectively 1.5 μ L, add distilled water to cumulative volume 50 μ L, 37 ℃, reaction 3-5h, after enzyme is cut, with the statin fusion gene fragment IS of low melting point agarose gel electrophoresis Separation and Recovery 858bp and the big fragment of eukaryon expression plasmid pVAX-asd of 3780bp.

Carry out ligation by following system then: 10 * connect damping fluid (available from Toyobo company) 2 μ L, the big fragment 3 μ L of IS 6 μ L, eukaryon expression plasmid pVAX-asd, T4 dna ligase (available from Toyobo company) 1 μ L, add distilled water to cumulative volume 20 μ L, 4 ℃ are spent the night.

4, with reference to general glycerine method (J. Sa nurse Brooker, D.W. La Saier, molecular cloning experiment guide [M]. the third edition, Beijing: Science Press, 2002) the fresh competence Salmonella choleraesuls C500 of preparation, concrete steps are as follows:

1) Salmonella choleraesuls C500 is placed on the LB substratum, 37 ℃ of following incubated overnight;

2) incubated overnight liquid is inoculated the 50mlLB liquid nutrient medium by 1: 100 volume ratio;

3) 37 ℃ of following 200r/min are cultured to OD ₆₀₀Reach 0.3-0.4, generally need 2.5-3 hour;

4) cell is under 4 ℃ of 5000g centrifugal 15 minutes, abandons supernatant liquor;

5) with the 50ml deionized water of the precooling thalline that suspends gently;

6) repeat step 2 time, the deionized water volume is respectively 25ml and 12.5ml;

7) cell is under 4 ℃ of 5000g centrifugal 15 minutes, abandons supernatant liquor and collects thalline;

8) 10% glycerine after that sterilize with 50ml, ice-cold is suspension cell gently;

9) cell is under 4 ℃ of 5000g centrifugal 15 minutes, abandons supernatant liquor;

10) according to top step triplicate, 10% glycerine volume is respectively 50,25,12.5ml;

11) at last in 4 ℃ of centrifugal 15min harvested cells of 7500r/min;

11) 10% glycerine of adding 0.25ml ice precooling is resuspended in the sedimentation cell;

12) with cell by the 40 μ l equal portions Eppendorf tube of packing into, standby in-80 ℃ of preservations.

5, will connect the competent cell that the product electricity is transformed into Salmonella choleraesuls, method is as follows:

1) the electroreception attitude cell that thaws was on ice cultivated about 5 minutes on ice;

2) add 10 μ l and connect product, cultivated on ice about 5 minutes;

3) shift mixture to cooled electric revolving cup;

4) electric revolving cup is carried out pulse, the 4 seconds time (pulse resistance 200 Ω, electric capacity 25 μ Fd, 2.0 kilovolts of voltages);

5) add SOC substratum (the peptone 20g of 1000 μ l immediately, yeast extract 5g, sodium-chlor 0.5g, 1mol/L Repone K 2.5ml is dissolved in the sterilization of 1L water mesohigh, just behind the substratum cool to room temperature, went out the 1mol/L magnesium chloride of bacterium except adding 10ml, the 1mol/L glucose (18g glucose is dissolved in the enough water, and water is supplied 100ml again, with the membrane filtration degerming of 0.22um) that adds the 20ml sterilization again.

6) cultivate 1 hour down to restore for 37 ℃;

7) transitional cell is cultivated to corresponding LB solid medium.

6, statin eukaryon expression plasmid (pXAIS) and be the screening and the evaluation of the inhibin DNA vaccine of carrier with Salmonella choleraesuls C500

The picking positive colony, be inoculated in the LB liquid culture, extract plasmid DNA, carry out enzyme and cut evaluation, identify recombinant plasmid with NheI and HindIII, double digestion respectively, the enzyme system of cutting is: recombinant plasmid 9 μ L, NheI and the HindIII of 10 * H buffer, 2 μ L, extraction are respectively 0.5 μ L, add ddH ₂O is to cumulative volume 20 μ L, 37 ℃, reaction 3-5h, 1.2% agarose electrophoresis detects, the electrophoretic band (see figure 5) that conforms to the purpose stripe size, serve extra large Ying Jun Bioisystech Co., Ltd and check order, obtain identifying correct statin eukaryon expression plasmid pXAIS and be the inhibin DNA vaccine of carrier with Salmonella choleraesuls C500.

7, with Salmonella choleraesuls C500 be the kalamycin resistance detection of the inhibin DNA vaccine of carrier

The Salmonella choleraesuls C500 that Screening and Identification is correct is that the inhibin DNA vaccine of carrier adds and do not add the kantlex cultivation respectively, the result shows, the Salmonella choleraesuls C500 that has added in the LB liquid nutrient medium of kantlex is the not growth of inhibin DNA vaccine of carrier, and the inhibin DNA vaccine that does not add Salmonella choleraesuls C500 in the LB liquid nutrient medium of kantlex and be carrier is the normal growth (see figure 6) then.

Embodiment 4: statin eukaryon expression plasmid pXAIS plasmid is in the detection of external protein expression

1, the detection of transcriptional level behind the statin eukaryon expression plasmid pXAIS transfectional cell:

With plasmid extraction kit (available from TIANGEN Biotech (Beijing) Co., Ltd.) extracting plasmid, treat that PK15 cell (cell of the normal kidney of wild boar) carries out transfection by lipofectamine box (available from Invitrogen company) specification sheets when individual layer grows to 60%-70%.Behind the transfection PK15 cell 48h, with trysinization and collecting cell.According to the mRNA of Trizol (available from Invitrogen company) working instructions extraction cell, reverse transcription obtains cDNA, increases according to the statin primer.

The cDNA that obtains with reverse transcription is a template, with INH primers (INHP1:TGCTGGATATCTGCAGAATTCCCT, INHP2:CTTCTCGAGATCTGTGGCAGTCGG.This primer is that to give birth to worker's biotechnology company limited by Shanghai synthetic) carry out the segmental amplification of INH purpose.Can find a fragment (see figure 7) about 858bp through the detection of 1% agarose electrophoresis, give birth to the order-checking of worker's biotechnology company limited through Shanghai and identify that this fragment is a statin purpose fragment that the fragment total length is 858bp.

2, the detection of statin eukaryon expression plasmid pXAIS vivoexpression

With cell to be detected (transient transfection cell) PBS damping fluid (8g NaCl, 0.2g KCl, 1.44g Na2HPO4 and 0.24g KH2PO4, be dissolved in the 800ml distilled water, pH value to 7.4 with the HCl regulator solution, last adding distil water is settled to 1L and gets final product) wash 3 times, 100% methyl alcohol room temperature is 10min fixedly, PBS damping fluid washing 3 times, with the PBS sealing 30min that contains 10% bovine serum, then successively respectively with mouse statin monoclonal antibody (available from Thermo company, article No. is MS-1863-SO) and each 30min of 37 ℃ of effects of sheep anti mouse two anti-(getting biotechnology company limited available from the Wuhan doctor) of FITC mark, therebetween with the washing of PBS damping fluid, washing 3 times with the PBS damping fluid with two anti-reaction backs, directly under fluorescent microscope microscopy (model Olympus, IX70).

Whether statin eukaryon expression plasmid pXAIS detects plasmid by indirect immunofluorescence method and expresses in eukaryotic cell behind transfection PK15 cell 48h.The result shows that PBS damping fluid and eukaryon expression plasmid pVAX-asd plasmid change then cell and do not find green fluorescence by fluorescent microscope after with the statin antibody test, and statin eukaryon expression plasmid pXAIS cells transfected back discovery green fluorescence (see figure 8) after testing.This result shows that the fusion rotein of expressing behind the statin eukaryon expression plasmid pXAIS transfecting eukaryotic cells has the statin immunocompetence.

3, the ELISA method detects the immunologic competence of cell expression product

Bag is treated the every hole 100 μ L of test sample, uses PH9.6, and 0.05M carbonate bag is cushioned (Na ₂CO ₃1.59 gram, NaHCO ₃2.93 gram, adding distil water is to 1000ml) the statin antigen concentration of liquid dilution is that 100ng/100 μ L bag is made positive control, 4 ℃ are spent the night; Use PBST damping fluid (KH on the automatic washer ₂PO ₄-0.2 gram, Na ₂HPO ₄12H ₂0-2.9 gram, the NaCl-8.0 gram, the KCl-0.2 gram, Tween-200.05%-0.5ml, adding distil water is to 1000ml) wash 6 times, every then hole adds confining liquid (bovine serum albumin-BSA 0.1 gram adds lavation buffer solution to 100ml) 200 μ L, 37 ℃ of reaction 1h; Washing adds mouse statin monoclonal antibody 100 μ L, 37 ℃ of reaction 1h; Washing adds rabbit anti-mouse igg-HRP (1: 5000, available from Wuhan doctor's moral company) 100ul/ hole, reacts 1 hour; Washing adds 150 μ L tmb substrate liquid (0.2M Na ₂HPO ₄(28.4g/L) 25.7ml, 0.1M citric acid (19.2g/L) 24.3ml, adding distil water 50ml is the substrate buffer solution of 100ml; 0.75%TMB promptly takes by weighing 37.5mg and is dissolved in the 5ml dehydrated alcohol; A liquid: 0.75ml TMB 0.75%+6.75mlddH ₂O-7.5ml; B liquid: 7.5ml substrate buffer solution+45ul urea peroxide-7.5ml) colour developing, 37 ℃ were reacted 25 minutes; Add 50 μ L 2mol/L H ₂SO ₄Termination reaction is surveyed OD on microplate reader ₄₅₀Value.

PK15 cell preparation behind the collection statin eukaryon expression plasmid pXAIS transfection 48h is treated test sample, and bag is treated the every hole 100 μ L of test sample, and bag is suppressed plain 100ng/100 μ L and makes positive control, OD ₄₅₀Value: PBS＜statin eukaryon expression plasmid pXAIS＜statin antigen 1 00ng, the measured OD value difference of statin eukaryon expression plasmid pXAIS and statin antigen is different less, and contrast greater than PBS and eukaryon expression plasmid pVAX-asd thin more than 2 times, the immunologic competence (table 1) that prompting statin eukaryon expression plasmid pXAIS all has statin.

Table 1 transfection detects the OD450 value of Recombinant Protein Expression situation by the ELISA method after 48 hours

Embodiment 4 the present invention on Kunming mouse to the influence of its reproductive performance and immunne response

1, vaccine production:

With Salmonella choleraesuls C500 is that the empty bacterium of the inhibin DNA vaccine of carrier and Salmonella choleraesuls C500 inoculates respectively in the LB liquid nutrient medium, is cultured to OD in 37 ℃, 150rpm jolting ₆₀₀0.3-0.4 4 ℃, the centrifugal 10min of 1500g abandon supernatant, adjust bacterial concentration to 10 with the PBS damping fluid of sterilization ¹⁰CFU/mL, the standby vaccine that obtains testing.

2, experimental animal immunity test:

Select totally 45 of the kunming mices of 50 ages in days, be divided into 3 groups, 15 every group, provide available from Hubei Province's Center for Disease Control, Animal Genetics, Breeding and Reproduction laboratory rearing at the applicant place enters test after one week, and each organizes test mice age and body weight basically identical.These three groups are respectively test group T1 (oral is the oral gene vaccine of statin of carrier with Salmonella choleraesuls C500), the empty bacterium control group T2 (oral Salmonella choleraesuls C500) of Salmonella choleraesuls C500, PBS control group T3 (only oral PBS damping fluid); Put into public mouse in rutting sedson and carry out mating,, observe its litter size, lactation power, the variation of newborn mouse surviving rate until farrowing; T12, T22, T32 group on booster immunization is once back is carried out same treatment; T13, T23, T33 carry out same treatment behind the booster immunization secondary.

3, immunization method:

Be subjected to animal (50 age in days sexual maturity kunming mice) immunity fasting in preceding 12 hours of immunity, exempt to prohibit in preceding 4 hours water, preceding 30 minutes of immunity, oral in advance 7.5% NaHCO ₃(200uL/ head) with in and hydrochloric acid in gastric juice, immunizing dose is 1 * 10 ¹⁰The CFU/ head.Three groups in each test group are immune different number of times respectively, exempt from for one group one, and with same dose, same procedure booster immunization once same, last was organized with same dose, same procedure booster immunization once 2 weeks behind two outer two groups of initial immunities.

4, feeding and management:

Test is per 5/cage with kunming mice, and room temp remains on about 28 degree always.Interior Animal House of phase trial period keeps clean always, health, in time removes fecaluria in the house, maintenance house inner drying.

5, blood plasma preparation:

With initial immunity was 0 week, carried out the tail vein in the 0th, 2,4,6,8 weeks respectively and taked blood sample, the heparin sodium anti-freezing, preparation blood plasma ,-20 ℃ of preservations, in order to detection of antibodies (with reference to Yang Liguo etc., press of enzyme immunoassay technique [M] Agricultural University Of Nanjing, Nanjing, 1998:138-139).

6, testing index:

(1) mouse litter size, lactation power, and newborn mouse surviving rate

Write down each test group mouse litter size, lactation power, and newborn mouse surviving rate.

With Salmonella choleraesuls C500 is the influence of the oral gene vaccine of statin of carrier to the kunming mice reproductive performance:

As described in Table 2, the average litter size of T1 group test mouse of the present invention, lactation power all is higher than sky bacterium group and control group, but does not have significant difference between test group and each control group.

(2) statin antibody test

Adopt indirect ELISA method to detect statin antibody in the blood plasma.Step is as follows:

Bag is suppressed the every hole of plain antigen 100ng/100 μ L, and 4C reacts 12h, abandons reaction solution, and washing is 6 times on automatic washer, each 30 seconds; Add confining liquid (5% skimmed milk solution) 200 μ L/ holes, 37 ℃ of reaction 1.5h abandon reaction solution, wash each 30 seconds 6 times; Kunming mice blood plasma was diluted 1: 2 successively, 1: 4,1: 5,1: 10,1: 20,1: 40,1: 50, establish negative control hole simultaneously, three repetitions of each sample, 37 ℃ of reaction 1h abandon reaction solution, wash each 30 seconds 6 times; Add sheep anti-mouse igg-HRP dilution (available from Wuhan doctor's moral company) in 1: 5000, every hole 100 μ L, 37 ℃ of reaction 1h abandon reaction solution, wash each 30 seconds 6 times; Add tmb substrate liquid, every hole 150 μ L, 37 ℃ of reaction 25min; Add 2mol/L sulfuric acid termination reaction, every hole 50 μ L; The OD450 reading.Be judged to the highest antibody titers with P/N value 〉=2, wherein P is the light absorption value of blood sample to be measured, and the negative contrast blood sample of N light absorption value the results are shown in Table 3 and Fig. 9.

Of the present invention with Salmonella choleraesuls C500 be the oral gene vaccine of the statin of carrier for the first time the 2nd all statin antibody titers values behind the oral immunity just reach the peak, and one exempts from the back test group is higher than other each group (P＜0.05), the antibody titers value of each group all descends to some extent subsequently, but test group all is higher than control group (P＞0.05).

Table 3: the antibody titers of statin in the oral DNA vaccine kunming mice blood plasma of the present invention's preparation

(3) the antibody horizontal somatotype detects:

It is identical with the method for measuring antibody that somatotype detects, and just carries out the somatotype detection of immune serum IgG antibody 1 and IgG2a with goat anti-mouse igg 1-HRP and the alternative goat anti-mouse igg-HRP of IgG2a-HRP when anti-adding two.Antibody typing detect to show: the OD value that immunity back two all IgG2a measure is higher than IgG1, and behind booster immunization the no significant difference of OD value of two kinds of somatotypes.The results are shown in Figure 10.

Claims (5)

1, a strain contains Salmonella choleraesuls (the Salmonella enterica sv.Choleraesuis) C500/pXAIS of statin eukaryon expression plasmid pXAIS, is deposited in Chinese typical culture collection center, and its preserving number is CCTCC NO:M208195.

2, the Salmonella choleraesuls inhibin DNA vaccine for preparing by the described Salmonella choleraesuls of claim 1.

3, a kind of expression plasmid pXAIS that expresses pig statin fusion gene IS, it is characterized in that, plasmid pVAX1 is digested with the pVUII restriction endonuclease, the nucleotide sequence that obtains aspartic acid beta galactose dehydrogenase gene that increases from plasmid PYA3493 is inserted in the pVAX1 plasmid, the pVAX-asd plasmid that is not contained kantlex, enzyme among the statin eukaryon expression plasmid PCIS is cut back to close the fusion gene that obtains hepatitis B surface antigen and statin α (1-32), this fusion gene is inserted in the pVAX-asd plasmid, plain eukaryon expression plasmid pXAIS is inhibited again.

4, a kind of preparation method who improves the Salmonella choleraesuls inhibin DNA vaccine of animal reproduction power is characterized in that:

(1) plasmid pVAX1 is digested with the pVUII restriction endonuclease, the nucleotide sequence that obtains aspartic acid beta galactose dehydrogenase gene that increases from plasmid PYA3493 is inserted in the pVAX1 plasmid, is not contained the pVAX-asd plasmid of kantlex;

(2) enzyme among the statin eukaryon expression plasmid PCIS is cut back to close the gene that obtains hepatitis B surface antigen and statin α (1-32), this fusion gene is inserted in the pVAX-asd plasmid, plain eukaryon expression plasmid pXAIS is inhibited;

(3) transform Salmonella choleraesuls C500 with plasmid pXAIS, obtaining preserving number is CCTCC NO:M208195 Salmonella choleraesuls (Salmonella enterica sv.Choleraesuis) C500/pXAIS;

(4) Salmonella choleraesuls of enlarged culturing step (3) obtain the Salmonella choleraesuls inhibin DNA vaccine.

5, the application of the described Salmonella choleraesuls of claim 1 in the Salmonella choleraesuls inhibin DNA vaccine of preparation raising animal reproduction power.

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