CN104531748A - Non-antibiotic screening KISS1 eukaryotic expression plasmid and application - Google Patents

Abstract

The invention belongs to the field of gene vaccines, and discloses a non-antibiotic screening KISS1 eukaryotic expression plasmid pVAX-KISS1-S-asd. The nucleotide sequence of the non-antibiotic screening KISS1 eukaryotic expression plasmid pVAX-KISS1-S-asd is shown as SEQ ID NO:1. The invention further discloses a recombined escherichia coli x6097/pVAX-KISS1-S-asd containing the non-antibiotic screening KISS1 eukaryotic expression plasmid pVAX-KISS1-S-asd; the recombined escherichia coli x6097/pVAX-KISS1-S-asd is preserved in the China Center for Type Culture Collection; and the preservation serial number is CCTCC NO: M2014503. A bacterial strain can be used for extracting a large number of plasmids pVAX-KISS1-S-asd, and an animal immune castration gene vaccine is prepared. A recombined vaccine can be used for Hu sheep male lamb immune; immune responses can be effectively induced; the gonad function and the sexual behavior are restrained; and the aim of immune castration is achieved. The non-antibiotic screening immune castration gene vaccine is low in cost, obvious in effect, convenient to use, safe, reliable, easy to store and capable of being used for immune castration of various mammals.

Description

A kind of non-resistance screening KISS1 eukaryon expression plasmid and application thereof

Technical field

The invention belongs to gene vaccine field.Be specifically related to a kind of non-resistance screening KISS1 eukaryon expression plasmid, recombination bacillus coli containing this eukaryon expression plasmid, and this recombination bacillus coli is preparing the application in animal immune castration gene vaccine, the invention still further relates to a kind of preparation method of animal immune castration gene vaccine.

Background technology

Domestic animal castration art has history remote in China, is recorded in the Xia dynasty " Xia little Zheng " the earliest, attacks coltfoal calling castrate or attacks spy.At present, domestic animal method of emasculation also mainly continues to use Surgical castration.Chinese Domestic Animals year, the amount of delivering for sale was huge, comprising 600,000,000 pigs and 300,000,000 bellwethers (meter Chun changes. national animal husbandry development overview. People's Republic of China's yearbook, 2012).Main to castrate in husbandry sector, dam not castration substantially.Annual approximately just have 300,000,000 boars and 100,000,000 rams to take castration of performing the operation, and workload is huge, and cost is very high, and easy infection.Meanwhile, scale standardization produce in the new demands such as animal welfare, cost control, skilled labor be rare and new situations force exploitation convenience and high-efficiency, castration new technology and method with low cost.In numerous castration scheme, hormone immunity castration has application potential most in the large production of scale standardization.The key problem in technology of immunocastration is the selection of immune targets.Hormone immunity castration mainly with the reproductive hormone member in hypothalamic pituitary gonadal axis for target (target gene or target protein).GnRH, FSH and LH be target that immunocastration is conventional (Han Yanguo, etc. the progress of animal GnRH-I immunocastration. physiological science is in progress, 2013,44 (5): 399-401; Jiang Xunping, etc. genetic immunization principle and method. Beijing: Science Press, 2004).On suppression animal testosterone secretion, spermatogenesis and semen quality, certain inhibition (Falvo RE is all had after the immunity of FSH or LH protein vaccine, Deng .Effect of active immunization against LHRH or LH in boars:reproductive consequences and performance traits.J Anim Sci, 1986,63 (3): 986-94; Ferro VA.Current advances in antifertility vaccines for fertility control and noncontraceptive applications.Expert Rev Vaccines, 2002,1 (4): 443-52).But their common issue is that after immune animal, antibody horizontal is not high, and castration effect is not obvious.So investigator improves except the method for immune effect except finding, and more perspective in research is turned to the upper level member GnRH of Reproductive Axis.GnRH protein vaccine (as Improvac and Bopriva) for animals can reach castration object (Brunius C, wait .Early immunocastration of male pigs with Improvac ( )-effect on boar taint, hormones and reproductive organs.Vaccine, 2011,29 (51): 9514-20).But this protein type castration vaccine complicated operation, cost is high.Given this, investigator constructs the GnRH gene vaccine of low cost for animal immune castration, test-results shows to be exempted from that mouse androgen levels reduces, testicular atrophy and sperm development are obstructed etc. (the .GnRH gene vaccine such as Ye Wenjian builds and immunocastration effect assessment. Wuhan: Library of Hua Zhong Agriculture University, 2012; Khan MAH, Deng .Immunisation with a plasmid DNA vaccine encoding gonadotrophin releasing hormone (GnRH-I) and T-helper epitopes in saline suppresses rodent fertility.Vaccine, 2008,26 (10): 1365-1374; Khan MAH, Deng .Immunisation of male mice with a plasmid DNA vaccine encoding gonadotrophin releasing hormone (GnRH-I) and T-helper epitopes suppresses fertility in vivo.Vaccine, 2007,, but there is no the generation of complete Inhibit sperm 25 (18): 3544-53).Therefore, in order to the object making the gene vaccine of exploitation reach immunocastration, gene vaccine expression efficiency or find new immunocastration target in body must be improved.KISS1 is secreted by hypothalamus, be in the most upstream that Reproductive Axis controls, the decisive role of Reproductive manipulation switch is played by the secretion of control GnRH, pounded out or seriously reduced after suddenling change reproductive performance (the Suzuki S of animal, Deng .Direct kisspeptin-10stimulation on luteinizing hormone secretion from bovine and porcine anterior pituitary cells.Animal Reproduction Science, 2008,103 (3-4): 360-5; Clarke IJ, waits .Kisspeptin stimulates ovulation in seasonally acylic ewes.In Proc 89th Annual Meeting of the Endocrine Society.Toronto, Ontario, Canada, 2007; Lapatto R, waits .Kiss1-/-mice exhibit more variable hypogonadism than Gpr54-/-mice.Endocrinology, 2007,148 (10): 4927-36).For the target of immunocastration vaccine, the effect upstream that the hormone member of hypothalamic pituitary gonadal axis upstream is used for immunocastration is better than downstream, such as good just than FSH or LH of GnRH immunocastration effect.Inference thus, may be not worse than GnRH with the KISS1 genetic immunization castration effect being in Reproductive Axis most upstream.For this reason, the research of KISS1 new target drone immunocastration effect need be launched further by the novel method of genetic immunization.

In the carrier of immunocastration gene vaccine (as GnRH), ubiquity resistance screening marker gene (Amp or Kan).Need to add this type of resisting substance to screen recombinant bacterium in the process of culturing bacterium, not only increase cost, also sending out and potential trace allergic phenomena (Han YG in gene vaccine product preparation process of clinical drug resistance strain is likely caused, Deng .Efficacy and safety of an oral somatostatin DNA vaccine without antibiotic resistance gene in promoting growth of piglets.Scand J Immunol, 2014,, thus be unfavorable for applying of vaccine 79 (4): 244-50).Therefore, to still need further Improvement about the carrier carrying goal gene.In addition, molecular weight is little usually for the target (as GnRH) in immunocastration gene vaccine, and immunogenicity is weak, and in order to ensure its good immunogenicity, the target in immunocastration gene vaccine needs to couple together with macromolecular carrier.Large quantity research proves, the target of gene vaccine is combined immunogenicity (the Liang AX that effectively can improve vaccine with macromolecular HBsAg Gene, Deng .Construction and evaluation of the eukaryotic expression plasmid encoding two copies of somatostatin genes fused with hepatitis B surface antigen gene S.Vaccine, 2008,26:2935 – 41).

Summary of the invention

The object of the invention is to build a kind of newly, for the gene vaccine of immunocastration target upstream KISS1, the present invention also provides a kind of non-resistance screening KISS1 eukaryon expression plasmid, and contains the recombination bacillus coli of this eukaryon expression plasmid.

The building process of animal immune castration gene vaccine of the present invention is: first by synthesis people KISS1 gene clone in HindIII and the EcoRI restriction enzyme site of eukaryotic expression vector pVAX1, obtain plasmid pVAX-KISS1, again the HBsAg Gene increased from plasmid pCMV-S is cloned in NheI and the HindIII restriction enzyme site of plasmid pVAX-KISS1, obtains middle interstitial granules pVAX-KISS1-S, use non-resistance screening-gene afterwards---the kanamycin gene (Kan) in aspartic acid beta galactose dehydrogenase gene (asd) replacement in interstitial granules pVAX-KISS1-S, obtain non-resistance screening KISS1 eukaryon expression plasmid pVAX-KISS1-S-asd (its nucleotide sequence is as shown in SEQ ID NO:1), again eukaryon expression plasmid pVAX-KISS1-S-asd is converted in the intestinal bacteria of disappearance asd gene, screening positive clone, namely the coli strain x6097/pVAX-KISS1-S-asd containing non-resistance screening KISS1 eukaryon expression plasmid pVAX-KISS1-S-asd is obtained, by this bacterial strain enlarged culturing, with alkaline lysis large quantity extracting plasmid pVAX-KISS1-S-asd, namely obtain animal immune castration gene vaccine, and this gene vaccine is applied to sheep ram carries out clinical verification, thus complete the present invention.

Applicant has prepared intestinal bacteria (Escherichia coli) the bacterial strain x6097/pVAX-KISS1-S-asd that a strain contains non-resistance screening KISS1 eukaryon expression plasmid pVAX-KISS1-S-asd, deliver China typical culture collection center (CCTCC) preservation in the Wuhan University of Wuhan City, Hubei Province on October 21st, 2014, its deposit number is CCTCC NO:M2014503.

The invention has the beneficial effects as follows:

1) the public lamb of recombiant vaccine immunity sheep of the present invention being prepared, can effectively induce its immunne response, suppress its gonad function and sexual behaviour, reach the object of immunocastration.Non-resistance screening immunocastration gene vaccine prepared by the present invention is with low cost, and Be very effective is easy to use, safe and reliable, is easy to preserve, and can be used for various mammiferous immunocastration.

2) present invention obtains a kind of non-resistance screening KISS1 eukaryon expression plasmid pVAX-KISS1-S-asd immunocastration vaccine, compared to FSH, LH, GnRH vaccine in past, KISS1 is a new immunocastration DNA vaccination target, this target is in Reproductive Axis most upstream, and its immunocastration effect is not worse than GnRH.

3) present invention obtains a kind of non-resistance screening KISS1 eukaryon expression plasmid pVAX-KISS1-S-asd, this plasmid is not containing resistance screening gene, trace allergic phenomena potential in the propagation of drug resistance strain and gene vaccine product preparation process can not be caused, and ubiquity resistance screening marker gene (Amp or Kan) in GnRH immunocastration gene vaccine in the past, need to add this type of resisting substance to screen recombinant bacterium in the process of culturing bacterium, not only increase cost, also sending out and potential trace allergic phenomena in gene vaccine product preparation process of clinical drug resistance strain is likely caused.

Accompanying drawing explanation

Fig. 1 is the process flow sheet building animal immune castration gene vaccine of the present invention.

Fig. 2 is that the KISS1 nucleotide sequence figure that the present invention synthesizes, ▼ and ▲ expression HindIII and EcoRI enzyme are cut and connection site, and taa is terminator codon.

Fig. 3 is the HBsAg Gene AFLP system in the present invention, and swimming lane 1-8 is pcr amplification product S fragment, and swimming lane 9 is negative controls, and M swimming lane is 100bp ladder DNA Marker (2000bp).

Fig. 4 is that the enzyme of interstitial granules pVAX-KISS1-S in the present invention cuts qualification collection of illustrative plates.In figure, each swimming lane is as follows: 1-2.pVAX-KISS1-S/ (NheI+EcoRI); M.100bp ladder DNA Marker (5000bp).

Fig. 5 is that the enzyme of the present invention's non-resistance screening KISS1 eukaryon expression plasmid pVAX-KISS1-S-asd cuts qualification collection of illustrative plates.In Fig. 5 a, each swimming lane is as follows: 1.pVAX-KISS1-S-asd/ (NheI+EcoRI); M.100bp ladder DNA Marker (5000bp); In Fig. 5 b, each swimming lane is as follows: 1.pVAX-KISS1-S-asd/ (pVUII+pVUII); M.100bp ladder DNA Marker (5000bp).

Fig. 6 is the immunne response of inducing after non-resistance screening animal immune castration DNA gene vaccine sheep ram of the present invention, and T is vaccine immunity group, and C is empty control plasmid group.

Fig. 7 be non-resistance screening animal immune castration gene vaccine of the present invention on the impact of sheep testosterone secretion, T is vaccine immunity group, and C is empty control plasmid group.

Embodiment

Embodiment 1: the structure of non-resistance screening KISS1 eukaryon expression plasmid pVAX-KISS1-S-asd with prepare in a large number

First synthesize people KISS1 gene, this gene 5 ' end contains HindIII restriction enzyme site, 3 ' end containing EcoRI restriction enzyme site, and is cloned in HindIII and the EcoRI restriction enzyme site of eukaryotic expression vector pVAX1 (purchased from Invitrogen company), obtains plasmid pVAX-KISS1; Again with plasmid pCMV-S for template, amplification HBsAg Gene, be then cloned in NheI and the HindIII restriction enzyme site of plasmid pVAX-KISS1, obtained middle interstitial granules pVAX-KISS1-S; Use non-resistance screening-gene---the kanamycin gene (Kan) in aspartic acid beta galactose dehydrogenase gene (asd) replacement in interstitial granules pVAX-KISS1-S, and the Plastid transformation after replacing extremely is lacked in the intestinal bacteria of asd gene, screening positive clone x6097/pVAX-KISS1-S-asd, namely obtains the coli strain x6097/pVAX-KISS1-S-asd containing non-resistance screening KISS1 eukaryon expression plasmid pVAX-KISS1-S-asd; By positive colony x6097/pVAX-KISS1-S-asd enlarged culturing, with alkaline lysis large quantity extracting plasmid pVAX-KISS1-S-asd, namely obtain non-resistance screening animal immune castration gene vaccine pVAX-KISS1-S-asd.The process flow sheet building animal immune castration gene vaccine of the present invention is shown in accompanying drawing 1.

1.1KISS1 gene clone is to pVAX1 carrier

1.1.1KISS1 the acquisition of gene

(GenBank accession number is NM_002256 to the KISS1 gene of synthesis people, proteins encoded Kisspeptin-54, synthesized by Shanghai Sheng Gong biotechnology company limited), its two ends comprise HindIII and EcoRI endonuclease digestion site (see accompanying drawing 2), and the nucleotide sequence of this gene is as shown in SEQ ID NO:2.

1.1.2 KISS1 gene is connected to pVAX1 carrier

By eukaryotic expression vector pVAX1 (purchased from Invitrogen company, its nucleotide sequence is as shown in SEQ ID NO:5) carry out double digestion with HindIII and EcoRI restriction endonuclease (purchased from NEB company), the enzyme system of cutting is: NEBuffer22 μ L, pVAX112 μ L, HindIII restriction endonuclease 0.5 μ L, EcoRI restriction endonuclease 0.5 μ L, BSA 0.2 μ L, add water to cumulative volume 20 μ L, 37 DEG C of reaction 2-3h, reclaim test kit (purchased from TIANGEN Biotech (Beijing) Co., Ltd.) with glue afterwards and reclaim purifying pVAX1 carrier large fragment (reclaiming test kit specification sheets with reference to glue), afterwards with connecting test kit (purchased from precious biotechnology (Dalian) company limited) 16 DEG C of connections of spending the night, in reaction system, the mole number of KISS1 fragment should control 3 ~ 10 times at pVAX1 carrier large fragment mole number, specific as follows:

Linked system is:

Add sterilizing deionized water to cumulative volume 10 μ L, 16 DEG C of connections are spent the night.

1.1.3 be converted into DH5 α

DH5 α competent cell, purchased from TIANGEN Biotech (Beijing) Co., Ltd., is stored in-80 DEG C, for subsequent use.Adopt conventional heat-shock transformed method that the connection product 10 μ L in 1.1.2 is converted into competence bacterium DH5 α.(step of converting reference J. Pehanorm Brooker, D.W. Russell, Molecular Cloning: A Laboratory guide [M]. the third edition, Beijing, Science Press, 2002,96-99).

1.1.4 positive colony screening, qualification with check order

By the competence bacterium of transform plastids in 1.1.3 in LB solid medium (the 1L LB solid medium moiety: 10g calcium chloride containing 50 μ g/mL kantlex, 5g yeast extract, 10g Tryptones, 15g agar powder, pH7.0) bed board is cultivated, in LB liquid nutrient medium (the 1L LB liquid nutrient medium moiety: 10g calcium chloride containing 50 μ g/mL kantlex after picking positive colony, 5g yeast extract, 10g Tryptones, pH 7.0) middle cultivation, with plasmid extraction agent box (purchased from TIANGEN Biotech (Beijing) Co., Ltd.) extracting plasmid (reference reagent box working instructions) in a small amount, serve the order-checking of Hai Shenggong biotechnology company limited, sequencing result display is without base mispairing and phase shift mutation, by the positive colony called after pVAX-KISS1 obtained, and be stored in-80 DEG C (bacterium liquid is containing 20% glycerine).

1.2 HBsAg Genes are cloned into pVAX-KISS1

1.2.1 the acquisition of HBsAg Gene

With plasmid pCMV-S (being given by Hua Zhong Agriculture University's agricultural animal genetic breeding and key lab of breeding the Ministry of Education professor Yang Liguo) for template, amplification hepatitis B surface antigen S fragment, primer sequence (primer sequence reaches dry etc. with reference to cogongrass. the construction and expression of Gonadostatin and hepatitis B surface antigen blending gene expression plasmid. and China Immunology Journal, 2003,19 (11): 775-778) be:

Upstream primer: 5'-TAATATT gCTAGCnheI site shown in CCTGCGCTGAACATGG-3'(underscore)

Downstream primer: 5'-GC aAGCTThindIII site shown in TACCCAAAGAC-3'(underscore)

PCR reaction system (20 μ L) is as follows:

2 × PCR Buffer 2 μ L, upstream, downstream primer each 0.5 μ L, dNTPs 0.5 μ L, Taq enzyme 0.5 μ L, template 1 μ L, finally adds sterilizing distilled water to 20 μ L.

PCR reaction conditions is as follows:

94 DEG C of denaturation 5min, then 94 DEG C of sex change 40sec, 57 DEG C annealing 40sec, 72 DEG C extend 45sec, 35 circulation latter 72 DEG C extend 10min again.

Finally, PCR primer is used 1.0% agarose gel electrophoresis, using 100bp ladder DNA Marker as molecular weight standard, electrophoresis result is presented between 500-750bp band, (see accompanying drawing 3) in the same size with specificity object strips S (705bp).The nucleotide sequence of the HBsAg Gene of amplification is as shown in SEQ ID NO:3.

1.2.2 carrier T is connected to

Purify by PCR primer and reclaim test kit (purchased from TIANGEN Biotech (Beijing) Co., Ltd.) recovery and purifying pcr amplification product (concrete operations reference reagent box working instructions).PCR primer after purifying is directly connected with pMD-18T Vector (purchased from precious biotechnology (Dalian) company limited).

Linked system is:

S 7.2μL

T-Vector 0.3μL

Ligation Solution 2.5μL

Cumulative volume 10 μ L, 16 DEG C of connections are spent the night.

1.2.3 be converted into DH5 α

DH5 α competent cell, purchased from TIANGEN Biotech (Beijing) Co., Ltd., is stored in-80 DEG C, for subsequent use.Adopt conventional heat-shock transformed method that the connection product 10 μ L in 1.2.2 is converted into competence bacterium DH5 α.(step of converting reference J. Pehanorm Brooker, D.W. Russell, Molecular Cloning: A Laboratory guide [M]. the third edition, Beijing, Science Press, 2002,96-99).

1.2.4 positive colony screening, qualification with check order

By the competence bacterium of transform plastids in 1.2.3 in LB solid medium (the 1L LB solid medium moiety: 10g calcium chloride containing 50 μ g/mL kantlex, 5g yeast extract, 10g Tryptones, 15g agar powder, pH 7.0) bed board cultivation, in LB liquid nutrient medium (the 1L LB liquid nutrient medium moiety: 10g calcium chloride containing 50 μ g/mL kantlex after picking positive colony, 5g yeast extract, 10g Tryptones, pH 7.0) middle cultivation, with plasmid extraction agent box (purchased from TIANGEN Biotech (Beijing) Co., Ltd.) extracting plasmid (reference reagent box working instructions) in a small amount, NheI and HindIII restriction endonuclease (purchased from NEB company) is used to carry out double digestion again, the enzyme system of cutting is: NEBuffer2 2 μ L, recombinant plasmid 2 μ L, NheI and HindIII restriction endonuclease is 0.5 μ L, BSA 0.2 μ L, adding ddH2O to cumulative volume is 20 μ L, 37 DEG C of reaction 2-3h, observe enzyme and cut result, electrophoretic band conforms to goal gene stripe size, serve the order-checking of Hai Shenggong biotechnology company limited, sequencing result display is without base mispairing and phase shift mutation, by the positive colony called after T-S obtained, and be stored in-80 DEG C (bacterium liquid is containing 20% glycerine).

1.2.5 plasmid extraction and purifying

With plasmid extraction agent box (purchased from TIANGEN Biotech (Beijing) Co., Ltd.) extracting T-S plasmid in a small amount.

1.2.6 double digestion be connected

Respectively by pVAX-KISS1 and T-S plasmid after NheI and HindIII double digestion (enzyme cuts the same 1.2.4 of system), reclaim test kit (purchased from TIANGEN Biotech (Beijing) Co., Ltd.) with glue and reclaim purifying object fragment (reclaiming test kit specification sheets with reference to glue), afterwards with connecting test kit (purchased from precious biotechnology (Dalian) company limited) 16 DEG C of connections of spending the night, in reaction system, the mole number of hepatitis B surface antigen S fragment should control 3 ~ 10 times at pVAX-KISS1 large fragment mole number, specific as follows:

Linked system is:

Add sterilizing deionized water to cumulative volume 20 μ L, 16 DEG C of connections are spent the night.

1.2.7 be converted into DH5 α

DH5 α competent cell, purchased from TIANGEN Biotech (Beijing) Co., Ltd., is stored in-80 DEG C, for subsequent use.Adopt conventional heat-shock transformed method that the connection product 10 μ L in 1.2.6 is converted into competence bacterium DH5 α.(step of converting reference J. Pehanorm Brooker, D.W. Russell, Molecular Cloning: A Laboratory guide [M]. the third edition, Beijing, Science Press, 2002,96-99).

1.2.8 positive colony screening, qualification with check order

By the competence bacterium of transform plastids in 1.2.7 in LB solid medium (the 1L LB solid medium moiety: 10g calcium chloride containing 50 μ g/mL kantlex, 5g yeast extract, 10g Tryptones, 15g agar powder, pH 7.0) bed board cultivation, in LB liquid nutrient medium (the 1L LB liquid nutrient medium moiety: 10g calcium chloride containing 50 μ g/mL kantlex after picking positive colony, 5g yeast extract, 10g Tryptones, pH 7.0) middle cultivation, with plasmid extraction agent box (purchased from TIANGEN Biotech (Beijing) Co., Ltd.) extracting plasmid (reference reagent box working instructions) in a small amount, NheI and EcoRI restriction endonuclease (purchased from NEB company) is used to carry out double digestion again, the enzyme system of cutting is: NEBuffer22 μ L, recombinant plasmid 2 μ L, NheI and EcoRI restriction endonuclease is 0.5 μ L, BSA 0.2 μ L, add ddH ₂o is 20 μ L to cumulative volume, 37 DEG C of reaction 2-3h, observe enzyme and cut result, electrophoretic band conforms to goal gene band (fusion gene KISS1-S) size, serve the order-checking of Hai Shenggong biotechnology company limited, sequencing result display, without base mispairing and phase shift mutation, by the positive colony called after pVAX-KISS1-S obtained, and is stored in-80 DEG C (bacterium liquid is containing 20% glycerine) (see accompanying drawings 4).

The structure of 1.3 coli strain x6097/pVAX-KISS1-S-asd

1.3.1 the extraction and purification of plasmid

With plasmid, extraction agent box (purchased from TIANGEN Biotech (Beijing) Co., Ltd.) difference extracting pVAX-KISS1-S plasmid and pVAX-asd plasmid (are given by Hua Zhong Agriculture University's agricultural animal genetic breeding and key lab of breeding the Ministry of Education teacher Han Li in a small amount, Han Li. the structure of statin novel gene engineered vaccine and immune effect research thereof. [Ph.D. Dissertation]. Wuhan: Library of Hua Zhong Agriculture University, 2009), concrete operation step reference reagent box working instructions.Asd gene nucleotide series in pVAX-asd plasmid is 1107bp (GenBank accession number is AE006468.1 (between 3708628-3709734)), this sequence is as shown in SEQ ID NO:4, and the nucleotide sequence of pVAX-asd plasmid is as shown in SEQ ID NO:6.

1.3.2 double digestion be connected

By middle interstitial granules pVAX-KISS1-S and pVAX-asd plasmid through pVUII restriction endonuclease (purchased from NEB company) double digestion, the enzyme system of cutting is: NEBuffer22 μ L, pVAX-asd plasmid (or pVAX-KISS1-S plasmid) 2 μ L, pVUII restriction endonuclease 1 μ L, BSA 0.2 μ L, add water to cumulative volume 20 μ L, reclaim test kit (purchased from TIANGEN Biotech (Beijing) Co., Ltd.) with glue afterwards and reclaim purifying object fragment (reclaiming test kit specification sheets with reference to glue), afterwards with connecting test kit (purchased from precious biotechnology (Dalian) company limited) 16 DEG C of connections of spending the night, in reaction system, the mole number of asd fragment should control 3 ~ 10 times at pVAX-KISS1-S large fragment mole number, specific as follows:

Linked system is:

Add sterilizing deionized water to cumulative volume 10 μ L, 16 DEG C of connections are spent the night.

1.3.3 heat shock transforms and connects product

The competent escherichia coli cell of disappearance asd gene by Hua Zhong Agriculture University's agricultural animal genetic breeding give with key lab of breeding the Ministry of Education teacher Liang Aixin (beam love. the structure of non-resistance screening somatostatin DNA vaccine and immune efficacy and safety research. [Ph.D. Dissertation]. Wuhan: Library of Hua Zhong Agriculture University, 2009).Connection product heat shock in 1.3.2 to be converted in the competent escherichia coli cell of disappearance asd gene (concrete operation step with reference to J. Pehanorm Brooker, D.W. Russell, Molecular Cloning: A Laboratory guide [M]. the third edition, Beijing, Science Press, 2002,96-99).

1.3.4 the screening of positive colony

The competent cell of transform plastids in 1.3.3 is cultivated in the LB solid medium bed board not containing any allogenic material, cultivate in not containing the LB liquid nutrient medium of any allogenic material after picking positive colony, with plasmid extraction agent box (purchased from TIANGEN Biotech (Beijing) Co., Ltd.) extracting pVAX-KISS1-S-asd plasmid (reference reagent box working instructions) in a small amount, pVUII restriction endonuclease (enzyme cuts the same 1.3.2 of system) and NheI/EcoRI restriction endonuclease (enzyme cuts the same 1.2.8 of system) is used to carry out double digestion afterwards respectively, 37 DEG C of reaction 2-3h, observe enzyme and cut result.Band is there is at 1000bp and 750bp place in result display plasmid after NheI/EcoRI enzyme is cut, (see accompanying drawing 5a) in the same size with object fragment KISS1-S (861bp), band is there is at 1500bp and 1000bp place in plasmid after pVUII enzyme is cut, (see accompanying drawing 5b) in the same size with object fragment asd (1141bp), serve the order-checking of Hai Shenggong biotechnology company limited, sequencing result display is without base mispairing and phase shift mutation.So far, the coli strain x6097/pVAX-KISS1-S-asd containing non-resistance screening KISS1 eukaryon expression plasmid pVAX-KISS1-S-asd has built, and is stored in-80 DEG C (bacterium liquid is containing 20% glycerine).

A large amount of preparations of 1.4 non-resistance screening eukaryon expression plasmid pVAX-KISS1-S-asd

By streak culture on the solid medium not adding any allogenic material for the coli strain x6097/pVAX-KISS1-S-asd preserved in 1.3.4, cultivate in 6mLLB liquid nutrient medium after picking positive colony, the 1ml bacterium liquid plasmid a small amount of that takes a morsel extraction agent box (purchased from TIANGEN Biotech (Beijing) Co., Ltd.) extracting plasmid carries out enzyme and cuts qualification and order-checking, after plasmid identification is correct, remaining 5mL LB liquid nutrient medium is put into 250mL LB liquid nutrient medium, 12-16h is cultivated in 37 DEG C of concussions, the centrifugal 10min of 8000r/min, collect thalline, precipitate and wash once with 100mlSTE, the centrifugal 10min of 6000r/min.With phenol/chloroform/isoamyl alcohol extraction plasmid, then add isopyknic PEG-MgCl2 solution purification plasmid (concrete operation step reference J. Pehanorm Brooker, D.W. Russell, Molecular Cloning: A Laboratory guide [M]. the third edition, Beijing, Science Press, 2002).Plasmid Endotoxin removal test kit (purchased from Pu Boxin bio tech ltd, Beijing) is used to remove the intracellular toxin (reference reagent box working instructions) extracting and produce in plasmid process afterwards, finally will the plasmid pVAX-KISS1-S-asd after intracellular toxin be gone to serve the order-checking of Hai Shenggong biotechnology company limited, sequencing result display is without base mispairing and phase shift mutation, and its nucleotide sequence is as shown in SEQ ID NO:1.So far, prepared by non-resistance screening KISS1 eukaryon expression plasmid pVAX-KISS1-S-asd (i.e. non-resistance screening animal immune castration gene vaccine).

Embodiment 2: the application of animal immune castration gene vaccine of the present invention on sheep

The immunity of 2.1 vaccines and sample collecting

Public for sheep about 68 week ages lamb is divided at random 2 groups (purchased from the permanent safe sheep cultivation company limiteds in Hubei), often organizes 3.24h before immunity, the animal vovocan of 4ml 0.25% divides 3 intramuscular injection to carry out pre-treatment at quadriceps muscle of thigh.T group is vaccination group, intramuscular injection pVAX-KISS1-S-asd plasmid, dosage is 1mg/, concrete operations divide 3 injections at sheep quadriceps muscle of thigh after 1mg plasmid vaccine is dissolved in 4ml physiological saline, C group is empty control plasmid group, intramuscular injection gymnoplasm grain pVAX-asd, dosage is 1mg/, and concrete operations are with vaccination group.Immunity three times altogether, three weeks, interval.

Within 0,2,4,6,10 and 14 week after immunity, take a blood sample, separation of serum, is placed in-20 DEG C, for subsequent use.Within 14 weeks after immunity, butcher, measure testis weight, testis is long and testis is wide.

The immune efficacy of 2.2 vaccines detects

Indirect ELISA method is adopted to detect anti-KISS1 antibody in serum.With the Kisspeptin-54 of synthesis (being synthesized by Chu Tai bio tech ltd, Shanghai), for envelope antigen, the minimum thinning ratio being greater than negative control mean OD value+2 × standard deviation with OD value is judged to the positive, the results are shown in accompanying drawing 6.Concrete steps are as follows: wrap by Kisspeptin-54 every hole 100ng/100 μ L, 4 DEG C of reaction 12h; Abandon reaction solution, wash 3 times, each 5min; Add confining liquid (PBST containing 0.5%BSA) 200 μ L/ hole, 37 DEG C reaction 1h after, washing, add test serum 100 μ L (with confining liquid to serum by 1:25; 1:50; 1:100; 1:200; 1:400; 1:800; 1:1600; 1:3200 carries out doubling dilution), establish negative control hole, zeroing hole (not adding any reaction solution), 37 DEG C of reaction 1h simultaneously; Abandon reaction solution, washing; Add the anti-sheep IgG-HRP of donkey (1:5000 dilution), every hole 100 μ l, 37 DEG C of reaction 1h; Abandon reaction solution, washing; Add tmb substrate liquid, every hole 150 μ L, 37 DEG C of reaction 25min; Add 2mol/L sulfuric acid termination reaction; OD450 reading.

KISS1 antibody test result (see accompanying drawing 6) display in immune serum, after immunity the 4th week, compared with empty control plasmid group (T group), vaccination group (T group) creates antibody, is continued until the 14th week after immunity.Result shows, after the public lamb of non-resistance screening animal immune castration gene vaccine pVAX-KISS1-S-asd immunity sheep, creates effective immunne response.

2.3 gonad functions detect

Adopt the testosterone levels (test kit is purchased from Wuhan Sino-American Biotechnology Company) in indirect ELISA method detection immunity latter 0,2,4,6, the 10 and 14 week public lamb serum of sheep, reference reagent box working instructions.Testosterone detected result (see accompanying drawing 7) display in immune serum, after immunity the 4th week, the testosterone concentration of vaccination group (T group), significantly lower than the testosterone concentration of empty control plasmid group (C group), is continued until the 14th week after immunity.Result shows, after the public lamb of non-resistance screening animal immune castration gene vaccine pVAX-KISS1-S-asd immunity sheep, significantly reduces the testosterone levels in serum.

After immunity 14 weeks, all experimental animals are butchered, measure that testis is heavy, testis is long and testis is wide.The slaughter results (see table 1) shows, and the testis of vaccination group (T group) is heavy and testis is long all remarkable in empty control plasmid group (C group).Result shows, after the public lamb of non-resistance screening animal immune castration gene vaccine pVAX-KISS1-S-asd immunity sheep, restrained effectively the growth of testis.

Rear immune group butchered by table 1 and control group sheep testis is heavy, testis is long and testis is wide

^*same row represent significant difference (P<0.05).

In sum, after the public lamb of non-resistance screening animal immune castration gene vaccine pVAX-KISS1-S-asd immunity sheep, restrained effectively the gonad function of animal.

The detection of 2.3 sexual behaviour

The last week of butchering, observe the sexual behaviour (comprise mounting, smell and hear and answer back to) of all experimental animals.Concrete grammar, for together with being closed with the ewe that oestruses by ram, is observed 30 minutes, Continuous Observation one week, until butcher.Observations (see table 2) shows, the mounting number of times of vaccination group (T group), smell hear number of times and answer back to number of times all remarkable in empty control plasmid group (C group).Result shows, after the public lamb of non-resistance screening animal immune castration gene vaccine pVAX-KISS1-S-asd immunity sheep, restrained effectively the sexual behaviour of sheep, reaches the effect of immunocastration.

Table 2KISS1 DNA gene vaccine is on the impact of sheep sexual behaviour

Group	Mounting number of times	Smell news number of times	Answer back to number of times
				T	0.33±0.58 *	2.00±1.00 *	0 *
C	4.67±2.08	11.00±3.61	6.00±1.73

^*same row represent significant difference (P<0.05).

Claims (5)

1. a non-resistance screening KISS1 eukaryon expression plasmid pVAX-KISS1-S-asd, its nucleotide sequence is as shown in SEQ ID NO:1.

2. a strain contains the recombination bacillus coli x6097/pVAX-KISS1-S-asd of non-resistance screening KISS1 eukaryon expression plasmid pVAX-KISS1-S-asd described in claim 1, be deposited in China typical culture collection center, its deposit number is CCTCC NO:M2014503.

3. recombination bacillus coli x6097/pVAX-KISS1-S-asd according to claim 2 is preparing the application in animal immune castration gene vaccine.

4. an animal immune castration gene vaccine, it contains the non-resistance screening KISS1 eukaryon expression plasmid pVAX-KISS1-S-asd extracted in the recombination bacillus coli x6097/pVAX-KISS1-S-asd described in Accessory Right requirement 2.

5. the preparation method of an animal immune castration gene vaccine, it is characterized in that, by the KISS1 gene clone of synthesis in eukaryotic expression vector pVAX1, obtain plasmid pVAX-KISS1, the HBsAg Gene of amplification is cloned in plasmid pVAX-KISS1, obtain middle interstitial granules pVAX-KISS1-S, use non-resistance riddled basins afterwards---the kanamycin gene in the replacement of aspartic acid beta galactose dehydrogenase gene in interstitial granules pVAX-KISS1-S, obtain non-resistance screening KISS1 eukaryon expression plasmid pVAX-KISS1-S-asd, again this Plastid transformation is extremely lacked in the intestinal bacteria of asd gene, screening positive clone, obtain the recombinant escherichia coli strain x6097/pVAX-KISS1-S-asd containing non-resistance screening KISS1 eukaryon expression plasmid pVAX-KISS1-S-asd, by this bacterial strain enlarged culturing, with alkaline lysis large quantity extracting plasmid pVAX-KISS1-S-asd, obtain described animal immune castration gene vaccine.

Described non-resistance screening KISS1 eukaryon expression plasmid pVAX-KISS1-S-asd, its nucleotide sequence is as shown in SEQ ID NO:1; Described recombinant escherichia coli strain x6097/pVAX-KISS1-S-asd, is deposited in China typical culture collection center, and its deposit number is CCTCC NO:M2014503.