CN102191272A - Antifertility recombinant plasmid and genetic vaccine and preparation method and applications thereof - Google Patents
Antifertility recombinant plasmid and genetic vaccine and preparation method and applications thereof Download PDFInfo
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Abstract
The invention provides an antifertility recombinant plasmid and genetic vaccine, and a preparation method and applications thereof. The recombinant plasmid consists of Cyp26a1 gene cDNA fragment and eukaryotic expression vector pCR 3.1. The preparation method of the recombinant plasmid and genetic vaccine comprises the following steps: extracting and purifying the total RNA of rat pregnant uterus, designing a specific primer, adopting the temperature reducing reverse transcription-polymerase chain amplification method to clone and amplify the Cyp26a1 gene cDNA fragment and recombining the fragment in eukaryotic expression plasmid. The prepared genetic vaccine can be used to inhibit the fertility of mammals and prevent and/or control rats, have the advantages of safety, long-term effectiveness, stability and convenient operation and have higher immune effect than other immune means; and the genetic vaccine can be used to effectively reduce the fertility of rats and obviously reduce newborn offspring rats.
Description
Technical field
The invention belongs to biological and modern agricultural technology field, relate to a kind of antifertility gene vaccine, more particularly, the present invention relates to a kind of antifertility gene vaccine, Preparation Method And The Use that contains rat cell cytochrome p 450 family 26 superfamily A polypeptide 1 (Cyp26a1).
Background technology
Mouse and other small-sized harmful Mammals, annual agriculture forest and husbandry to the whole world causes serious loss.Home mouse (Mus domesticus) plague of rats in cereal grain-producing area, Australian southeast loses 2106 ton-grain food every year on average.The eighties, general big generation of China's pesticide herd forestry plague of rats, the area of the plague of rats takes place up to tens million of hectares in annual farming and pastoral area, reduces more than 100 hundred million kilograms in grain, the tens billion of kilograms of herbage loss, direct economic loss over ten billion Yuan.Estimate that according to Food and Argriculture OrganizationFAO the grain that the whole world is produced has 10% to be trampled on by mouse.In every year, mouse and other small-sized harmful Mammals are not only caused heavy losses to the agriculture forest and husbandry output in the whole world, also propagate hemorrhagic fever, the plague etc. cause serious threat to human body health even life disease.Though mouse killing agent lethality commonly used at present is strong but residual hazard is very big, serious environment pollution.In addition, the reproductivity of muroid is extremely strong, even a deratization rate reaches 95%, last 5% also can make the mankind will improve constantly mouse medicine toxicity, year in year out dispensing one to returning to previous level in two years, form the vicious cycle of " going out-grow-go out ", cause large-area residual hazard to pollute.Therefore, can control the plague of rats long-term effectively, can keep the eubiosis again, reduce environmental pollution, become the human target of in plague of rats Control Study process, pursuing.
Along with developing rapidly of gene recombination technology and genophore transfer system, the beginning of the nineties, the genetic immunization technology was come out.The core of technology is that the nucleotide sequence with coding for antigens is assembled into to contain in the carrier for expression of eukaryon that is necessary expression regulation element and makes up gene vaccine, direct inoculation is in animal body then, the gene vaccine of reorganization can utilize the synthetic foreign gene encoded protein of the intravital enzyme of host system, thereby induce host's body that this foreign protein is produced antigen-specific immune responses, thereby reach immune purpose.Many animal models and human experimentation have proved that the nuclear gene vaccine both can activate humoral immunization; again can the activating cells immunity, gene vaccine compare with traditional inactivated vaccine, attenuated vaccine have good immune effect, security is higher, persistence, the cross immunity provide protection of different strain of the same race, preparation are easy, with low cost, be convenient to preserve and many brand-new potential advantages such as transportation.Over past ten years, gene vaccine has important breakthrough in the fundamental research aspect many such as disease preventing and treating, control animal reproduction etc.The research of the development and application of nucleic acid vaccine has caused the great attention of countries in the world, and many enterprises drop into huge fund in succession and make it commercialization.
Cyp26a1 (retinoic acid hydroxylase) claim P450RA1 again, be Cytochrome P450 (CYP) family member, be Cytochrome P450 family 26 superfamily A polypeptide 1, although CYP26 has similar structure to the member of Cytochrome P450 family, but any member of it and Cytochrome P450 family does not have homology, therefore is considered to a newcomer of the gene family of Cytochrome P450.White etc. cloned the newcomer P450RA1 that obtains this Cytochrome P450 family in 1996 first from zebra fish, its cDNA is known as CYP26 at first, and the back is by name Cyp26a1.Afterwards, the researchist is cloned into Cyp26a1 in succession from the organ-tissues such as liver of people, mouse and chicken.Rna blot analysis shows that Cyp26a1 mRNA has expression at liver, duodenum, colon, placenta and brain.Discover Cyp26a1 vitamin A acid (RA) optionally can be converted into the polar metabolite of RA (4-OH-, 18-OH-, 4-oxo-RA).RA is the main active metabolite of vitamin A, and energy phylloquinone oxide phylloquinone oxide A changes the 4-oxygen Vogan-Neu of biologically active into, and Cyp26a1 is by regulating RA level and then control retinoid signal.There is report to point out that the retinoid signal has played vital role in fetal development.Feed the feed of raising the A that is deficient in vitamin to animal, fetal development occurs unusual, and the embryo accepts too much retinoid signal also can destroy embryo's normal development.Also have document announcement, picked-up RA too much during the mouse pregnancy may cause dysontogenesis, and the odd-shaped type is relevant with dosage with degree, and the Cyp26a1 afunction can cause mice embryonic death.
Summary of the invention
The inventor adopts the subtractive hybridization technology (SSH) that suppresses, uterus difference expression gene before and after the screening rat is implanted, and by RT-PCR and Northern blotting experiment confirm: Cyp26a1 gene embryo is a difference expression gene before and after implanting.Clone the cDNA total length of SD rat uterus Cyp26a1 gene first with the RACE method, and logined GenBank (accession number: DQ317305).This gene and construction of eukaryotic expression vector go out immune female mice behind the gene vaccine, show that the Cyp26a1 gene vaccine has tangible antifertility action.
Therefore, the objective of the invention is to, a kind of gene vaccine that is used for the recombinant plasmid of Mammals antifertility gene vaccine and comprises this recombinant plasmid is provided.
Another object of the present invention is, the Preparation method and use of above-mentioned recombinant plasmid and gene vaccine is provided.
The objective of the invention is to realize by the following technical solutions.On the one hand, the invention provides a kind of recombinant plasmid that is used for Mammals antifertility gene vaccine, its cDNA fragment and carrier for expression of eukaryon by the Cyp26a1 gene is formed, and carrier for expression of eukaryon is preferably pCR 3.1.
Preferably, the cDNA fragment of described Cyp26a1 gene is derived from the Spreqne-Dawley rat uterus, and its base sequence is preferably shown in SEQ ID NO.1.
On the other hand, the invention provides the bacterial strain that comprises above-mentioned recombinant plasmid, preferably, described bacterial strain is selected from intestinal bacteria competence DH5 α bacterial strain.
The present invention also provides the cell that comprises above-mentioned recombinant plasmid, and preferably, described cell is selected from human cervical carcinoma's (HeLa) cell and Chinese hamster ovary (CHO) cell.
Another aspect the invention provides a kind of gene vaccine of Mammals antifertility, wherein comprises above-mentioned recombinant plasmid.
Preferably, described gene vaccine also comprises adjuvant; More preferably, it is 0.25% PROCAINE HCL, PHARMA GRADE that described adjuvant is selected from percentage concentration, the volume mass proportioning of per injection adjuvant consumption and gene vaccine consumption is 2 (V/ μ l): 1 (W/ μ g), concrete preferred adjuvant consumption and gene vaccine consumption are respectively 100 μ l and 50 μ g.
Also on the one hand, the invention provides a kind of method for preparing above-mentioned recombinant plasmid, the cDNA fragment of Cyp26a1 gene as described in the base sequence of this method employing shown in SEQ ID NO.2 and SEQ ID NO.3 cloned as primer; Preferably, adopt temperature fall reverse transcription-polymerase chain amplification method to clone the cDNA fragment of described Cyp26a1 gene in the described method.
Again on the one hand, the invention provides the purposes of above-mentioned recombinant plasmid in preparation Mammals antifertility gene vaccine, preferably, described Mammals is selected from rat, mouse, monkey, rabbit and pig.
The present invention also provides the purposes of said gene vaccine in the Mammals antifertility drug, and preferably, described Mammals is selected from rat, mouse, monkey, rabbit and pig.
The present invention also provides the purposes of said gene vaccine in the prevention and/or the control plague of rats in addition.
In sum, the present invention overcomes the blank that prior art is not also used gene vaccine the antifertility control plague of rats, a kind of gene vaccine of the obviously antifertility control plague of rats is provided, this vaccine is made up of the carrier for expression of eukaryon PCR 3.1 that contains the proteic gene of the 26 superfamily A polypeptide 1-Cyp26a1 of Codocyte cytochrome p 450 family and filter out, the proteic gene of the described Codocyte cytochrome p 450 26 superfamily A polypeptide 1-Cyp26a1 of family is the total length complementary DNA (cDNA) gene of SD rat uterus, and this gene has been logined gene library (Cyp26a1:DQ317305).The present invention pushes the antifertility gene vaccine of laboratory development to industrialization, controls the plague of rats effectively, keeps the eubiosis, provides new way for the mankind control fertility simultaneously.
This shows that the advantage of pCR3.1-Cyp26a1 gene vaccine provided by the present invention is: (1) required immunity amount is less, and 20～50 micrograms get final product; (2) gene vaccine can efficiently excite humoral immunization and cellular immunization simultaneously, and is stronger than other immune means immune efficacy; (3) not only have the effect of effective reduction mouse fertility, and can also obviously reduce newborn newborn mouse effect; (4) genetic immunization safety, long-acting (immunological competence has long memory), stable and simple operation.
Description of drawings
Fig. 1 is the total RNA electrophorogram in pregnant rat uterus in the embodiment of the invention 1.
Fig. 2 is by the cDNA fragment of Cyp26a1 gene and the recombinant plasmid spectrogram of carrier for expression of eukaryon pCR 3.1 structures in the embodiment of the invention 2.
Embodiment
Below the invention will be further described by specific embodiment.Should be understood that following examples only are used to illustrate the present invention, and be not used in the scope of the present invention that limits.
In following each embodiment, related extraction cell total rna, enzyme cut, the concrete operation method of plasmid purification, RT-RCR method, ELISA method, cell transfecting equimolecular biological means and technology is referring to works such as " fine works molecular biology experiment guide " F. Ao Sibai, Yan Ziying etc. translate, Science Press, 1998.
In following each embodiment, employed laboratory animal Spreqne-Dawley rat, BaLb/c mouse and Kunming white (KMB) mouse are all available from Beijing Vital River Experimental Animals Technology Co., Ltd..
Embodiment 1: the cDNA total length of clone's rat uterus Cyp26a1
Present embodiment is the cDNA full-length clone of rat uterus Cyp26a1, and concrete steps are as follows:
(1) animal tissues draws materials: choose sexual maturity, the Spreqne-Dawley female rats of body weight 220-260g mates mating in oestrus and male rat (1: 1), gets pregnant D3-D6 uterus, picks clean mesenteric tissue and embryo.
(2) extraction of the total RNA of gravid uterus and purifying: adopt the Promega RNAgents of company
Total RNA extraction system (Total RNA Isolation System kit) is extracted the total RNA of gravid uterus, use the TRIzol single stage method to extract total tissue RNA, concrete operations are as follows: the about 50-100mg of 1ml TRIzol and uterine cancer cell adds in the homogenate pipe of sterilization, shreds tissue with the sterilization scissors, changes the Eppendorf pipe after the homogenate fragmentation over to, add the 0.2ml chloroform, shake 15sec, room temperature leaves standstill 2-3min, and 12, the centrifugal 15min of 000rpm (4 ℃), get supernatant to new Eppendorf pipe, add the 0.5ml Virahol, gently mixing, room temperature leaves standstill 10min, 12, the centrifugal 10min of 000rpm (4 ℃) abandons supernatant, add 1ml 75% ethanol washing and precipitating, 7, the centrifugal 5min of 500rpm (4 ℃) (this step repeats twice), vacuum seasoning, the water 500 μ L dissolving that adds nuclease free (Nuclease-free) ,-80 ℃ of preservations.Getting 5 μ L sample solutions carries out 1.5% denaturing formaldehyde sepharose (containing 0.2 μ g/mL EB) electrophoresis detection (concrete grammar is referring to Jian-Jun Chang, Jing-Pian Peng
*YingYang, Jing-LingWang, LiXu (2006) Study on the Anti-fertility Effects ofthe Plasmid DNA Vaccine Expressing Partial brLDH-C4.Reprod.131:183-192).Gel band analytical results shows in prepared sample that as shown in Figure 1 the amount of 28SrRNA is about two times of 18S rRNA.
(3) design Auele Specific Primer: according to the highly conserved sequence of people, mouse Cyp26a1 gene order beginning codon and terminator codon outer end, the Auele Specific Primer (having restriction enzyme site) of design amplification total length Cyp26a1 gene, its sequence is as follows:
Upstream primer (SEQ ID NO.2):
5′-CG
A?AGC?TT(HindIII)A?TGG?GGC?TCC?CGG?CGC?TGC?T-3′；
Downstream primer (SEQ ID NO.3):
5′-CG
CTC?GAG(XhoI)TCAGATATCTCCCTGGAAGTGG-3′。
(4) clone and amplification Cyp26a1 gene: adopt temperature fall reverse transcription-polymerase chain amplification method (TDRT-PCR, Touch-down reverse transcription polymerase chainreaction) clonal expansion rat uterus Cyp26a1 gene cDNA sequence, concrete operations are as follows: the reaction system of 50 μ L is by AMV/Tf1 5 * reaction buffer of 10 μ L, the dNTPs mixture of 2mM, the MgSO of 2mM
4, the upstream primer of 1 μ M and upstream primer, the AMV reversed transcriptive enzyme (the promega product is available from the flat science and technology limited Company in pool, Beijing) of 0.1U/ μ L, the RNasin of 0.1U/ μ L
The water of nucleic acid inhibitor (available from Sigma company), the total RNA of 2 μ g and nuclease free is formed, and covers 50 μ L nuclease free mineral oil.Reverse transcription reaction is at 48 ℃ of reaction 45min, then at 95 ℃ of deactivation 5min, start the TD-PCR amplified reaction, concrete operations are as follows: add the Tf1 archaeal dna polymerase (the promega product is available from Beijing Yili Fine Chemicals Co., Ltd.) of 0.1U/ μ L when first denaturing step, in the landing TD-PCR amplification stage, denaturation temperature is 94 ℃ of 1min, annealing temperature is reduced to 55 ℃ of 1min from beginning 65 ℃ of 1min with the speed of 1 ℃ of per two circulation decline, and elongating temperature is 68 ℃ of 1.5min; Continuing 15 circulations of amplification, extend 10min in 72 ℃ more at last with 58 ℃ of annealing temperatures.(5) reclaim purifying Cyp26a1 cDNA: utilize Wizard
PCR Preps.DNA resin purification system reagent box (the promega product is available from Beijing Yili Fine Chemicals Co., Ltd.) reclaims the Cyp26a1 gene cDNA fragment of purifying TDRT-PCR method amplification.
The present invention clones the full-length gene of rat uterus Cyp26a1, and its sequence is shown in SEQ ID NO.1, and its GeneBank accession number is DQ317305.
Embodiment 2: make up the recombinant plasmid that comprises Cyp26a1
Present embodiment is for the present invention comprises the construction of recombinant plasmid of Cyp26a1, is with the Cyp26a1 full-length cDNA fragment PCR that recombinates
3.1 in the eukaryon expression plasmid (the Invitrogen product is available from the leading Science and Technology Ltd. in Central Plains, Beijing), concrete plasmid map is seen Fig. 2, the recombinant plasmid that is built into is the PCR3.1-Cyp26a1 gene vaccine of antifertility, and concrete operations are as follows:
Embodiment 1 purifying is reclaimed back amplification PCR products Cyp26a1 cDNA fragment, be connected into pMD18-T carrier (the Takara product is available from Beijing six directions trade company limited of stimulating the menstrual flow) and transform DH5 α competent cell (available from the Beijing Quanshijin Biotechnology Co., Ltd).The picking positive colony shakes bacterium, after order-checking is identified correctly, and little upgrading grain pMD18-T-Cyp26a1 (adopt little upgrading grain test kit, available from TIANGEN Biotech (Beijing) Co., Ltd., concrete operation method is referring to the test kit specification sheets).With Hind III/Xba I at 37 ℃ of double digestion pMD18-T-Cyp26a1 and pCR3.1, product purification reclaims and adopts Qia28704 glue to reclaim test kit (available from Beijing North instrument great waves commerce and trade company limited, concrete operation method is referring to the test kit specification sheets) after, connection under dna ligase (the promega product is available from Beijing Yili Fine Chemicals Co., Ltd.) effect obtains pCR3.1-Cyp26a1 with Cyp26a1 fragment and pCR3.1.Through enzyme cut and check order identify correct after, extract pCR3.1-Cyp26a1 in a large number according to the big upgrading grain of Qiagen test kit operation steps, and utilize ultraviolet spectrophotometer (U.S. Beckman, model DU530) quantitative.
Embodiment 3: the vivoexpression of pCR3.1-Cyp26a1 gene vaccine is identified
Present embodiment is the pCR3.1-Cyp26a1 gene vaccine constructed to embodiment 2, carry out the evaluation of outer transient expression of external source Cyp26a1 genosome and expression contents, be divided into Cyp26a1 gene in vitro mRNA and express and the external protein expression two portions of external source Cyp26a1.
(1) Cyp26a1 gene in vitro mRNA expresses:
Set up the external transient expression system of human cervical carcinoma's (HeLa) cell and Chinese hamster ovary (CHO) cell (available from basis institute of Beijing consonance medical university cell centre), concrete operations are as follows: the PCR3.1-Cyp26a1 eukaryon expression plasmid is through liposome transfection, respectively at 24 hours, 48 hours, 72 hours collection nutrient solutions, extract the transfection HeLa cell of collection and total RNA of Chinese hamster ovary celI, adopt RT-PCR methods analyst Cyp26a1 gene in vitro to express.The RT-PCR method amplifies Cyp26a1 cDNA band, the result shows: the pCR3.1-Cyp26a1 eukaryon expression plasmid can efficiently express on the mRNA level in HeLa cell and the external transient expression system of Chinese hamster ovary celI, and the expression amount of mRNA level exceeds 186% and 172% respectively than control group (empty plasmid that does not contain the Cyp26a1 gene).
(2) the external protein expression of external source Cyp26a1:
Adopt the method detection transfection HeLa cell of immunofluorescence and the proteic expression of Cyp26a1 of Chinese hamster ovary celI, concrete operations are as follows: HeLa cell and the Chinese hamster ovary celI got behind the transfection 48h add fixedly 1h of 4% Paraformaldehyde 96 room temperature, 0.1%Triton X-100 adds the penetrating 10min of 0.1% trisodium citrate, PBS rinsing 3 times, 1%BSA room temperature sealing 1h then adds 4 ℃ of overnight incubation of antiserum(antisera) (immune serum be anti-) of dilution in 1: 50 again.After PBS thoroughly cleans, add the sheep anti mouse two anti-(the Jackson product is available from Bioisystech Co., Ltd of China fir Golden Bridge in Beijing) of FITC mark, hatch 1h for 37 ℃, after the PBS rinsing with 20 μ g/mL propidium iodides (PI) at 4 ℃ of dyeing 2min, PBS rinsing, anti-quencher mounting.Observation analysis under laser confocal microscope (German ZEISS company, model LSM 510META) 488nm and the 564nm excitation wavelength.Immunofluorescence result confirms that PCR3.1-Cyp26a1 can produce the Cyp26a1 albumen of biologically active in Hela cell and the external transient expression system of Chinese hamster ovary celI, and the expression amount of protein level is than control group (empty plasmid) difference high 172% and 146%.
Embodiment 4: the expression in vivo of pCR3.1-Cyp26a1 gene vaccine is identified
Present embodiment is the pCR3.1-Cyp26a1 gene vaccine constructed to embodiment 2, carries out the evaluation of Cyp26a1 genetic expression of source, inside and outside and expression contents, and concrete operations are as follows:
The BALB/C mice machine is divided into 3 groups, 25 every group, experimental group intramuscular injection 50 μ gpCR3.1-Cyp26a1 gene vaccines; Control group 1 intramuscular injection 50 μ g PCR3.1 empty plasmid DNA; Control group 2 intramuscular injection 100 μ L physiological saline.Muscle, liver and the spleen tissue of the 3rd week back collection pCR3.1-Cyp26a1 immune balb/c mice, extract total tissue RNA and (adopt conventional Trizol method, the Invitrogen product, available from the luxuriant strong couple stars in Beijing Science and Technology Ltd., concrete operation method is referring to the test kit specification sheets), RT-PCR detects external source Cyp26a1 gene at the intravital expression of BALB/C mice.Detected result shows, behind the pCR3.1-Cyp26a1 dna gene vaccine in muscle, liver and the spleen tissue cell Cyp26a1 on the mRNA level, express, the average expression amount of mRNA level exceeds 286% than control group (empty plasmid).
Embodiment 5: ELISA detects the detection of pCR3.1-Cyp26a1 gene vaccine humoral immunoresponse(HI)
Present embodiment is the pCR3.1-Cyp26a1 gene vaccine constructed to embodiment 2, carries out ELISA and detects the BALB/C mice humoral immunoresponse(HI), and concrete operations are as follows:
The experiment grouping is identical with embodiment 4 with PCR3.1-Cyp26a1 gene vaccine injection system and position.With the serum of the intramuscular injection 50 μ g PCR3.1 empty plasmid DNA of the normal serum (not penetrating any plasmid) of 1: 100 times of dilution and 1: 100 times of dilution respectively in contrast, collect the dilution proportion that the serum of PCR3.1-Cyp26a1 dna gene vaccine BALB/C mice after three weeks was pressed 1: 100 to 1: 6000, BALB/C mice uterogolbin with 2 μ g/ holes is made envelope antigen, goat anti-mouse igg-HRP 50 μ L/ holes of 1: 2000 are done two and are resisted, and measure 490nm OD value with microplate reader (U.S. Bio-Rad3550Microplate Reader).Adopt the ELISA method to detect humoral immunoresponse(HI), detected result shows that body can produce high titre antibody (antibody titers＞1: 4500) behind the pCR3.1-Cyp26a1 dna gene vaccine.
Go out to comprise the recombinant plasmid of Cyp26a1 respectively with pCMV4 and two kinds of vector constructions of pVAX1 with reference to the method described in the embodiment 2, carry out immune mouse ELISA according to the method described above and detected pCMV4-Cyp26a1, the detection of pVAX1-Cyp26a1 gene vaccine humoral immunoresponse(HI), detected result shows, the antibody titers that body can produce behind pCMV4-Cyp26a1 and the pVAX1-Cyp26a1 dna gene vaccine was respectively 1: 3500 and 1: 2800, therefore, determine that the obtained immunne response positive effect of pCR3.1-Cyp26a1 gene vaccine is better than the gene vaccine of other carriers and Cyp26a1 gene cDNA structure
Embodiment 6: the safety evaluation of pCR3.1-Cyp26a1 gene vaccine
The constructed pCR3.1-Cyp26a1 gene vaccine of present embodiment assessment embodiment 2 is to the security of immune animal, and concrete operations are as follows:
Detect various dose (20 μ g-300 μ g) 35 BALB/C mice of PCR3.1-Cyp26a1 dna gene vaccine, none is only dead after 180 days; Carrying out the toxicity behind the various dose PCR3.1-Cyp26a1 gene vaccine injection animal and the observation of conventional physical signs measures, (concrete grammar is referring to " practical immunocytochemistry and making nucleic acid molecular hybridization technology " Cai Wenqin; Wang uncle Yun chief editor to utilize histochemical method, Sichuan science tech publishing house, 1994) determine animal tissues's pathological change after the vaccinate: to brain, the heart, liver, spleen, lung, kidney, respective organization such as uterus and muscle tissue organ carries out frozen section, at microscope (Japanese Nikon instrument model Nikon 80i Microscope System; Cold CCD SPOT) observes down, do not find that there is pathological change in animal tissues.
Embodiment 7: the experiment of pCR3.1-Cyp26a1 dna gene vaccine antifertility
Present embodiment carries out immune antifertility experiment for using the constructed pCR3.1-Cyp26a1 gene vaccine of embodiment 2 to different mouse kinds:
(1) immune antifertility experiment-I
Choose sexual maturity, 45 of the Spreqne-Dawley of body weight 220-260g (SD) female rats, 45 of male rats.Animal rearing under manually operated condition, raising temperature about 25 ℃, 12h:12h periodicity of illumination, free choice feeding, drinking-water.45 female sd inbred rats are divided into three groups at random, 15 every group.24h injects 100 μ L immunological adjuvants, 0.25% PROCAINE HCL, PHARMA GRADE before the immunity plasmid, alcohol partly sterilised before the injection, 3 injections of inoculation position leg muscle.
Control group 1: inoculate 100 μ l physiological saline.
Control group 2: inoculate 100 μ l pCR3.1 empty plasmids (containing pCR3.1 empty plasmid 50 μ g).
Experimental group: inoculate 100 μ l PCR3.1-Cyp26a1 gene vaccines (containing PCR3.1-Cyp26a1 gene vaccine 50 μ g),
Above-mentioned 3 groups of interval 1 all immunity once are total to immunoprophylaxis three times.Mate mating at 1: 1 with male rat after three immunity, testing bolt positive is gestation the 1st day (concrete grammar of testing bolt is referring to people such as " mice embryonic operation experiments guide " A. Na Ji work, Science Press, 2004).
In the experiment of immunity antifertility, the fertility of control group 1 (physiological saline treatment group) is 93%, and the fertility of control group 2 (pCR3.1 treatment group) is 100%, and the fertility of experimental group (PCR3.1-Cyp26a1 gene vaccine treatment group) is 35.7%.Compare with control group, the experimental group fertility significantly reduces (P＜0.01), and experimental group neonate rat number of elements (F1) also significantly is lower than control group.Concrete experimental result statistics, as shown in table 1.
Table 1 gene vaccine of the present invention is to the immune antifertility experimental result of SD female rats
* represents difference extremely significantly (P＜0.01)
Conclusion: the pCR3.1-Cyp26a1 dna gene vaccine has the effect that reduces the fertility of Spreqne-Dawley female rats, and the effect of comparing antifertility with control group 2 with control group 1 is (p＜0.01) significantly.
(2) immune antifertility experiment-II
Choose 75 of sexually matured female BaLb/c mouse, 75 of male BaLb/c mouse.Animal rearing under manually operated condition, raising temperature about 25 ℃, 12h:12h periodicity of illumination, free choice feeding, drinking-water.75 female BaLb/c mouse are divided into three groups at random, 25 every group.24h injects 100 μ L immunological adjuvants, 0.25% PROCAINE HCL, PHARMA GRADE before the immunity plasmid, alcohol partly sterilised before the injection, 3 injections of inoculation position leg muscle.
Control group 1: inoculate 100 μ l physiological saline.
Control group 2: inoculate 100 μ l pCR3.1 empty plasmids (containing pCR3.1 empty plasmid 50 μ g).
Experimental group: inoculate 100 μ l pCR3.1-Cyp26a1 gene vaccines (containing pCR3.1-Cyp26a1 gene vaccine 50 μ g),
Above-mentioned 3 groups of interval 1 all immunity once are total to immunoprophylaxis three times.Mate mating at 1: 1 with male BaLb/c mouse after three immunity, testing bolt positive is gestation the 1st day.
Immunity antifertility experimental result statistics
In the experiment of immunity antifertility, the fertility of control group 1 (physiological saline treatment group) is 82.6%, and the fertility of control group 2 (pCR3.1 treatment group) is 81.8%, and the fertility of experimental group (pCR3.1-Cyp26a1 gene vaccine treatment group) is 45.8%.Compare with control group, the fertility of experimental group obviously reduces (P＜0.05), and experimental group newborn mice number of elements (F1) significantly is lower than control group (P＜0.01).Concrete experimental result statistics, as shown in table 2.
Table 2 gene vaccine of the present invention is to the immune antifertility experimental result of BaLb/c female mice
* represents difference extremely significantly (P＜0.01)
Conclusion: the pCR3.1-Cyp26a1 dna gene vaccine has the effect that reduces female BaLb/c mouse fertility, and the effect of comparing antifertility with control group 2 with control group 1 is (p＜0.01) obviously.
(3) immune antifertility experiment-III
Choose 75 of sexually matured female Kunming white (KMB) mouse, 75 of male Kunming white mouse.Animal rearing under manually operated condition, raising temperature about 25 ℃, 12h:12h periodicity of illumination, free choice feeding, drinking-water.75 female Kunming white mouse are divided into three groups at random, 25 every group.24h before the immunity plasmid injects 100 μ L immunological adjuvants, 0.25% PROCAINE HCL, PHARMA GRADE, 3 injections of inoculation position leg muscle.
Control group 1: inoculate 100 μ l physiological saline.
Control group 2: inoculate 100 μ l pCR3.1 empty plasmids (containing pCR3.1 empty plasmid 50 μ g).
Experimental group: inoculate 100 μ l PCR3.1-Cyp26a1 gene vaccines (containing PCR3.1-Cyp26a1 gene vaccine 50 μ g).
Above-mentioned 3 groups of interval 1 all immunity once are total to immunoprophylaxis three times.Mate mating at 1: 1 with male mice after three immunity, testing bolt positive is gestation the 1st day.
Immunity antifertility experimental result statistics
In the experiment of immunity antifertility, the fertility of control group 1 (physiological saline treatment group) is 100%, and the fertility of control group 2 (pCR3.1 treatment group) is 92%, and the fertility of experimental group (processing of PCR3.1-Cyp26a1 gene vaccine) is 37.5%.Compare with control group, the experimental group fertility obviously reduces (P＜0.01), and experimental group newborn mice number of elements (F1) significantly is lower than control group (P＜0.01).Concrete experimental result statistics, as shown in table 3.
Table 3 gene vaccine of the present invention is to the immune antifertility experimental result of female KMB female mice
* represents difference extremely significantly (P＜0.01)
Conclusion: the pCR3.1-Cyp26a1 dna gene vaccine has the effect that reduces the fertility of female Kunming white (KMB) mouse, and the effect of comparing antifertility with control group 2 with control group 1 is (p＜0.01) significantly.
Sequence table
＜110〉Institute of Zoology, Academia Sinica
＜120〉antifertility recombinant plasmid, gene vaccine and its production and use
<130>DIC09110031
<160>3
<170>PatentIn?version?3.3
<210>1
<211>1766
<212>DNA
＜213〉SD rat Cyp26a1 cDNA
<400>1
acgcgggggc?gagggcggcg?gcggcaggtg?gcgcgggagg?cttgctgcgt?gccatggggc 60
tcccggcgct?gctggccagt?gctctctgca?ccttcgtgct?gccgctgctg?ctcttcctgg 120
cggcgctcaa?gctctgggac?ctgtactgtg?tgagcagccg?cgatcgcagc?tgcgctctcc 180
ccttgccccc?gggtaccatg?ggcttcccat?tctttgggga?aacattgcag?atggtgctgc 240
agcggaggaa?gtttctgcag?atgaagcgca?ggaaatacgg?cttcatctac?aagacgcatc 300
tgtttgggcg?gcccacggtg?cgagtgatgg?gcgcggataa?tgtgcggcgc?atcttgctgg 360
gggagcaccg?gttggtgtca?gtgcactggc?ctgcttcggt?gcgcaccatc?ctgggcgccg 420
gctgcctctc?caacctgcat?gattcctcgc?acaagcagcg?aaagaaggtg?attatgcagg 480
ccttcaaccg?agaggcgctt?cagtgctacg?tgccagtgat?tgctgaagaa?gtgagcggtt 540
gtctggagca?gtggctaagc?tgcggcgagc?gcggcctcct?ggtctacccc?gaggtgaagc 600
gcctcatgtt?ccgcatcgcc?atgcgcatcc?tgctgggctg?cgagccgggt?ccagcgggcg 660
gcggggaaga?cgagcagcag?ctagtggagg?ctttcgagga?gatgacccgc?aatctcttct 720
ctctccccat?tgacgtgccc?tttagcgggc?tgtaccgggg?cgtgaaggcg?cggaacctta 780
tccacgcgcg?catcgaggag?aacattcggg?ccaagatccg?ccggcttcag?gccgcagagc 840
cggatgcggg?ctgcaaggac?gcactgcagc?tcttgattga?gcactcatgg?gagagaggag 900
agaggctgga?tatgcaggca?ctaaaacaat?cgtccacaga?gctcctcttt?ggtggccatg 960
aaactacagc?cagtgcagcc?acatcactga?tcacctacct?aggactctac?ccacatgtcc 1020
tccaaaaagt?tcgagaagag?ataaagagca?agggcttact?ttgcaagagc?catcacgagg 1080
acaagttaga?catggaaact?ttggaacagc?tcaaatacat?tgggtgtgtt?attaaggaga 1140
cccttcgatt?gaatcctccg?gttccgggag?ggtttcgggt?ggctctgaag?acttttgagc 1200
tgaacggtta?ccagattccc?aaggggtgga?atgttattta?cagtatctgt?gacacccatg 1260
acgtggcaga?cagcttcact?aacaaggagg?agtttaatcc?cgaccgattt?acatcgcttc 1320
atccagagga?cacctccagg?ttcagtttca?ttccatttgg?aggaggcctt?cggagctgcg 1380
taggcaaaga?gtttgcaaaa?attcttctta?agatatttac?cgtggagctg?gccagacgtt 1440
gtgactggca?gctgctaaat?ggacctccta?caatgaagac?aagccccacc?ttctaccctg 1500
tggacaatct?tcctgcaaga?ttcacccact?tccagggaga?tatctgacag?ctatttcagt 1560
tcttggactc?atttgaagtg?tacattgttt?ttttttttta?aatagtgtca?tgttgccttt 1620
atttaatttc?taaatgtata?gtataatatt?tatatgtctc?tactacagcc?ccatggtctt 1680
taaatattaa?aataatgaat?ttgtatgatt?tcccaataaa?gtaaaatttt?aaagtgtaaa 1740
aaaaaaaaaa?aaaaaaaaaa?aaaaaa 1766
<210>2
<211>28
<212>DNA
＜213〉artificial sequence
<400>2
cgaagcttat?ggggctcccg?gcgctgct 28
<210>3
<211>30
<212>DNA
＜213〉artificial sequence
<400>3
cgctcgagtc?agatatctcc?ctggaagtgg 30
Claims (10)
1. recombinant plasmid that is used for Mammals antifertility gene vaccine, its cDNA fragment and carrier for expression of eukaryon by the Cyp26a1 gene is formed, and carrier for expression of eukaryon is preferably pCR 3.1.
2. recombinant plasmid according to claim 1 is characterized in that, the cDNA fragment of described Cyp26a1 gene is derived from the Spreqne-Dawley rat uterus, and its base sequence is preferably shown in SEQ IDNO.1.
3. the bacterial strain that comprises claim 1 or 2 described recombinant plasmids; Preferably, described bacterial strain is an intestinal bacteria competence DH5 α bacterial strain.
4. the cell that comprises claim 1 or 2 described recombinant plasmids; Preferably, described cell is selected from human cervical carcinoma cell and Chinese hamster ovary cell.
5. Mammals antifertility gene vaccine, wherein said vaccine comprises claim 1 or 2 described recombinant plasmids.
6. gene vaccine according to claim 5 is characterized in that described vaccine also comprises adjuvant; Preferably, to be selected from percentage concentration be 0.25% PROCAINE HCL, PHARMA GRADE to described adjuvant.
7. a method for preparing claim 1 or 2 described recombinant plasmids is characterized in that, the cDNA fragment of Cyp26a1 gene as described in the base sequence of described method employing shown in SEQ ID NO.2 and SEQ ID NO.3 cloned as primer; Preferably, this method adopts temperature fall reverse transcription-polymerase chain amplification method to clone the cDNA fragment of described Cyp26a1 gene.
8. claim 1 or the 2 described recombinant plasmids purposes in preparation Mammals antifertility gene vaccine; Preferably, described Mammals is selected from rat, mouse, monkey, rabbit and pig.
9. claim 5 or the 6 described gene vaccines purposes in preparation Mammals antifertility drug; Preferably, described Mammals is selected from rat, mouse, monkey, rabbit and pig.
Claim 5 or 6 described gene vaccines in prevention and/or control the purposes in the plague of rats.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1425769A (en) * | 2002-12-26 | 2003-06-25 | 复旦大学附属妇产科医院 | Process for preparing human chorionic gonadotropin beta subunit |
| CN1440811A (en) * | 2002-07-09 | 2003-09-10 | 复旦大学附属妇产科医院 | Contraception vaccine cross-linked with molecular adjuvant |
| CN101161287A (en) * | 2006-10-12 | 2008-04-16 | 复旦大学附属妇产科医院 | A anastomosing protein contraception vaccine carrying human molecule adjuvant and its preparing method |
-
2010
- 2010-03-18 CN CN 201010129212 patent/CN102191272A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1440811A (en) * | 2002-07-09 | 2003-09-10 | 复旦大学附属妇产科医院 | Contraception vaccine cross-linked with molecular adjuvant |
| CN1425769A (en) * | 2002-12-26 | 2003-06-25 | 复旦大学附属妇产科医院 | Process for preparing human chorionic gonadotropin beta subunit |
| CN101161287A (en) * | 2006-10-12 | 2008-04-16 | 复旦大学附属妇产科医院 | A anastomosing protein contraception vaccine carrying human molecule adjuvant and its preparing method |
Non-Patent Citations (2)
| Title |
|---|
| 《Genbank》 20051228 Xia,H.-F.等 DQ317305.1 第1页 2-10 , * |
| 《JOURNAL OF CELLULAR PHYSIOLOGY》 20100128 BING-CHEN HAN等 Retinoic Acid-Metabolizing Enzyme Cytochrome P450 26a1 (Cyp26a1) Is Essential for Implantation: Functional Study of Its Role in Early Pregnancy 第471-479页 1-10 第223卷, * |
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Application publication date: 20110921 |