CN100404679C - GRF(1-32)and SS-HbsAg fusion gene combination and its constructed expression system and pharmaceutical preparation - Google Patents

GRF(1-32)and SS-HbsAg fusion gene combination and its constructed expression system and pharmaceutical preparation Download PDF

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CN100404679C
CN100404679C CNB2005100171542A CN200510017154A CN100404679C CN 100404679 C CN100404679 C CN 100404679C CN B2005100171542 A CNB2005100171542 A CN B2005100171542A CN 200510017154 A CN200510017154 A CN 200510017154A CN 100404679 C CN100404679 C CN 100404679C
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grf
hbsag
gene
growth
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CN1769453A (en
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张永亮
刘松财
戴建威
任晓慧
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Guangzhou Chuang Xi Wan biological science and Technology Co., Ltd.
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张永亮
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Abstract

The present invention relates to a combination method for promoting genes which are related to the animal growth, and a constructed eukaryotic expression vector. The genes which are related to the growth comprise growth hormone releasing factors GRF (1 to 32), growth hormone release inhibiting hormone SS and hepatitis B virus surface antigen (HBdAg) fusion gene. The GRF (1 to 32) and the SS-HBsAg fusion genes are combined and constructed into an expression plasmid pIRES-GRF-HBsAg-SS, or cloned into an adeno-associated virus vector. The bare plasmids and plasmids wrapped by PLGA microspheres are injected into the animal muscular tissues, and both the bare plasmids and plasmids wrapped by PLGA microspheres can promote the animal growth. The two genes are combined, and therefore, the growth promotion effect is superior to the effect of each individual gene. The PLGA microsphere wrap improves the genic expression level, and has an effect which is superior to the effect of bare plasmid injection. The present invention provides a novel and effective method for applying gene transfer technique to promoting the animal growth.

Description

GRF(1-32) with SS-HBsAg fusion gene combination and the expression system and the pharmaceutical preparation that are built into
Technical field:
The present invention relates to a kind of assortment of genes that promotes growth of animal, with GRF (1-32) and the combination of SS-HBsAg fusion gene, and be built into expression system, also having made has the pharmaceutical preparation of actuating thing and people's growth effect, belongs to the genomic medicine and the applied technical field thereof that promote growth of animal.
Background technology:
Tethelin (growth hormone, hereinafter to be referred as: be the major hormone of reconciling animal and human's growth GH), have the effect that promotes growth, raising lean ratio and suppress lipogenesis, in livestock industry, have important use to be worth.It is somatotropin releasing factor (growth hormone releasing factor that the level height of GH mainly is subjected to the positivity regulatory factor, hereinafter to be referred as: GRF) or claim growth hormone releasing hormone (growth hormone releasinghormone, hereinafter to be referred as: GHRH) and the negativity regulatory factor be somatostatin (somatostatin, hereinafter to be referred as: SS) or be called for short the regulation and control of Somatostatin.GRF is controlling the pulsatile secretion of GH, and SS is controlling basal secretion, and SS has antagonistic action to GRF, influences the GRF expression of gene.
Carrier for expression of eukaryon is arrived in the gene clone of GRF, and transfer to the animal expression in vivo, or SS fusion gene induced animal is produced SS antibody, can promote the release of GH, thereby play promotes growth of animal and the effect that improves production performance.With GRF and SS and combination of HBsAg fusion gene and structure coexpression system, can make the two play synergy, both express GRF and HBsAg-SS simultaneously and induced the antibody that produces SS, and strengthened the positive regulating and controlling effect of GH and face upward the effect of system negative regulation, played the promotes growth effect that is better than every individual gene.
Summary of the invention
The invention provides a kind of GRF (1-32) and the combination of SS-HBsAg fusion gene.
The present invention also provides and has used the eukaryotic expression system that the said gene combination makes up.
The present invention further provides the above-mentioned eukaryotic expression system of application and made pharmaceutical preparation.
Technical solution of the present invention may further comprise the steps:
With the combination between GRF and SS and hepatitis B surface antigen (HBsAg) fusion gene.
1, through the improved GRF of gene structure (1-32) gene clone, its improved gene order is as follows:
ATG CTG CTC TGG GTG TTC TTC CTC GTC ACC CTCACC CTC AGC ACC GGC TCC CTC AGC TCC CTG CCCTCC CAG CGC CTC AGG ATG CCG CGG CAC GTG GATGCC ATC TTC ACC CAG AAC TAC CGG AAG GTG CTGGCA CAG CTC TCT GCC CGA AAG CAC CTC CAG GACATC TCT AGC AGG CAG CAG GAG
2, will transform back GRF (1-32) gene clone to pcDNA3, make up carrier for expression of eukaryon pcDNA3GRF (1-32);
Clone SS gene with the HBsAg gene fusion, and is cloned into pIRES, obtains pIRES-HBsAg-SS;
GRF (1-32) and HBsAg-SS gene are cloned into pIRES simultaneously, obtain double gene coexpression carrier pIRES-GRF-HBsAg-SS.
Or above-mentioned dual-gene combination clone carried (rAAV2) to adeno-associated virus, obtain the rAAV2 expression system rAAV2-GRF-HBsAg-SS of dual-gene combination.
A kind of eukaryotic expression system is made pharmaceutical preparation, it is characterized in that: the plasmid that the expression system that is built into by the combination of GRF (1-32) and SS-HBsAg fusion gene is made, use the above-mentioned plasmid of a large amount of extractions of alkaline denaturation and make pharmaceutical preparation, be expelled in the animal body by following mode:
The gymnoplasm grain directly or by PLGA microballoon parcel is expelled to animal muscle tissue:
1 hind leg forelimb muscle direct injection is expressed in muscle tissue;
2 are prepared into poly(lactic acid) and polyglycolic acid (PLGA) microballoon (about diameter 3um), intramuscular injection with plasmid.
Show through experimentation on animals: above-mentioned plasmid can both promote the growth of animal, and particularly two kinds of double gene expression modes promptly have better promotes growth effect with pIRES-GRF (1-32)-PACAP.Concrete experimental result is seen attached list.
Different plasmids are to the growth promoting function (is example with the rabbit experiment) of animal:
Table 1PLGA parcel and naked GRF gene expression plasmid are to the influence of rabbit average daily gain
Figure C20051001715400051
Annotate: GRF-MS:PLGA microballoon parcel pcDNA3-GRF (1-32) plasmid, GRF:pcDNA3-GRF (1-32) gymnoplasm grain.A: with empty microballoon group than difference remarkable (P<0.05); B: with the salt solution group than difference remarkable (P<0.05); C: organize than difference remarkable (P<0.05) with GRF.
Table-2GRF and HBsAg/SS gene and combination thereof are to the influence (g) of rabbit growth
Time (d) Physiological saline p-GRF p-HBsAg/SS p-GRF-HBsAg/SS
15 333.75± 36.84 500.00±25.35** 413.25±29.61* 528.75±29.69**
30 488.75± 32.92 767.50±33.58** 785.00±69.26** 848.75±54.95**
45 871.25± 58.23 1170.00±38.91** 1135.00±65.44** 1221.25±59.00**
Annotate: p-GRF:PLGA parcel GRF expression plasmid; P-HBsAg/SS:PLGA parcel HBsAg/SS gene expression plasmid; P-GRF-HBsAg/SS:PLGA parcel GRF and HBsAg/SS double gene expression plasmid.
**P<0.01
*P<0.05
The animal of above-mentioned indication comprises mouse, rabbit, pig, ox, sheep and furbearer etc.
Positively effect of the present invention is: GRF is carried out genetic modification to strengthen its biologic activity, two kinds of monogenic expression vectors and a kind of dual-gene expression vector have been made up, proposed the novel method of GRF and HBsAg-SS fusion gene coexpression, above-mentioned three kinds of plasmids all have growth promoting function to animal.The new ideas that plasmid promotes growth of animal have been proposed to use, for the growth and the raising production performance of protecting the precession thing provides new method.
Description of drawings
Fig. 1 is constructed plasmid figure of the present invention.
Fig. 2 is the PLGA parcel plasmid microballoon of the present invention's preparation.
Fig. 3 is the cloning process synoptic diagram of plasmid of the present invention.
Fig. 4 identifies figure for PCR.
Fig. 5 identifies figure for double digestion.
Embodiment:
By following examples the present invention is described for example further, and do not limit the present invention in any way, under the prerequisite that does not deviate from technical solution of the present invention, any change or change that those of ordinary skills that the present invention did are realized easily all will fall within the claim scope of the present invention.
Embodiment 1
Press the sequence of GRF (1-32), method synthetic gene segment with chemosynthesis, two ends are provided with the restriction enzyme site of Eco RI and Hind III, cut GRF (1-32) gene and pcDNA3 with above-mentioned enzyme enzyme simultaneously, connect and conversion JM109 competent cell through the T4 ligase enzyme, identifying positive colony, both had been carrier for expression of eukaryon pcDNA3-GRF (1-32).
Embodiment 2
The gene of chemosynthesis SS, with pcr amplification HBsAg gene, preparation fusion gene HBsAg-SS.With the method (with embodiment 2) that Sma I, Xba I enzyme are cut HBsAg-SS being cloned into pIRES, identifying positive colony, both had been carrier for expression of eukaryon pIRES-HBsAg-SS.
Embodiment 3
With the GRF gene among the pcDNA3-GRF (1-32) with double digestion (with reference to embodiment 1) thereby and subclone obtain GRF (1-32) and HBsAg-SS double gene expression vector pIRES-GRF (1-32)-HBsAg-SS to pIRES-HBsAg-SS.
The cloning process of plasmid is seen Fig. 3.
Method by PCR and double digestion is identified the plasmid result.
See Fig. 4; PCR identifies figure.
Fig. 5: double digestion is identified figure.
Embodiment 4
With the method amplification of dual-gene combination GRF-HBsAg-SS with PCR, enzyme is cut, and makes up shuttle vectors pSNAV-GRF-HBsAg-SS.Design primer two ends add two restriction enzyme sites (Smal I and EcoR I) and the protection base in the shuttle vectors multiple clone site; the PCR reaction of standard corresponding gene of amplification from GRF, SS-HBsAg and GRF-SS-HBsAg plasmid; wherein GRF-SS-HBsAg comprises GRF gene, IVS (intron), IRES (ribosome entry site(RES)) and SS-HBsAg, expresses dual-gene the time guaranteeing.Cut the gene of shuttle vectors and pcr amplification with corresponding restriction endonuclease (SmalI and EcoR I) enzyme, respectively with the agarose purifying and reclaim endonuclease bamhi, with the T4 ligase enzyme corresponding endonuclease bamhi is connected, be transformed into the Bacillus coli cells (JM109) of sensitization, corresponding positive colony is screened and identified to overnight incubation with PCR and double digestion.Extract and the above-mentioned shuttle plasmid that contains range gene of purifying,, after screening, obtain rAAV2-GRF-SS-HBsAg with the cultured BHK-21 cell of Lipofectinamine transfection.
Embodiment 5
Microballoon preparation technology is as follows:
(1) gets three kinds of plasmid p-GRF, p-HBsAg/SS, each 500 μ l of p-GRF-HBsAg/SS solution, put respectively in the 25ml beaker, add CH 2Cl 21.25ml, acetone 0.15ml, PLGA 0.25g, fully dissolving.
(2) ultrasonic power is 15W, the ultrasonic 3S of interval 3S, and ultrasonic time 40~70S makes solution even.
(3) PVA4ml of adding 4% in each beaker, ultrasonic 60~120S.(get 1 suspension and splash into gently in the aqueous solution, can observation sink diffusing).
(4) in each beaker, add 0.4%PVA20ml, room temperature magnetic agitation 8~10h.
(5) with 4 layers of sterilization nylon cloth above-mentioned solution is filtered to-the aseptic centrifuge tube of 40ml (accurately taking by weighing centrifuge tube weight) in, allow solution filter to the greatest extent as far as possible.
(6) with the centrifugal 10min of solution 10000~12000rpm that collects.
(7) get supernatant, the estimation volume carries out nucleic acid content to supernatant simultaneously and measures.
(8) with the resuspended precipitation of 20ml distilled water, by (5) (6) step operation 2 times.
(9) discard supernatant, precipitation is placed-20 ℃ of refrigerators and is preserved.
Be white powder, prepare suspension liquid with physiological saline during injection.
Embodiment 6
With above-mentioned plasmid by different way (with the gymnoplasm grain directly or by PLGA microballoon parcel) be expelled to animal muscle tissue, can promote the growth of animal, the concentration of the intravital IGFI of raising animal.And GRF and SS and HBsAg fusion gene coexpression, the promotes growth effect is higher than individual gene; Intramuscular injection PLGA microballoon parcel plasmid is more effective than gymnoplasm grain promotes growth.Concrete outcome sees Table 1, table 2.
Sequence table
<110〉Zhang Yongliang
<120〉GRF (1-32) and the combination of SS-HBsAg fusion gene and the expression system that is built into and about thing preparation
<130>1
<160>1
<170>PatentIn version 3.3
<210>1
<211>186
<212>DNA
<213〉people (Homo sapiens)
<220>
<222>(1)..(186)
<223>
<400>1
atgctgctct gggtgttctt cctcgtcacc ctcaccctca gcaccggctc cctcagctcc 60
ctgccctccc agcgcctcag gatgccgcgg cacgtggatg ccatcttcac ccagaactac 120
cggaaggtgc tggcacagct ctctgcccga aagcacctcc aggacatctc tagcaggcag 180
caggag 186

Claims (4)

1. a GRF (1-32) gene clone has following gene order:
ATG CTG CTC TGG GTG TTC TTC CTC GTC ACC CTC
ACC CTC AGC ACC GGC TCC CTC AGC TCC CTG CCC
TCC CAG CGC CTC AGG ATG CCG CGG CAC GTG GAT
GCC ATC TTC ACC CAG AAC TAC CGG AAG GTG CTG
GCA CAG CTC TCT GCC CGA AAG CAC CTC CAG GAC
ATC TCT AGC AGG CAG CAG GAG。
2. the described GRF of claim 1 (1-32) makes up and the expression system of structure with the SS-HBsAg fusion gene.
3. claim 2 fusion gene makes up the expression system that is built into, and comprises the pIRES-GRF-HBsAg-SS plasmid expression system, or the AAV2-GRF-HBsAg-SS virus expression systems.
4. preparing the purposes that promotes the growth of animal medicine by the described GRF of claim 1 (1-32) with the expression system that the combination of SS-HBsAg fusion gene is built into.
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CN101804209B (en) * 2010-03-31 2012-07-04 四川大学 PEDF (Pigment Epithelial Derived Factor) gene PLGA nano compound as well as preparation method and use thereof
CN105194662B (en) * 2015-09-16 2018-08-28 华北制药金坦生物技术股份有限公司 Sustained release microsphere agents and preparation method thereof containing recombination hepatitis B surface antigen

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0459747A1 (en) * 1990-05-29 1991-12-04 Eli Lilly And Company Precursor forms of porcine growth hormone releasing factor and related DNA compounds
CN1201831A (en) * 1997-06-05 1998-12-16 中国科学院微生物研究所 Method for preparation of recombined human luteinizing-hormone releasing factor

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0459747A1 (en) * 1990-05-29 1991-12-04 Eli Lilly And Company Precursor forms of porcine growth hormone releasing factor and related DNA compounds
CN1201831A (en) * 1997-06-05 1998-12-16 中国科学院微生物研究所 Method for preparation of recombined human luteinizing-hormone releasing factor

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
. 动物生理生化第八次学术会议论文摘要汇编. 2004
. 动物生理生化第八次学术会议论文摘要汇编. 2004 *
生长激素释放因子(GRF)的基因改造及化学合成. 冯立文等.中国兽医学报,第18卷第6期. 1998
生长激素释放因子(GRF)的基因改造及化学合成. 冯立文等.中国兽医学报,第18卷第6期. 1998 *

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Assignee: Shenzhen agriculture and animal husbandry industry Co., Ltd.

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