CN1316021C - Nerve growth factor cDNA, clone and expresion of Chinese cobra - Google Patents
Nerve growth factor cDNA, clone and expresion of Chinese cobra Download PDFInfo
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- CN1316021C CN1316021C CNB2004100215137A CN200410021513A CN1316021C CN 1316021 C CN1316021 C CN 1316021C CN B2004100215137 A CNB2004100215137 A CN B2004100215137A CN 200410021513 A CN200410021513 A CN 200410021513A CN 1316021 C CN1316021 C CN 1316021C
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Abstract
The present invention relates to a China cobra nerve growth factor NGF cDNA and the cloning and expression thereof, particularly to cDNA and cloning for recombining the China cobra nerve growth factor, the expression of the nerve growth factor, a method for preparing the nerve growth factor by using an expression carrier thereof and the pharmacy use of protein. Total RNA is separated and extracted from a China cobra poison gland, total cDNA is reversely transcribed by an RT-PCR method, a specific cDNA sequence is provided by a primer designed by people, the length of the specific cDNA sequence is 348 bp polynucleotide sequence, the present invention also provides small molecular weight protein successfully expressed in Pichia microzyme, and the molecular weight of the small molecular weight protein is approximately 13000. The recombinant nerve growth factor has the pharmacy use on the aspects of blood sugar reduction, islet function improvement and the treatment of tip end spirit phlegmasia caused by diabetes.
Description
Technical field
The invention belongs to biological technical field, relate to reorganization Chinese cobra (Chinesecobra[Naja naja atra]) nerve growth factor (nerve growth factor, NGF) cDNA and clone, the expression of nerve growth factor and utilize its expression vector to prepare the method and the described proteinic pharmaceutical use of nerve growth factor.
Technical background
(nerve growth factor NGF) is one of most important biological activity protein of neural system to nerve growth factor.It mainly acts on neural system, and as sympathetic neuron, Sensory neurone and forebrain cholinergic neuron, NGF participate in to regulate their growth and differentiation, keeps their normal function, promotes the reparation after their damage.At present NGF reparation and some neuronal degeneration diseases such as treatment presenile dementia after promoting nerve injury have demonstrated important clinical application value.Go out NGF from multiple biomaterial separation and purification at present, but the abundantest being still in multiple snake venom and the male mice submaxillary gland of content.
Diabetes are because the caused disease of the relative or absolute hypoinsulinism of beta Cell of islet.At present, the treatment diabetes adopt methods such as insulin injection or oral hypoglycemic other drug more, and cooperate means such as sitotherapy.In case suffer from diabetes, patient has to take medicine throughout one's life.Attempted by people though the method for engineered method or Transplanted cells is also many, all do not have to find basic solution.The complication of diabetes is a lot, as arteriosclerosis, hypertension, blood dirtyly get involved, infection, peripheral neuritis etc.Though NGF is known by people, and utilize NGF to carry out the treatment of peripheral neuritis, obtained certain achievement.The someone finds Brain Derived Neurotrophic Factor (Brain-derived neurotrophicfactor in recent years, BDNF) can reduce the blood sugar of laboratory animal by the metabolism of regulating body, but do not see the report of relevant NGF reducing blood glucose so far, especially adopt snake venom gland NGF to carry out the report of lowering blood glucose, more unmanned utilization reorganization snake NGF carries out the work of this respect.
Summary of the invention
First purpose of the present invention provides the cDNA sequence of Chinese cobra snake venom reorganization nerve growth factor (NGF).
Second purpose of the present invention provides reorganization NGF cDNA and compiles proteinic aminoacid sequence.
The 3rd purpose of the present invention provides the method for preparing the nerve growth factor of recombinating.
The 4th purpose of the present invention provides the reorganization nerve growth factor at lowering blood glucose and improve islet function, and the pharmaceutical use of the scorching aspect of tip spirit that caused by diabetes of treatment.
The detailed description of technology of the present invention:
Separation and Extraction goes out total RNA from the NNAV gland, utilize the method reverse transcription of RT-PCR to go out total cDNA, the primer that utilizes us to design, a special cDNA sequence is provided, its length is the 348bp polynucleotide sequence, and we also provide a part amount of a successful expression in Pichia yeast to be about 13000 small molecular weight protein.
Chinese cobra nerve growth factor NGF cDNA sequence contains 348 base pairs for to clone from the NNAV gland, nucleotides sequence is classified as:
1 gaagatcatc?ctgtgcataa?cctaggggaa?cattctgtgt?gtgacagtgt?cagtgcctgg
61 gttactaaaa?ctacagcaac?agacatcaaa?ggcaatacgg?tgactgtgat?ggaaaatgta
121?aatcttgata?acaaagtcta?caagcagtac?ttttttgaaa?ccaagtgcaa?aaatccaaat
181?ccagaaccga?gtgggtgcag?gggcattgat?tccagccatt?ggaattctta?ttgcaccgaa
241?acagacacat?ttatcaaggc?attaaccatg?gaaggcaatc?aggcatcctg?gcgcttcatt
301?cggattgaaa?ctgcctgtgt?gtgtgtaatc?actaaaaaaa?aggggaac
This cDNA sequence construct is gone into plasmid pET23b and pPIC9K, be transformed into intestinal bacteria and yeast respectively, induce, give expression to a kind of recombinant protein.Protein is made up of 116 amino-acid residues, and its sequence is:
1 edhpvhnlge?hsvcdsvsaw?vtkttatdik?gntvtvmenv?nldnkvykqy
51 ffetkcknpn?pepsgcrgid?sshwnsycte?tdtfikaltm?egnqaswrfi
101?rietacvcvi?tkktgn
The preparation of nerve growth factor, the cloning vector of this gene is pET23b and pPIC9K, step is:
A, Chinese cobra cut head and puts to death after, isolate poison gland as early as possible, and it is frozen to put into liquid nitrogen.
B, get above-mentioned poison gland, add TRIzol reagent, chloroform, jolting is placed on ice, the centrifuging and taking supernatant moves into supernatant in another centrifuge tube, adds Virahol, place centrifugal again in above-mentioned condition then on ice, add 75% washing with alcohol in will precipitating, mixing, centrifugal again that RNA precipitates, after air-dry, add an amount of TE solution or do not have in the water of RNase standby.
C, employing RNAPCR Kit (AMV) carry out reverse transcription PCR, carry out RT-PCR as template with the primer that contains BamHI and HindIII restriction enzyme site with above-mentioned total RNA and prepare the nerve growth factor mrna exon fragment, the reflection product carries out 1% agarose electrophoresis and identify that it is the band of goal gene that a band is arranged near 348bp.
D, be built into plasmid pET23b: utilize above-mentioned double digestion the genes identified fragment be built in the multiple clone site of plasmid pET23b, and in bacillus coli DH 5 alpha, increase, the preparation competence is carefully used Calcium Chloride Method, making up the back adopts PCR and double digestion fragment electrophoresis to identify that the pET23b that will contain NGF cDNA with aforesaid method is transformed into e. coli bl21 (DE
3) under the inducing of IPTG expression of NGF albumen.
E, goal gene structure and the screening in expression plasmid of yeast pPIC9K: utilize the primer that contains SnaBI and Eco521 double enzyme site that the pET23b that contains goal gene is carried out PCR, utilize test kit that electrophoresis identified and utilized that Qiagen company provides with the PCR product purification, utilize this two enzyme that the pPIC9K enzyme is cut again, insert the goal gene that purifying is crossed, thereby obtain containing the pPIC9K of goal gene.
Utilize the pPIC9K plasmid that electroporation will contain goal gene to be converted into Pichia yeast pichia pastoris GS115, under the condition that Histidine lacks, utilize the G418 of different concns that recon is screened, obtain G418 is had the bacterial strain of resistance, this bacterial strain is cultivated under the stable condition, at the NGF of the yeast expressed justacrine reorganization of methanol induction.
This protein is expressed in Pichia yeast GS115, with DEAE-Sephadex25 and SdphadexG-50 it is carried out purifying, carries out affinity chromatography with His-Bind Column70971-3.
Studied the Histological change that nerve growth factor makes blood sugar reduce and improve pancreas islet in addition, the reorganization nerve growth factor NGF of the feature that machine is mentioned in the demonstration can be used for drug manufacture, in treatment type ii diabetes and the complication peripheral neuritis process thereof, lowering blood glucose, improve islet function etc.
Embodiment
Further specify the present invention below in conjunction with embodiment, but be not limited to embodiment.
The clone of embodiment 1:NGF cDNA:
A, Chinese cobra cut head and puts to death after, isolate poison gland as early as possible, and it is frozen to put into liquid nitrogen.
B, get 0.1～1g tissue, add 1～5mlTRIzol reagent, chloroform, jolting 15～30sec is placed on 5～10min on ice.In 4 ℃ of centrifugal 10000～30000xg, 10～30min, get supernatant.Supernatant is moved in another centrifuge tube, add 0.5～2ml Virahol, and place 10～20min on ice.Centrifugal again in above-mentioned condition then.Add 75% washing with alcohol in will precipitating, mixing, centrifugal that RNA precipitates in 4 ℃ again.After air-dry, add an amount of TE solution or do not have in the water of RNase standby.
C, RT-PCR: adopt RNAPCR Kit (AMV) to carry out reverse transcription PCR.Its bare bones is: add MgCl in the reaction solution
2, RNA PCR damping fluid, deoxymononucleoside acid mixture, ribonuclease inhibitor, 9 polymers, sample RNA at random.Reaction total amount 20～100 μ l react laggard performing PCR reaction.Contain MgCl in the reactant
2, PCR damping fluid, Tap enzyme and primer (containing BamHI and HindIII restriction enzyme site).Reaction cumulative volume 20～100 μ l.The reflection product carries out 1% agarose electrophoresis and identify that it is the band of goal gene that a band is arranged near 348bp.
PT-PCR: TaKaRa RNA PCRKit (AMV) Ver.2.1 that adopts precious biotechnology company limited to provide carries out reverse transcription PCR.Its bare bones is: add in the reaction solution
25mmol/L?MgCl
2 4μl
10×RNA?PCR?buffer 2μl
Rnase?Free?dHO 8.5μl
dNTP?Mixture 2μl
Rnase?Inhibitor 0.5μl
Reverse?Transcriptase 1μl
Random?9mers 1μl
Sample?RNA 1μl
(oisutuve?cibreik?RNA) (1μl)
Cumulative volume 20 μ l
The reflection condition is: 30 ℃ of 10min
55℃ 30min
99℃ 5min
5℃ 5min
PCR reaction: get above-mentioned reaction solution 5 μ l, add in the Eppendorf pipe, add following reaction solution:
25mmol/L?MgCl
2 1.5μl
10×RNA?PCR?buffer 2μl
Sterile distilled water 16 μ l
TaKarA?taq 0.125μl
Primer?I 0.25μl
Primer?II 0.25μl
Cumulative volume 25 μ l
Reflection condition: 94 ℃ of 2min
Reaction with primer is:
F5’-GGGGATCCGGAAGATCATCCTGTGCA-3’
R5’-CCCAAGCTTGTTCCCCTTTTTTTTAGTGATTAC-3
Reaction product is carried out 1% agarose electrophoresis and is identified.It is the gene fragment band of purpose that one band is arranged near 348bp, and its cDNA sequence contains 348 base pairs for to clone from the NNAV gland, and nucleotides sequence is classified as:
1 gaagatcatc?ctgtgcataa?cctaggggaa?cattctgtgt?gtgacagtgt?cagtgcctgg
61 gttactaaaa?ctacagcaac?agacatcaaa?ggcaatacgg?tgactgtgat?ggaaaatgta
121?aatcttgata?acaaagtcta?caagcagtac?ttttttgaaa?ccaagtgcaa?aaatccaaat
181?ccagaaccga?gtgggtgcag?gggcattgat?tccagccatt?ggaattctta?ttgcaccgaa
241?acagacacat?ttatcaaggc?attaaccatg?gaaggcaatc?aggcatcctg?gcgcttcatt
301?cggattgaaa?ctgcctgtgt?gtgtgtaatc?actaaaaaaa?aggggaac
Embodiment 2: the expression of reorganization NGF in intestinal bacteria:
Be built into plasmid pET23b: the genes identified fragment is built in the multiple clone site of plasmid pET23b to utilize above-mentioned double digestion to incite somebody to action, and in bacillus coli DH 5 alpha, increase, the preparation competence is carefully used Calcium Chloride Method, making up the back adopts PCR and double digestion fragment electrophoresis to identify that the pET23b that will contain NGF cDNA with aforesaid method is transformed into e. coli bl21 (DE
3) under the inducing of IPTG expression of NGF albumen.
Embodiment 3: the expression of reorganization NGF in Pichia yeast:
Structure and the screening of goal gene in expression plasmid of yeast pPIC9K.The primer that utilization contains SnaBI and Eco521 double enzyme site carries out PCR to the pET23b that contains goal gene, utilizes test kit that electrophoresis identified and utilized that Qiagen company provides with the PCR product purification.Utilize this two enzyme that the pPIC9K enzyme is cut again, insert the goal gene that purifying is crossed.Thereby obtain containing the pPIC9K of goal gene.
The primer that contains the His label:
F5’-GCTACGTAGAAGATCATCCTGT-3’
R5’-TTCGGCCGGTGGTGGTGGTGGTGGTGCGCAAGCTT-3’
The primer that does not contain the His label:
F5’-GCTACGTAGAAGATCATCCTGT-3’
R5’-TTCGGCCGGTGGTGGTGGTGGTGGTGCGCAAGCTT-3’
Utilize the pPIC9K plasmid that electroporation will contain goal gene to be converted into Pichia yeast pichia pastoris GS115.The condition of electroporation is 2.5KV, 800-1000 Ω, and 25 μ F, the time is the 14-21 millisecond.Instrument is Bio-Rad E.colipulser, the U.S..
This protein is plucked among the Pichia yeast GS115 and is expressed, and with DEAE-Sephadex25 and SdphadexG-50 it is carried out purifying, carries out affinity chromatography with His-Bind Column70971-3.
Under the condition that Histidine lacks, utilize the G418 of different concns that recon is screened.Obtain G418 is had the bacterial strain of resistance.This bacterial strain is cultivated under the stable condition, at the NGF of the yeast expressed justacrine reorganization of methanol induction.
Protein is made up of 116 amino-acid residues, and its sequence is:
1 edhpvhnlge?hsvcdsvsaw?vtkttatdik?gntvtvmenv?nldnkvykqy
51 fetkcknpn pepsgcrgid?sshwnsycte?tdtfikaltm?egnqaswrfi
101?rietacvcvi?tkktgn
Embodiment 4: reorganization NGF lowering blood glucose and improve islet function:
Use the SD rat experiment and show, make diabetes model to the disposable injection strepto-assistant of rat abdominal cavity rhzomorph 55mg/kg body weight.After determining that blood sugar is higher than 16mmol/L, NGF and reorganization Chinese cobra each 100U/kg of NGF and 50U/kg are extracted in the injection of experimental group difference every day hindlimb muscle from Chinese cobra-venom, once in a week from the afterbody measuring blood sugar of blood extracting.The result shows, from the Histological change that the Chinese cobra NGF that extracts and the NGF that recombinates all can make blood sugar reduce and improve pancreas islet.The NGF of people's type ii diabetes support person intramuscular injection state approval listing after two months, visible blood sugar obviously reduces.
Claims (7)
1, Chinese cobra nerve growth factor NGF cDNA is characterized in that: clone from the NNAV gland, contain 348 base pairs, its nucleotides sequence is classified as:
1 gaagatcatc?ctgtgcataa?cctaggggaa?cattctgtgt?gtgacagtgt?cagtgcctgg
61 gttactaaaa?ctacagcaac?agacatcaaa?ggcaatacgg?tgactgtgat?ggaaaatgta
121 aatcttgata?acaaagtcta?caagcagtac?ttttttgaaa?ccaagtgcaa?aaatccaaat
181 ccagaaccga?gtgggtgcag?gggcattgat?tccagccatt?ggaattctta?ttgcaccgaa
241 acagacacat?ttatcaaggc?attaaccatg?gaaggcaatc?aggcatcctg?gcgcttcatt
301 cggattgaaa?ctgcctgtgt?gtgtgtaatc?actaaaaaaa?aggggaac
2, the nerve growth factor of the described Chinese cobra nerve growth factor of claim 1 NGF cDNA sequence expression, it is characterized in that: the recombinant protein of expression is made up of 116 amino-acid residues, and its sequence is:
1 edhpvhnlge?hsvcdsvsaw?vtkttatdik gntvtvmenv?nldnkvykqy
51 fetkcknpn?pepsgcrgid?sshwnsycte?tdtfikaltm egnqaswrfi
101?rietacvcvi?tkktgn
3, the preparation method of the described nerve growth factor of claim 2 is characterized in that:
A, Chinese cobra cut head and puts to death after, isolate poison gland as early as possible, and it is frozen to put into liquid nitrogen;
B, get above-mentioned poison gland, add TRIzol reagent, chloroform, jolting is placed on ice, the centrifuging and taking supernatant moves into supernatant in another centrifuge tube, adds Virahol, place centrifugal again in above-mentioned condition then on ice, add 75% washing with alcohol in will precipitating, mixing, centrifugal again that RNA precipitates, after air-dry, add an amount of TE solution or do not have in the water of RNase standby;
C, employing RNAPCR Kit carry out reverse transcription PCR, carry out RT-PCR as template with the primer that contains BamHI and HindIII restriction enzyme site with above-mentioned total RNA and prepare the nerve growth factor mrna exon fragment, the reflection product carries out 1% agarose electrophoresis and identify that it is the band of goal gene that a band is arranged near 348bp;
D, be built into plasmid pET23b: utilize above-mentioned double digestion the genes identified fragment be built in the multiple clone site of plasmid pET23b, and in bacillus coli DH 5 alpha, increase, the preparation competence is carefully used Calcium Chloride Method, making up the back adopts PCR and double digestion fragment electrophoresis to identify that the pET23b that will contain NGF cDNA with aforesaid method is transformed into e. coli bl21 (DE
3) under the inducing of IPTG expression of NGF albumen;
E, goal gene structure and the screening in expression plasmid of yeast pPIC9K: utilize the primer that contains SnaBI and Eco521 double enzyme site that the pET23b that contains goal gene is carried out PCR, utilize test kit that electrophoresis identified and utilized that Qiagen company provides with the PCR product purification, utilize this two enzyme that the pPIC9K enzyme is cut again, insert the goal gene that purifying is crossed, thereby obtain containing the pPIC9K of goal gene;
Utilize the pPIC9K plasmid that electroporation will contain goal gene to be converted into Pichia yeast pichia pastoris GS115, under the condition that Histidine lacks, utilize the G418 of different concns that recon is screened, obtain G418 is had the bacterial strain of resistance, this bacterial strain is cultivated under the stable condition, at the NGF of the yeast expressed justacrine reorganization of methanol induction.
4, the preparation method of nerve growth factor according to claim 3 is characterized in that: this protein is expressed in Pichia yeast GS115, with DEAE-Sephadex25 and SdphadexG-50 it is carried out purifying.
5, the preparation method of nerve growth factor according to claim 3 is characterized in that: this protein is expressed in Pichia yeast GS115, carries out affinity chromatography with His-Bind Column70971-3.
6, Chinese cobra nerve growth factor NGF cDNA according to claim 1 is characterized in that: is used for lowering blood glucose and improves islet function, and the pharmaceutical use for the treatment of the scorching aspect of tip spirit that causes by diabetes.
7, Chinese cobra nerve growth factor according to claim 2 is characterized in that: is used for lowering blood glucose and improves islet function, and the pharmaceutical use for the treatment of the scorching aspect of tip spirit that causes by diabetes.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB2004100215137A CN1316021C (en) | 2004-07-19 | 2004-07-19 | Nerve growth factor cDNA, clone and expresion of Chinese cobra |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB2004100215137A CN1316021C (en) | 2004-07-19 | 2004-07-19 | Nerve growth factor cDNA, clone and expresion of Chinese cobra |
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| CN1597952A CN1597952A (en) | 2005-03-23 |
| CN1316021C true CN1316021C (en) | 2007-05-16 |
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Families Citing this family (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103159843B (en) * | 2011-12-19 | 2014-11-05 | 中国科学院生物物理研究所 | Preparation method and drug application value of nerve growth factor mutant with low immunization side effects |
| CN102552881A (en) * | 2012-03-02 | 2012-07-11 | 广西医科大学 | Application of cobra venom nerve growth factor in preparing the medicine of preventing and treating liver fibrosis and liver cirrhosis |
| CN102816766A (en) * | 2012-05-04 | 2012-12-12 | 沈阳医学院 | Method for screening snake venom nerve growth factor (vNGF) active polypeptides |
| CN106939041B (en) * | 2017-04-14 | 2020-10-16 | 奔驰生物科技（云南）有限公司 | Method for purifying cobra venom nerve growth factor by using polyclonal antibody affinity column |
| CN111000984A (en) * | 2019-11-16 | 2020-04-14 | 祁展楷 | Application of snake nerve growth factor and snake nerve growth factor precursor in treating senile dementia |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5229365A (en) * | 1991-05-15 | 1993-07-20 | President And Fellows Of Harvard College | Endocrine cell stimulation by neurotrophic agents |
| CN1074448A (en) * | 1992-09-29 | 1993-07-21 | 中国医科大学 | The method of separation and purification of nerve growth factor from snake venom |
| CN1246483A (en) * | 1998-08-28 | 2000-03-08 | 大连卫康生物科技开发有限公司 | Process for extracting nerve growth factor from poisonous snake |
-
2004
- 2004-07-19 CN CNB2004100215137A patent/CN1316021C/en not_active Expired - Fee Related
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5229365A (en) * | 1991-05-15 | 1993-07-20 | President And Fellows Of Harvard College | Endocrine cell stimulation by neurotrophic agents |
| CN1074448A (en) * | 1992-09-29 | 1993-07-21 | 中国医科大学 | The method of separation and purification of nerve growth factor from snake venom |
| CN1246483A (en) * | 1998-08-28 | 2000-03-08 | 大连卫康生物科技开发有限公司 | Process for extracting nerve growth factor from poisonous snake |
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