CN101613679A - A kind of baculovirus of recombination superoxide dismutase gene and preparation thereof and application - Google Patents
A kind of baculovirus of recombination superoxide dismutase gene and preparation thereof and application Download PDFInfo
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Abstract
The present invention relates to a kind of recombinant baculovirus and preparation thereof and application.The present invention makes up and contains the recombinant baculovirus of having a liking for high temperature bacterium superoxide-dismutase sod gene, can efficiently express superoxide-dismutase SOD in silkworm, and described albumen keeps higher SOD activity through high temperature purification.The present invention has a liking for high temperature bacterium superoxide-dismutase sod gene and silkworm baculovirus BmBacPAK6 recombinates with described, obtain recombinant silkworm baculovirus BmBacPAK-SOD, be deposited in China Committee for Culture Collection of Microorganisms common micro-organisms center, deposit number is CGMCC No.2769.The present invention also discloses the method that the described recombinant baculovirus expression of a kind of usefulness and purifying are had a liking for high temperature bacterium superoxide-dismutase.Compared with prior art, raw material is easy to get, and production cost is low, has important industrialization value.
Description
Technical field
The genetically engineered that the present invention relates in the biotechnological pharmaceutics engineering is produced the polypeptide drug technical field.
Background technology
Superoxide-dismutase (SOD, Superoxide dismutase) is a kind of antioxidase that extensively exists in vivo.Mann in 1938 and Keilin find a kind of nattier blue cuproprotein that contains, but its physiological function be it be unclear that when carrying out ox blood red corpuscle fractional separation.Nineteen sixty-eight, Fricovich finds: " O
2-make the reduction of cytochrome C be subjected to a kind of protein factor resistance.McCord confirmed with Fridouich in 1969: this supressor is identical with superoxide dismutase.And find that superoxide dismutase, hepatocuprein, cerebrocuprein all have ultra-oxygen anion free radical (O
2-) the disproportionation activity, so the called after superoxide-dismutase (SuperoxideDismutase, SOD).SOD is the focus of domestic and international experts and scholars' research always over past ten years.SOD is a kind of important oxygen free radical scavenger in the body, oxyradical that can the balance body, thus avoid the untoward reaction that causes during the ultra-oxygen anion free radical excessive concentration when in the body, physiological function such as have organ damage, the skin care of prevention, delay senility.SOD is a kind of medicinal enzyme on way of great use simultaneously.
The research of relevant SOD is subjected to the extensive concern of Chinese scholars, relates to chemistry, biology, medicine, daily-use chemical industry, all fields of food.The SOD clinical application mainly concentrates on anti-inflammatory aspect (based on the inflammation patient who causes behind similar rheumatism and the radiotherapy), in addition some autoimmune disorder (as lupus erythematosus, dermatomyositis), pulmonary emphysema, anticancer and oxygen intoxication etc. is all had certain curative effect; Mainly be used as foodstuff additive and important function base-material in foodstuffs industry; In others related application is arranged also.Multiple diseases such as oxygen intoxication, senile cataract, diabetes, cardiovascular disorder, various inflammation have been treated both at home and abroad with SOD; also can be used as radio-protector; be used for the protection and the transplanting of organs such as auxiliary radiotherapy and chemotherapy and kidney, liver, heart, to reduce the caused side effect of high dose radiation.Food, beverage, makeup, the skin care product of many SOD of being rich in are all developed in countries in the world, and have brighten, renew one's youth, the healthcare products of delaying senility function.
The production technique of present domestic SOD is a raw material with animal blood or plant basically, extracts SOD with Mccord and Fridovich method, and comprise following three steps: ethanol-chloroform removes dehemoglobinize; Organic solvent and ammonium sulfate precipitation; The ion-exchange chromatography purifying.These method securities are not high, easily cause the animal virus cross infection, and European Union promulgated a decree in 1999, forbid that the SOD that extracts from animal blood is used for the mankind.In addition, it is few that these methods obtain the final product amount, and generally speaking, 1 kilogram of blood extracts about SOD 0.08 gram only.
This bio-reactor of baculovirus expression system is that the eighties is set up.(smith since nineteen eighty-three utilizes baculovirus expression system to efficiently express people's alpha-interferon first, Mol CellBiol.3:2156.2165,1983), existing dozens of foreign gene has obtained efficiently expressing, only just there are alpha-interferon (Yang Guanzhen etc. in China, Acta Biochimica et Biophysica Sinica, 22:355-361,1990), arrowhead proteinase inhibitor (season equality, silkworm industry science, 21:223-227,1995), Mareks disease virus Glycoprotein B (Xiao Qingli etc., silkworm industry science, 23:104-108,1997) etc. multiple.Baculovirus comprises BmNPV, AcMNPV, ApNPV, BssNPV, EOSNPV, HaNPV, HzNPV, LdMNPV, MbMNPV, UpMNPV, SlMNPV, SeMNPV, TnNPV etc., and insect host comprises silkworm, wild silkworm etc.At present, baculovirus expression system, baculovirus expression vector system especially wherein are one of individual expression systems of eukaryote that has most in the world business development value.
The present invention utilizes silkworm biological reactor to efficiently express to have a liking for the SOD of stable existence in the high temperature bacterium, after 70 ℃ of thermal treatment, just can remove most of foreign protein, obtain high vigor SOD, simplified the technical process that SOD produces, reduce its production cost, the expression amount of SOD in a silkworm chrysalis can reach 1mg at most, far above traditional mode of production, has important scientific research and industrialization value.
Summary of the invention
The invention provides a kind of recombinant baculovirus and preparation method thereof.
The present invention also provides recombinant baculovirus of the present invention to have a liking for application in the high temperature bacterium SOD albumen in preparation.
On the one hand, the invention provides a kind of recombinant baculovirus, this virus contains has a liking for high temperature bacterium sod gene, and described sod gene sequence is shown in SEQ ID NO.1.Described recombinant virus can be inoculated silkworm, expresses and has a liking for high temperature bacterium SOD albumen, and certainly, recombinant baculovirus of the present invention not only can be inoculated silkworm, also can inoculate other insect.
Equally, utilize other baculovirus expression system, as AcMNPv, ApNPv, etc. also can express production in the same way.Use gene recombination technology, sod gene under the control of polyhedron promotor or other virus and Eukaryotic strong promoter, by in the body or vitro recombination, is incorporated into sod gene on the genome of baculovirus, obtain recombinant virus.
In an embodiment, the present invention will have a liking for recombinating with silkworm baculovirus BmBacPAK6 of high temperature bacterium superoxide-dismutase sod gene, obtain recombinant silkworm baculovirus BmBacPAK-SOD, be deposited in China Committee for Culture Collection of Microorganisms common micro-organisms center, preservation date is on November 28th, 2008, deposit number is CGMCC No.2769, classification called after Bombyx mori nuclear polyhydrosis virus (Bombyx mori nucleopolyhedrovirus).Utilize recombinant silkworm baculovirus BmBacPAK-SOD inoculation silkworm of the present invention, can obtain silkworm biological reactor.This reactor can be mass-produced, and has resources advantage and cost advantage.
On the other hand, the invention provides a kind of method for preparing recombinant baculovirus, this method comprises: high temperature bacterium superoxide-dismutase sod gene is had a liking in preparation, and its gene order is shown in SEQ ID NO.1; The sod gene of preparation is imported in the baculovirus, form recombinant baculovirus.
In an embodiment, the invention provides the method for preparing the recombinant silkworm baculovirus, be to have a liking for high temperature bacterium sod gene by pcr amplification, after enzyme is cut, be connected and obtain recombinant transfer vector with transfer vector PVL1393 plasmid (as shown in Figure 1).Make this recombinant transfer vector and linearizing baculovirus BacBAK6 reorganization then, foreign gene SOD is inserted BacBAK6 in the baculovirus, obtain containing the recombinant baculovirus of having a liking for high temperature bacterium sod gene, make up principle as shown in Figure 2.
On the other hand, the invention provides a kind of proteic method of recombinant baculovirus preparation reorganization SOD of the present invention of utilizing.This method comprises with recombinate shape virus infection silkworm of the present invention, after the great expression, through high temperature purification.
In an embodiment, with recombinate shape virus infection bombyx mori cell of the present invention, cultivated 120 hours, behind the collecting cell medium centrifugal, collecting precipitation, with the PBS rupture of membranes that suspends, the centrifugal supernatant that regathers is and has a liking for high temperature bacterium sod gene expression product.This albumen has high thermotolerance and also has 80% enzyme activity after handling 2h at 70 ℃.
Optimized scheme is to separate the sod gene that obtains from have a liking for high temperature bacterium (Geobacillus kaustophilus HTA426) genome among the present invention, with the amplification after sod gene through BamH I, behind the EcoR I double digestion, by the continuous method of sticky end, with goal gene be connected with the transfer vector pVL1393 of EcoR I double digestion through BamH I, obtain recombinant transfer vector pVL1393-SOD.Cut with the PCR identified gene correct through enzyme.With recombinant transfer vector pVL1393-SOD DNA and linearizing viral Bm-BacPAK6DNA by liposome embedded back cotransfection silkworm cultured cell, cultivated 4-6 days, after treating to observe cell and infection symptoms occurring, by the plaque screening technology, obtain to carry a large amount of recombinant silkworm baculovirus BmBacPAK-SOD that have a liking for high temperature bacterium sod gene.The present invention is to having a liking for the activity identification of high temperature bacterium sod gene expression product.Record its enzyme activity 3505 ± 32U/2 * 10
6Cell.Have a liking for high temperature bacterium sod gene expression product and behind high temperature (70 ℃) purifying, can keep 80% activity.
The invention provides a kind of medicine, its activeconstituents be utilize that recombinant baculovirus prepares according to the method described above have a liking for high temperature bacterium SOD albumen.
The advantage of utilizing this method to produce SOD is:
1, silkworm disappearance complementary type linearizing baculovirus makes the recombinant celo virus high efficiency and time conservation.
2, the expression efficiency height of this expression system, reorganization SOD protein-active can reach 3505 ± 32U/2 * 10
6Expression amount in the cell levels, silkworm chrysalis can reach 1mg at most, and conventional prokaryotic expression, its expression amount is a 0.05-0.3mg/ml bacterium liquid.Thereby can reduce production costs greatly, and make scale operation become possibility.
3, baculovirus only is insect or arthropodan virus, and is nontoxic to people and animals, and the virogene after reorganization to lose polyhedrosis protection very weak in natural viability, can not cause public hazards.Utilize silkworm biological reactor to produce SOD and compare, can avoid the cross infection of animal virus with the method for extracting SOD from animal blood, safer reliable.
4, have a liking for 411 amino acid of high temperature bacterium SOD total length, this albumen has high thermotolerance and has higher enzyme activity at normal temperatures, therefore the high temperature bacterium SOD that has a liking for that produces with this method need not through complicated purification step, only need to have a liking for high temperature bacterium sod gene expression product pyroprocessing, get final product to such an extent that high temperature bacterium SOD albumen is had a liking in the reorganization of high vigor, simplify the technical process that SOD produces, reduced its production cost.
Compared with prior art, utilize silkworm biological reactor to efficiently express to have a liking for the SOD albumen of stable existence in the high temperature bacterium, raw material is easy to get, and production cost is low, has important scientific research and industrialization value.
Description of drawings
Fig. 1 is a transfer vector PVL1393 plasmid ring-type collection of illustrative plates;
Fig. 2 makes up schematic diagram for the reorganization silkworm baculovirus.
Embodiment
For achieving the above object, technical scheme of the present invention is achieved in that
1, design primer, amplification purpose fragment sod gene.
2, make up recombinant vectors.Sod gene fragment through EcoR I and BamH I double digestion is connected to by sticky end in the transfer vector of EcoR I and BamH I double digestion, is built into recombinant transfer plasmid.
3, preparation contains the recombinant baculovirus of sod gene.Get recombinant transfer vector, linearizing viral DNA, liposome, use the HBS mixing.Add the Bm N cell of attached cell, continue to cultivate, treat cell rupture after, collect supernatant, it is carried out plaque select, promptly get the recombinant virus that contains sod gene.
4, the expression and purification of sod gene.Behind the recombinant virus infection bombyx mori cell, cultivated 120 hours, the collecting cell nutrient solution, centrifugal collecting precipitation, with the PBS rupture of membranes that suspends, the centrifugal supernatant that regathers is the sod gene expression product.
The preparation of embodiment 1.SOD gene
According to the relevant homologous gene complete sequence of having delivered, design upstream primer P1 and downstream primer P2 contain BamH I restriction enzyme site, initiator codon ATG and EcoR I restriction enzyme site, termination codon TAA respectively, and cDNA is a template, and primer P1 and P2 design are as follows:
P1:5 '-CGGATCCATGCGTGGGGCAAGCACGGA-3 ' (shown in SEQ NO.2)
P2:5 '-ATTTGCGGCCGCTTTAAAACGGCTGCCAAC-3 (shown in SEQ NO.3)
Amplification purpose fragment sod gene from have a liking for high temperature bacterium (Geobacillus kaustophilus HTA426) genome.
The structure of embodiment 2. recombinant transfer vector pVL1393-SOD.
With the amplification after goal gene SOD through BamH I, behind the EcoR I double digestion, the method that links to each other by sticky end is connected acquisition recombinant transfer vector pVL1393-SOD with transfer vector pVL1393 (available from Introvigen company) through BamH I and EcoR I double digestion.
Embodiment 3. preparations contain the recombinant baculovirus of having a liking for high temperature bacterium sod gene
Getting 5 μ l recombinant transfer vector plasmids and 20 μ l mends cumulative volume to 50 μ l, mixing with HBS through Bsu36I linearization for enzyme restriction viral DNA.Get liposome 10 μ L, cumulative volume is mended to 50 μ l with HBS, mixing, and with both mixings.The supernatant of attached cell is removed in the culturing bottle in suction, and the Bm N cell of cultivating in advance (available from the biochemical cell in Shanghai institute) is washed twice with serum free medium TC-100, adds 100 μ l mixtures.Continue to cultivate after 4-6 days for 27 ℃, with the nutrient solution of cotransfection cell another bottle growth conditions good cell of transferring.After treating that cells infected breaks, collect supernatant, it is carried out plaque select, what the reorganization spot was arranged promptly gets recombinant virus BacPAK-SOD.With parental virus Bm-BacPAK6DNA and the negative contrast of the total DNA of bombyx mori cell, with the positive contrast of pVL1393-SOD, choose a recombinant virus BacPAK-SOD DNA as identifying sample, carry out pcr amplification, then electrophoresis detection.The result shows that the recombinant virus spot can amplify and positive control size identical segments, but negative control can not, illustrate that SOD has been reconstituted among the viral BacPAK-SOD.
Embodiment 4. has a liking for the expression of high temperature bacterium SOD in bombyx mori cell
Recombinant virus BacPAK-SOD and Bm-BacPAK6 virus are with the dosage infected silkworm cell of MOI=10, in 27 ℃ of incubators, cultivated 120 hours, the collecting cell nutrient solution, behind centrifugal 5 minutes of the 12000rpm, collecting precipitation is with PBS (0.02mol/L, pH 7.4) suspend, through ice-bath ultrasonic ripple rupture of membranes, the centrifugal supernatant that regathers is and has a liking for high temperature bacterium SOD expression product, and-20 ℃ of preservations are standby.
Embodiment 5, have a liking for the activity identification of high temperature bacterium sod gene expression product
Getting among the embodiment 4 gained, to have a liking for high temperature bacterium SOD expression product be sample.Draw 4.5ml TrisHCl-EDTA damping fluid (pH8.2) in the 10ml colorimetric cylinder; Put 25 ℃ of water-bath 20min; Add constant temperature to 25 ℃ sample solution 50 μ l, mixings immediately; The 45mmol/L pyrogallol 40 μ l (10mmol/L HCl solution allocation) that add 25 ℃ of preheatings, mixing immediately; Pour 1cm quartz cuvette 325nm wavelength photometry density value rapidly into; Survey an optical density value every 30s, survey 4min altogether, obtain pyrogallol autoxidation speed, ODB/min.The result shows: wild virus Bm-BacPAK6 cells transfected expression product SOD activity is 263 ± 3U/2 * 10
6Cell; Recombinant virus BacPAK-SOD cells transfected expression product SOD activity reaches 3505 ± 32U/2 * 10
6Cell.
The high temperature purification of embodiment 6, gene expression product
Gained in the example 4 is had a liking for high temperature bacterium SOD expression product after 70 ℃ of modules are bathed 2h, and the albumen of non-refractory precipitates 70 ℃ of sex change, and bite high temperature bacterium SOD albumen and still be dissolved state, centrifugal 3 minutes of 12000rpm, the gained supernatant is the sod gene expression product.According to method shown in the embodiment 5 gained sod gene expression product is carried out determination of activity, the result shows, no longer show the SOD activity after the wild virus Bm-BacPAK6 cells transfected expression product pyroprocessing, and recombinant virus BacPAK-SOD cells transfected expression product can keep 80% SOD activity, has confirmed that high temperature purification obtains the feasibility of high vigor SOD.
The above is preferred embodiment of the present invention only, is not to be used to limit protection scope of the present invention.
SEQUENCE?LISTING
＜110〉Zhongqi Biological Pharmaceutical Co., Ltd., Zhejiang
＜120〉a kind of baculovirus of recombination superoxide dismutase gene and preparation thereof and application
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gtatacactc?gggccgtccg?cctggctgat?tcgtcagctg?ccgatcggga?agaaggggcg 360
acgcaagaac?aagtcgaagg?ggaatcggtg?tcgcctgaac?tggagagtga?aaacaaagag 420
aacgaagacg?gatggcttga?caccagcggg?acggcagagc?gggttgagga?cgcaaaagag 480
ccggctttta?tggcggagct?ctccgactcg?ccgactgatg?cagctgacgg?cgagccggat 540
caagtggaca?acgtgaccga?tggaaagcga?cgctgggtgg?atgcggacga?cggcggtgag 600
ccgcgtcaac?aagtagcgcc?cggccgtcat?gtcttgccgc?cgctgccgta?ccgttatgat 660
gcgctcgagc?cgcatatttc?cgaagaaatc?atgcgccttc?accatacgaa?gcatcatcaa 720
agctatgtcg?acggtcttaa?tcaggcggag?cggatgatgg?ccgaggcgcg?ccggacgaac 780
aattttgatc?tgttgaaaca?ttgggagcgg?gaagcggcgt?tccacggctc?cgggcattat 840
ttgcatacaa?tcttttggta?caacatgcat?ccagagggcg?gcggcgagcc?gcgcggggag 900
ctgcgggcgc?aaatcgagcg?cgattttggc?agttttaccg?cctttcgccg?tcatttcacc 960
gaagcggcga?aaagcgccga?aggggtcggc?tgggcgctgc?tcgtttgggc?gccgcgagcg 1020
caccgtctgg?aaattttgca?ggcggaaaaa?caccagctca?tggcgcaatg?ggatgtgatc 1080
ccgcttctcg?tgcttgatgt?gtgggagcat?gcctactatt?tgcaatacaa?aaatgatcgg 1140
gcggcgtaca?tcgaacattg?gtggaacgtc?gtcaactggc?gcaacgttga?agaacgcttt 1200
catgaggcgc?agaagcttcg?ttggcagccg?ttttaa 1236
<210>2
<211>27
<212>DNA
＜213〉primer P1
<400>2
cggatccatg?cgtggggcaa?gcacgga 27
<210>3
<211>30
<212>DNA
＜213〉primer P2
<400>3
atttgcggcc?gctttaaaac?ggctgccaac 30
Claims (10)
1. a recombinant baculovirus is characterized in that, this virus contains the superoxide-dismutase sod gene.
2. recombinant baculovirus according to claim 1 is characterized in that, described sod gene is for having a liking for high temperature bacterium superoxide-dismutase sod gene, and its sequence is shown in SEQ ID NO.1.
3. recombinant baculovirus according to claim 1 and 2 is characterized in that, this virus is silkworm baculovirus.
4. recombinant baculovirus according to claim 2 is characterized in that this virus is BmBacPAK6, is deposited in China Committee for Culture Collection of Microorganisms common micro-organisms center, and deposit number is CGMCC No.2769.
5. method for preparing the described recombinant baculovirus of claim 1, this method comprises:
(1) preparation superoxide-dismutase sod gene;
(2) prepared sod gene is imported in the transfer vector, obtain recombinant transfer vector;
(3) recombinant transfer vector DNA that step (2) is obtained and baculovirus DNA reorganization obtain recombinant baculovirus.
6. method according to claim 5 is characterized in that, described transfer vector is pVL1393.
7. according to claim 5 or 6 described methods, it is characterized in that described baculovirus is silkworm baculovirus BmBacPAK6.
8. one kind prepares the proteic method of high temperature bacterium SOD of having a liking for, and this method comprises that after efficiently expressing, high temperature purification obtains to have a liking for high temperature bacterium SOD albumen with each described recombinant baculovirus inoculation silkworm of claim 1～4.
9. method according to claim 8 is characterized in that, described high temperature purification temperature is 70 ℃.
10. a medicine is characterized in that, its activeconstituents is for having a liking for high temperature bacterium SOD albumen according to the reorganization of claim 8 or 9 described method preparations.
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Cited By (1)
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| CN113215198A (en) * | 2021-04-07 | 2021-08-06 | 浙江安各洛生物技术有限公司 | Recombinant baculovirus and preparation method and application thereof |
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| CN113215198A (en) * | 2021-04-07 | 2021-08-06 | 浙江安各洛生物技术有限公司 | Recombinant baculovirus and preparation method and application thereof |
| CN113215198B (en) * | 2021-04-07 | 2023-09-22 | 浙江安各洛生物技术有限公司 | Recombinant baculovirus and preparation method and application thereof |
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