Baculovirus of a kind of recombined human growth hormone gene and preparation method thereof and application
Technical field
The genetically engineered that the present invention relates in the biotechnological pharmaceutics engineering is produced the polypeptide drug technical field.
Background technology
Human growth hormone (HGH, Human Growth Hormone) be a kind of by human body self excretory one peptide species, the strand globular preteins of forming by 191 amino-acid residues, molecular weight is 22000 dalton, secrete by pituitary body, discharge via hypothalamus, master control is grown, immunity and human body metabolism.HGH stimulates epiphysis end chondrocyte differentiation, propagation, stimulates the cartilage matrix cell enlargement, causes that linear growth quickens and bone broadens; Regulate growth hormone deficiency or adult's fat, bone metabolism, cardiorenal function, improve motor capacity and quality of life.
HGH has the effect that promotes steatolysis and blood plasma free fatty acid is raise to fatty tissue; HGH can promote RNA and proteinic synthetic in many tissues, positive nitrogen balance occurs, and cartilage, bone, muscle reticular tissue and internal organ are all increased.Tethelin is transported to liver by blood and stimulates liver to produce a kind of protein molecule (IGF-1) that is called as insulin-like growth factor, increase collagen and other protein, chondroitin sulfate, the effect of nucleic acid synthetic in the cartilage, thereby promote chondrocyte's division and matrix hyperplasia, promote the growth of bone and reticular tissue.Except these promoted effects of growth, tethelin also participated in the metabolic regulation of body, and it is stored in polymerization, the regulating and controlling blood sugar of a large amount of greases in the adipocyte, promotion intracellular protein molecule in minimizing and all plays an important role aspect horizontal.Therefore tethelin has many-sided influence to the configuration and the function of the body of growing, also can continue to play a role in adult's stage, repairing and safeguarding in the process of body different tissues and serving as important role, but its secretion of back of growing up increases with the age and reduces.The people arrives after 30 years old every secretion of HGH during the decade and just successively decreases with 15% speed, thereby causes human senescence.Especially in the living environment of today, various pressure, bad habit and the harm that sitotoxin caused make the HGH secretory volume reduce rapidly, and more therefore the mankind quicken old and feeble.
Nineteen twenty, scientist finds tethelin.1958, the HGH that Herber Boyer concentrated on studies 38 years utilized gene recombination technology successfully to come out, and obtained the prize of Nobel's biology in then.1996, drugs approved by FDA utilized genetic engineering technique synthetic HGH to be used for flight against senium of human body.A large amount of appearance along with this recombinant hormone become possibility with the growth hormone therapy disease, and the children and the adult that lack tethelin can treat by supplementation with growth hormones now.Owing to suffer certain disease (as acquired immune deficiency syndrome (AIDS)) to cause amyotrophy and weakly patient also can from this replenishing, benefit.At present HGH is extensive use of in Hormone Replacement Therapy, but because of its enteron aisle very easily metabolism lose activity, the HGH preparation on the market mainly is an injection.
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, S1MNPV, 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.
Summary of the invention
One object of the present invention be to provide a kind of recombinant baculovirus and preparation method thereof with and application in preparation reorganization HGH albumen.
Recombinant baculovirus provided by the invention, this virus contains the HGH gene, and described HGH gene order sees that this recombinant virus can be inoculated silkworm shown in the SEQ ID NO.1, express recombinant HGH albumen.Certainly, recombinant baculovirus of the present invention not only can be inoculated silkworm, also can inoculate other insect cell.
In an embodiment, recombinant baculovirus provided by the invention is a silkworm baculovirus.Utilize silkworm baculovirus inoculation silkworm of the present invention, can obtain silkworm biological reactor.This reactor can be mass-produced, and has resources advantage and cost advantage.This virus is deposited in China Committee for Culture Collection of Microorganisms common micro-organisms center, the address is the No. 13, North No.1 Row, Zhongguancun, Haidian District, Beijing City, deposit number is CGMCC No.2031, classification called after Bombyx mori nuclear polyhydrosis virus Bombyx morinucleopolyhedrovirus, preservation date is on April 28th, 2007.
On the one hand, the invention provides a kind of method for preparing recombinant baculovirus, this method comprises: preparation human growth hormone HGH gene; The HGH gene Fusion gene that contains of preparation is imported in the baculovirus, form recombinant baculovirus.In an embodiment, the invention provides the method for preparing recombinant baculovirus, be by pcr amplification human growth hormone HGH gene, after enzyme is cut, import transfer vector and obtain recombinant transfer vector.Make this recombinant transfer vector and linearizing baculovirus homologous recombination then, foreign gene is inserted in the baculovirus, obtain containing the recombinant baculovirus of HGH gene.
On the other hand, the invention provides the application of a kind of recombinant baculovirus in preparation reorganization HGH albumen.
On the other hand, the invention provides a kind of proteic method of recombinant baculovirus preparation reorganization HGH of the present invention of utilizing.This method comprises inoculates silkworm with recombinant baculovirus of the present invention, after the great expression, and separation and purification, lyophilize.
In an embodiment, with recombinant baculovirus inoculation silkworm of the present invention, the silkworm body is collected in inoculation back 5-7 days, and-20 ℃ frozen.Get frozen silkworm chrysalis sample, pulverize and centrifuging.Get the homogenate after the filtration, centrifugal removal virus particle ,-50 ℃ of lyophilizes promptly get the HGH lyophilized powder.
The baculovirus expression system of optimizing among the present invention is a baculovirus expression vector system, by gene recombination, silkworm baculovirus BmBacPAK6 is advanced in the HGH gene recombination, obtain recombinant silkworm baculovirus BmBacHGH, the BmBacHGH that carries the HGH gene is bred in the silkworm (larva, pupa etc.) of different developmental phases, use silkworm and produce HGH as host's bio-reactor.Equally, utilize other baculovirus expression system, as AcMNPv, ApNPv, etc. also can express production in the same way.Use gene recombination technology, the HGH gene under the control of polyhedron promotor or other virus and Eukaryotic strong promoter, by in the body or vitro recombination, to the genome of baculovirus, is obtained recombinant virus with the HGH gene integration.Insect larvae or the pupal cell of various means through epidermis infection 1-5 age (optimal time was four or five ages) be eaten down or be adopted to recombinant virus can by per os, expresses production reorganization HGH albumen.
Optimized scheme is that the HGH gene is inserted on the pAcHLT-B baculovirus transfer vector among the present invention, again by recombinating in the body the HGH transgenosis to the genome of silkworm baculovirus BmBacPAK6, by the plaque screening technology, obtain to carry a large amount of recombinant silkworm baculovirus BmBacHGH of HGH.With its inoculation 1-5 silkworm larva or the pupa in age, can breed in a large number.When duplicating in the silkworm body, the HGH gene is expressed under polyhedrin gene promoter control, produces HGH.Collect silkworm larva or the pupa that contains HGH after infecting 5-7 days, frozen, pulverizing is also passed through 100 order net centrifugings, and centrifugal removal virus particle is got supernatant, and-50 ℃ of lyophilizes promptly get the HGH albumen lyophilized powder of recombinating.The HGH gene obtains high expression level, and the proteic output of each pupal cell reorganization HGH can reach 0.5mg, and is active in 1 * 10
7IU/ml.And conventional prokaryotic expression, its expression amount is a 0.05-0.3mg/ml bacterium liquid.
The advantage of utilizing this system to produce is:
1. the expression efficiency of this expression system is high, and reorganization HGH protein yield can reach the level of 0.5mg/ worm, thereby can reduce production costs greatly, and carries out scale operation.
2. this expression system is an eukaryotic expression system, and the exogenous protein of its expression can carry out posttranslational modification, makes it similar to natural product with biological activity etc. at biochemical property, and this provides assurance for the HGH that gives expression to has normal biologic activity.
3. the HGH that produces with this system need not through complicated purification step, only needs to get final product to such an extent that bioactive reorganization HGH albumen is arranged with expressing larva or the pupa roughing of HGH, greatly reduces production cost.
In an embodiment, the present invention utilizes silkworm to be bio-reactor, expresses the reorganization HGH albumen with clinical value.Reorganization HGH albumen lyophilized powder oral administration, nasal feeding, filling stomach medication biologically active that present method is produced, nasal feeding and the test of filling stomach confirm reorganization HGH albumen lyophilized powder toxicological harmless effect in rat, macaque; Confirm that mouse and the oral pharmacodynamic experiment that carries out of fruit bat it has delaying senility function.Present method is applicable to scale operation, and cost is low, and the output height has potential applicability in clinical practice widely.
Description of drawings
Fig. 1. recombinant transfer plasmid pAcHLT-HGH ring-type collection of illustrative plates;
Fig. 2. the proteic evaluation figure of the reorganization HGH in the silkworm hemolymph sample;
(A) SDS-PAGE electrophoretic separation
M. molecular weight standard product;
1. infect the control group of wild baculovirus BmBacPAK6;
2. infect recombinant virus BmBacHGH group, arrow shows reorganization HGH albumen.
(B) Western blot identifies
1. infect the control group of wild baculovirus BmBacPAK6;
2. infect the sample group of recombinant virus BmBacHGH.
Embodiment
For achieving the above object, technical scheme of the present invention is achieved in that
1. design primer, pcr amplification human growth hormone HGH gene from recombinant plasmid PWR-HGH.
2. make up the pAcHLT-HGH carrier.Be connected by the T4DNA enzyme with the HGH gene fragment of Sac I double digestion through EcoR I and be cloned in the pAcHLT-B baculovirus transfer vector of EcoR I and Sac I double digestion, make the HGH gene place polyhedrin (polyhedrin, ph) under the gene promoter control, be built into recombinant transfer plasmid pAcHLT-HGH.
3. preparation contains the silkworm with recombinant baculovirus BmBacHGH of HGH gene.In the BmN cell of cultivating in advance, dropwise add the pAcHLT-HGH transferring plasmid, through linearizing virus BmBacPAK6DNA and Dosper mixture, cultivated 4-5 days for 27 ℃, collect supernatant and carry out first round plaque screening and second and take turns plaque screening.Get the supernatant infected silkworm cell amplification of positive colony, can obtain a large amount of recombinant baculovirus BmBacHGH that contains the HGH gene.
4. express recombinant HGH albumen in silkworm larva and pupa.Recombinant virus is inoculated in the silkworm larva or the silkworm chrysalis of surface sterilization, and silkworm chrysalis is collected in inoculation back 5-7 days, and-20 ℃ frozen.Get frozen silkworm chrysalis sample, pulverizing is also passed through 100 order net centrifugings.Get the homogenate after the filtration, centrifugal removal virus particle is got supernatant, and-50 ℃ of lyophilizes promptly get the HGH albumen lyophilized powder of recombinating.
Elaborate particular content of the present invention below in conjunction with embodiment:
The amplification of embodiment 1.HGH gene
With the HGH gene is a pair of primer of stencil design, and upstream primer contains EcoR I restriction enzyme site, and downstream primer contains Sac I restriction enzyme site, and design of primers is as follows:
Upstream primer P1:5 ' GGGAATTCAGTTTCCTAC-TATACCAC 3 ' (SEQ NO.2);
Downstream primer P2:5 ' TTGAGCTCCTAGAAGCC-ACAGCTGCC 3 ' (SEQ NO.3).
Pcr amplification human growth hormone HGH gene from recombinant plasmid PWR-HGH (Shanghai biochemical teacher Wu Xiangfu of institute be so kind as to give).With recombinant plasmid PWR-HGH is template, behind 94 ℃ of pre-sex change 5min, and 94 ℃ of sex change 1min; 57 ℃ of annealing 1min; 72 ℃ are extended 1min, 30 circulations; 72 ℃ are extended 10min.Amplification obtains purpose HGH gene fragment.
Embodiment 2. makes up the pAcHLT-HGH carrier that contains the HGH gene
Be connected by T4DNA enzyme (MBI) with the HGH gene fragment of Sac I double digestion through EcoR I and be cloned in the pAcHLT-B baculovirus transfer vector (available from BDBiosciences Pharmingen) of EcoR I and Sac I double digestion, make the HGH gene place polyhedrin (polyhedrin, ph) under the gene promoter control, be built into recombinant transfer plasmid pAcHLT-HGH (as Fig. 1), correct through restriction analysis and order-checking identified gene sequence.Recombinant protein HGH is with the formal representation of fusion rotein in the silkworm expression system.
The preparation of embodiment 3. silkworm with recombinant baculovirus BmBacHGH
Get 5 μ l recombinant transfer plasmid pAcHLT-HGH DNA and 6 μ l through linearizing virus BmBacPAK6DNA (available from the biochemical cell in Shanghai institute), add TC-100 substratum (available from the GIBCOBRL company) mixing of 100 μ l serum-frees.The TC-100 substratum of getting 6 μ l Dosper (Bao Ling Man) adding, 100 μ l serum-frees is mixed.The TC-100 substratum washed twice of BmN cell (available from the biochemical cell in Shanghai institute) the usefulness serum-free in the 35mm plate will be cultivated in advance, and dropwise add pAcHLT-HGH transferring plasmid and Dosper mixture, cultivated 4-5 days, and collected supernatant and carry out first round plaque screening for 27 ℃.Get the Bm N cell in the 5 μ l supernatants infection 35mm plate, supernatant discarded adds the TC-100 substratum and the low melting-point agarose of balanced mix after 1 hour.Picking plaque after 4-5 days, infected the BmN cell 3-4 days, preserve supernatant, cell is used for Southern blot dot hybridization with the NaOH cracking, make template random primer probe mark test kit (Bao Ling Man) label probe with the HGH gene, hybridizing method is according to " molecular cloning " (Science Press, 1995).
The supernatant of getting positive colony carries out second and takes turns plaque screening.Get the supernatant infected silkworm cell amplification of positive colony.Can obtain a large amount of recombinant baculovirus BmBacHGH that contains the HGH gene.Behind this virus infection bombyx mori cell, be the morbidity shape at the microscopically cell.This recombinant baculovirus has been submitted China Committee for Culture Collection of Microorganisms's common micro-organisms center preservation to, and preservation date is on April 28th, 2007, and deposit number is 2031.
Embodiment 4. reorganization proteic expression of HGH and evaluations
About 10
6The pfu recombinant virus by inoculating needle infect every five age silkworm, silkworm body fluid and pupal cell are taked in the about 120 hours larvas in inoculation back or almost all morbidities of pupa, (12000rpm 30min), gets supernatant and surveys the proteic expression of reorganization HGH in the hemolymph through high speed centrifugation.Protein sample carries out electrophoretic separation on 15%SDS-PAGE.The result shows: the hemolymph sample that infects recombinant virus at about 27KDa place has an obvious band, and (Fig. 2, A), size is suitable with the proteic theoretical value of reorganization HGH, and does not have this band in the reference protein sample that infects wild virus.For verify this specific proteins whether can with HGH antiserum(antisera) generation specific immune response, we have carried out the reaction of Western blot trace, shown in Fig. 2 (B), 2 for infecting the silkworm sample of recombinant virus BmBacHGH, show this protein band can with HGH antiserum(antisera) generation specific reaction, can judge that thus this albumen is reorganization HGH albumen.
Embodiment 5. reorganization HGH protein biological activities are measured
The HGH standard substance are mixed with solution from a series of concentration of 2.5 μ g/ml to 62ng/ml, carry out ELISA with reference to method shown in molecular cloning one book and detect.Protein sample is coated to 96 holes under 4 ℃ of conditions of spending the night, and seals 37 ℃ of 1h with the PBS solution that contains 1%BSA with PBST washing back; The HGH antiserum(antisera) is hatched 2h for 37 ℃ with coating protein after diluting by 1: 3000; PBST solution washing 3 times, each 5-10min; 1h is hatched in two anti-(the anti-HGH antiserum(antisera) of rabbit is available from sigma companies) 37 ℃ that add dilution in 1: 2000; OD492 is surveyed in OPD sodium citrate buffer solution colour developing back, and sets up the relation that typical curve is represented protein concentration and absorbancy with this.Cell expressing protein sample and silkworm hemolymph carry out ELISA under same condition, record the OD492 value respectively, according to typical curve can calculation sample in the proteic content of reorganization HGH and tiring.。The proteic output of each pupal cell reorganization HGH can reach 0.5mg, and is active in 1 * 10
7IU/ml.
The preparation and the experimentation on animals of embodiment 6. reorganization HGH oral pharmaceutical
Silkworm chrysalis soaks 3min through 70% edible ethanol before inoculation BmBacHGH, carry out surface sterilization.Infected 5-7 days, and collected silkworm chrysalis ,-20 ℃ frozen.Get an amount of frozen silkworm chrysalis sample, with the pulverizing of secondary colloidal mill and by 100 order nets centrifugal (600rpm, 10min) filtration.Get the homogenate after the filtration, the centrifugal 20-60min of 12000rpm gets supernatant, and the centrifugal 30-90min of 30000rpm removes virus particle again, after the centrifugal end, gets supernatant, and lyophilized powder is made in lyophilize in the vacuum freeze drier (50 ℃).
Auxiliary material is made: normal 9 age in days silkworm chrysalises, carried out surface sterilization in 3 minutes with the immersion of 70% edible ethanol.Pulverize homogenate through low temperature (0~-20 ℃), cross 100 order mesh screens (600rpm * 30min), get supernatant liquor lyophilize (lyophilisation condition is identical with the HGH raw material).The lyophilized powder calibrating that keeps sample, all the other refrigerate in 4 ℃, behind the assay approval as auxiliary material.
According to HGH content, add auxiliary material in proportion and make HGH content more than 13 μ g/100mg.Fully load capsule behind the mixing, the packing back is a finished product.Following experimentation on animals, dosage all are that the actual content of HGH calculates.
The toxicity test and the effect experiment of embodiment 7. reorganization HGH oral pharmaceutical
Laboratory animal: macaque (Macaca mulatta) is provided by Fujian Province's birth control Institute Of Science And Technology non-human primates experimental center, the laboratory animal certification of fitness (producing macaque): accurate No. 12 of the real kinoplaszm in Fujian.16 of macaques, body weight 3.6~5.8kg, male and female half and half, be divided into 4 groups at random, recombinant human somatropin (HGH) is low, in, a Senior Three dosage group is 100-300 μ g respectively, 400-800 μ g, 1100-1300 μ g/kg/d, other establishes distilled water and makes the solvent control group, adopt the nasal feeding gastric infusion, successive administration 52 days, cut open 8 animals (male and female half and half) extremely, all the other animal drug withdrawal 30 heaven-made decubations observations, respectively to the administration phase and decubation each dosage group macaque body weight, food-intake, breathe, blood pressure, electrocardiogram(ECG, hematology, urine, blood biochemical is learned, every index such as organ coefficient detects, and the result shows, relatively shows no obvious abnormalities before above-mentioned detection index of each medication group and the medication.Each group macaque main organs is carried out histopathologic examination, find no the Histological change of toxicity meaning.Macaque is irritated tethelin (HGH) high dosage that stomach gives recombinant human, and continuous 52 days, do not find the overt toxicity reaction, multinomial detection all belongs to normally.
Long poison experiment: SD rat oral gavage administration recombinant human somatropin capsule 1250 μ g/kg/d, continuous 60 days, the result shows: toxic reaction was that blood-sugar content increases, and drug withdrawal recovered normal level in 30 days, and toxic reaction is a reversibility.The proteic rat oral gavage experimental result of reorganization HGH shows that the safe dose of no toxic side effects is below the 250 μ g/kg/d.Anxious malicious result shows, reorganization HGH to NIH mouse and SD rat through the maximum tolerated dose of gastric infusion all greater than 3300ug/kg.
The pharmacodynamic experiment (referring to table 1) of the reorganization HGH administration of mouse: this capsule of mouse orally give was compared with the blank group after 50 days, (0.05g/ day/60kg) the heart lipofuscin content descends 22% to low dose group, liver SOD improves 16% than vigor, in dosage group (0.1g/ day/60kg) heart lipofuscin content 13% (P<0.05) that descends, liver SOD improves 19% (P<0.01) than vigor, (0.3g/ day/60kg) heart lipofuscin content 12% (P<0.05) that descends, liver SOD improves 28% (P<0.001) than vigor to high dose group.
The fruit bat experimental result shows with the blank group compares (referring to table 2), the male fruit bat mean lifetime of low dose group has prolonged 11% (P<0.05), the middle male fruit bat mean lifetime of dosage group has prolonged 12% (P<0.05), the half death time prolongs 8 days, female fruit bat mean lifetime prolongs 8%, the male fruit bat mean lifetime of high dose group has prolonged 14% (P<0.05), and the half death time prolongs 7 days, and female fruit bat mean lifetime prolongs 10%.
|
The blank group |
Low dose group |
Middle dosage group |
High dose group |
Heart lipofuscin (μ g/g wet tissue) |
7.02±0.4 |
5.48±0.35 |
6.11±0.41 |
6.18±0.37 |
Liver SOD is than vigor (Nu/ml) |
58.0±16.4 |
67.3±18.1 |
69.02±17.5 |
74.24±17.1 |
The proteic drug effect result of various dose reorganization HGH of table 1. mouse
The proteic drug effect result of various dose reorganization HGH of table 2. fruit bat
As seen, this reorganization HGH protein product has no side effect in animal body, and safe and reliable, oral absorption is effective, delaying sanility.
SEQUENCE LISTING sequence table
<110〉Zhongqi Biological Pharmaceutical Co., Ltd., Zhejiang
<120〉baculovirus of a kind of recombined human growth hormone gene and preparation method thereof and application
<130>081003-I-CP-NZJ
<160>3
<170>PatentIn version 3.5
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tatatcccaa aggaacagaa gtattcattc ctgcagaacc cccagacctc cctctgtttc 240
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