CN1410541A - Expression quantity of increasing insect rhabdovirus system exogenous gene by utilizing insect hormone - Google Patents
Expression quantity of increasing insect rhabdovirus system exogenous gene by utilizing insect hormone Download PDFInfo
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Abstract
A process for increasing the expression to exogenous gene in the insect baculovirus system by insect hormones features that the molting hormone (MH), the juvenile hormone (JH) and their mixture are used to increase the expression efficiency of exogenous gene and polyhedron protein gene in the insect baculovirus system and the copying efficiency of insect baculovirus in insect cells.
Description
One, technical field
The present invention relates to biotechnology genetically engineered field, be specifically related to use the expression amount that insect hormone improves insect rhabdovirus system exogenous gene.
Two, background technology
The insect baculovirus expression system of using mainly contains two at present, one is with autographa california californica nuclear polyhedrosis virus (Autographa Californica Nuclear Polyhedrosis Virus, AcMNPV) be carrier, with autumn mythimna separata (Spodoptera frugiperda, Sf) cell, cabbage caterpillar (Trichopiusta ni, Ai-5) cell or insect are the host; Another is that (Bombyx mori Nuclear Polyhedrosis Virus BmNPV) is carrier, is expressive host with bombyx mori cell or silkworm polypide and pupal cell with Bombyx mori nuclear polyhydrosis virus.Two expression systems are all so that the polyhedron gene or the strong promoters such as P10 gene or utmost point early gene IE-1 of virus vector are the controlling element of exogenous gene expression separately, by the external source goal gene being placed the recombinant virus infection host cell under the strong promoter control, in host and cell paste, carry out massive duplication by recombinant baculovirus, in virus infection host's late period, the transcription product of foreign gene accumulates in a large number, and efficiently expressed within a short period of time.And the silkworm expression system is the safest I level (Nong Jian examines word 2000B-03-007).
The genomic size of baculovirus is about 90-230kb, and wherein the gene through identifying known function has more than 80 at present.Contain the steroidal uridine diphosphoglucose transferase gene (egt) of casting off a skin in most of nuclear polyhedrosis virus genomes of Rhabdoviridae.The EGT enzyme molecular weight of egt genes encoding is 60ku in AcMNPV and BmNPV, and each is made up of 506 amino-acid residues, and homology between the two is about 95%.It can catalysis uridine diphosphoglucose and the glycosyl shift reaction between the steroidal compounds of casting off a skin in insect body, with moulting hormone and UDP-glucose is substrate, then the product of shift reaction is moulting hormone 22-O-β-D-glycopyranoside (O`Reilly et a1.Biotechnology, 9:1086-1089,1991), find also that simultaneously the EGT enzyme that AcMNPV encodes can not be to lack the steroidal compounds of casting off a skin of hydroxyl as substrate on 22 carbon atoms.
Insect moulting hormones and neotonin are that the control polypide grows and the abnormal major decision factor, also are simultaneously the triggering devices (trigger) that triggers a series of (set) genetic transcription and translation regulation process switch.Use moulting hormone, neotonin or moulting hormone and neotonin coupling and also can increase the synthetic of silk-protein.Host to baculovirus infection uses insect hormone, might recover host's the normal physiological balance and the efficient of raising protein synthesis system, but does not have specifically to use to the host who is subjected to baculovirus infection the method for insect hormone both at home and abroad so far.
Three, summary of the invention
The purpose of this invention is to provide a kind of method that insect hormone improves baculovirus expression system exogenous gene expression amount of using, be applied to fields such as agricultural, medicine, health, food, environment, zymin industry to produce the range gene engineering product.
The object of the present invention is achieved like this:
Insect moulting hormones (MH), corpus allatum hormone (JH) and moulting hormone (MH) and neotonin (JH) are united use to improve foreign gene and the expression efficiency of polyhedron gene in insect baculovirus expression system.
Described baculovirus is: BmNPV, AcMNPV, ApNPV, BsSNPV, CfMNPV, EoSNPV, HaNPV, HzNPV, LdMNPV, MbMNPV, OpMNPV, SIMNPV, SeMNPV, and TnNPV.
Described insect host is: silkworm (Bombyx mori), wild silkworm (Bombyx mandarina), Semen Ricini silkworm (Philosomia cynthia ricini), wild silkworm (Dictyoploca japonica), Philosamia cynthia (Philosamia cynthiapernyi), tussah (Antheraea pernyi), Japan giant silkworm (Antheraea yamamai), wild giant silkworm (Antheraeapolyphymus), autographa california (Autographa californica), tea geometrid (Ectropis obliqua), cabbage army worm (Mamestra brassicae), prodenia litura (Spodoptera littoralis), autumn mythimna separata (Spodoptera frugiperda), cabbage looper (Trichoplusia ni), armyworm (Thaumetopoea wilkinsoni), bollworm (Heliothisarmigera), U.S. bollworm (Heliothis zea), oriental tobacco budworm (Heliothis assulfa), cigarette beetle (Heliothisvirescens), oriental armyworm (Pseudaletia separata), and gypsymoth (Lymantria dispar).
Described insect is a silkworm, and described baculovirus is a silkworm baculovirus.
Described insect moulting hormones is: α-ecdysone, and β-ecdysone and 20,26-dihydroxyl ecdyson is planted the ecdysteroids compound of source property, synthetic ecdysteroids compound.Described corpus allatum hormone is: terpenoid compound JH-0 (C19 neotonin), JH-I (C18 neotonin), JH-II (C17 neotonin); JH-III (C16 neotonin) and derivative 738 thereof, ZR515, ZR512, ZR619, ZR777.
Described insect hormone using method is that puncture injection, mouthful food and body surface spray use.
Described insect hormone is five length of times and the pupa time at silkworm larva duration of service.
The amount ranges that described insect moulting hormones MH is used in silkworm larva is every silkworm of 1～8 μ g/; The amount ranges that is used in silkworm pupa is every pupa of 1～6 μ g/; The concentration range that described corpus allatum hormone JH is used in silkworm larva is the spray of 25～150ppm body; The amount ranges that described insect moulting hormones MH and neotonin JH unite use is 1～8 every of μ g MH/ and the spray of 25～150ppm JH body.
The optimum consumption that described insect moulting hormones MH is used in silkworm larva is every silkworms of 4 μ g/; The optimum consumption that is used in silkworm pupa is every pupas of 2 μ g/; It is 100ppm that described corpus allatum hormone JH is used in the silkworm larva optimal concentration; Described insect moulting hormones MH and neotonin JH unite use, and optimum consumption is the JH body spray of 4 every of μ g MH/ and 100ppm.
Described insect moulting hormones MH is used in bombyx mori cell, and its concentration range that makes an addition in the cell culture medium is 1～5 μ g/ml substratum.
Scope of the present invention is not limited to optimization range, can contain other baculovirus expression system and insect hormone and derivative thereof from mechanism.
The present invention is further elaborated by the following drawings and enforcement, but does not limit the scope of the invention.
Four, description of drawings: Fig. 1, part steroid structural representation Fig. 2, structural representation Fig. 3 of part neotonin and derivative thereof, MH is to the Fig. 4 that influences of phytase gene expression and sick body weight of silkworm larva, the application process of different insects hormone is to the Fig. 5 that influences of phytase gene expression, different MH consumptions are to the Fig. 6 that influences of silkworm phytase gene expression, different MH consumptions are to the Fig. 7 that influences of silkworm chrysalis phytase gene expression, phase Fig. 8 when MH handles the expression of back phytase gene in the silkworm body, phase graphic representation 9 during sick silkworm mortality ratio that MH handles behind the 24h behind the virus infection, MH handles time phase curve Figure 10 of the polyhedron number of postoperative infection Wt BmNPV silkworm and sick body weight of silkworm larva, MH handles influences Figure 11 to what Wt BmNPV bred in the BmN cell of cultivating, different concns JH handle to phytase gene in silkworm, express and sick body weight of silkworm larva influence Figure 12, phase Figure 13 when JH handles the silkworm body weight change of postoperative infection Bm-phy, phase Figure 14 when JH handles expression in silkworm of phytase gene down, phase Figure 15 when JH handles the expression of back Wt BmNPV polyhedrosis gene in silkworm, JH handles infecting the Figure 16 that influences of Wt BmNPV silkworm mortality ratio, Figure 17 mutually during the expression of phytase gene in silkworm under MH and the JH combination treatment, MH and JH combination treatment are to infecting the Figure 18 that influences of Bm-phy family's body weight of silkworm larva, Figure 19 mutually during the expression of polyhedron gene in silkworm under MH and the JH combination treatment, MH and JH combination treatment are to infecting the influence of Wt BmNPV family body weight of silkworm larva
Five, embodiment experiment material:
1. virus stain: the escherichia coli vector pyy-cl (Chinese patent, the publication number: CN1184156A) preserve that carry acid phytase gene by Institute of Feeds,China Academy of Agriculture Sciences; Carry the recombinant silkworm caryogram polyhedron rBmNPV (phyA2 of acid phytase gene, brevity code is Bm-phy, Chinese patent application number: 99103564.X) and the Bombyx mori nuclear polyhydrosis virus Degt of laboratory cultivated silkworm breed variety JYI and egt genetically deficient (season equality, 2000, viral journal 16 (1): 54-59) preserve by the Ministry of Agriculture of Inst. of Silkworm, Chinese Academy of Agricultural Sciences silkworm biotechnology emphasis open laboratory; Bombyx mori cell strain BmN and wild-type polyhedron Zhenjiang strain (BmNPV-ZJ8) are provided by Shanghai Inst. of Biochemistry, Chinese Academy of Sciences animal virus laboratory.
2. Summers is pressed in the daily cultivation of bombyx mori cell BmN, M.D. wait " A Manual of Methods forBaculovirus Vectors and Insect Cell Culture Procedures " (the Texas Agricultural ExperimentStation Bulletin that compiles, No.1555,1987) handbook carries out.
The raising of silkworm is pressed the ordinary method of " Chinese sericulture " (Shanghai science tech publishing house, 1991) of Lv Hongsheng chief editor and is carried out.
3. used substratum TC-100 of cell cultures and foetal calf serum (FBS) are available from Gibco-BRL company, and reagent such as other inorganic salt are all available from Sigma company.The used substratum of silkworm BmN cell is the TC-100 substratum that contains 10% foetal calf serum.
4. insect moulting hormones (Ecdysone) developed by Inst. of Silkworm, Chinese Academy of Agricultural Sciences for Molting hormone, MH by 20-beta-hydroxy ecdyson; (Juvenile hormone, JH) to be Cui for the full professor synthetic and (Cui Weizheng etc., silkworm industry science 22 (2): 125-127,1996) is provided by Shandong Agricultural University's bombycology for Z-512 for corpus allatum hormone.Embodiment one, use the influence of moulting hormone to the sick body weight of silkworm larva of silkworm phytase gene expression amount
The JY kind is raised according to a conventional method to five ages and is played silkworm, and 15 identical boss silkworms of 48h selection mean body weight are one group behind the first feeding, every silkworm inoculation 1.0 * 10
5(5 μ l inoculation liquid contain 2.0 * 10 to pfu Bm-phy
7The Bm-phy of pfu/ml and 0.8mg MH/ml), totally 12 groups.The most of morbidity of silkworm of waiting to infect Bm-phy is collected blood sample and is stored in-20 ℃ of phytase vigor to be determined during to death situation attitude frequently.After MH being mixed the method processing silkworm of in viral liquid, injecting together, the phytase vigor comparison of average every silkworm unit volume hemolymph is according to high by 59.80%, weight ratio contrast weightening finish 5.2%, through significance analysis, both difference reaches extremely and to show (Fig. 3), the MH that uses 4 μ g is described after, can significantly increase the expression amount of unit volume, increase the body weight of every silkworm simultaneously, both increase effects multiply each other, and counting every silkworm increase expression amount is about 68%.The using method of embodiment two, different moulting hormones and of the influence of different cultivated silkworm breed varieties to silkworm phytase gene expression amount
With high expression level kind A1, A15, A16, S16, S17 and JY1 are experiment material (Zhang, Zhifang et al., Symposium of the 10
ThGene, Gene families, lszoyme.1999 Beijing), selects the silkworm that body weight is close in each kind to make the experiment material.The method of injection MH is: containing 1.0 * 10
5Add the MH of 4 μ g/ heads in the Bm-phy diluent of pfu, this mixed solution together is injected into the silkworm body.The method of adding food MH is: infect 1.0 * 10
524h behind the Bm-phy, average every silkworm is added the MH of the about 4 μ g of food, after the mulberry leaf of promptly waiting to spray 90ppm MH are eaten to the greatest extent substantially, feeds new fresh mulberry leaf.Later on by five age silkworm the raising rules carry out.
Phytase vigor (7 the kind mean value) comparison of adding unit hemolymph volume behind the food 4 μ g MH by every silkworm is according to significantly high by 20%, and every body weight of silkworm larva increases by 4.7%; And the phytase vigor of the unit hemolymph volume of injection MH is all compared according to significantly high by 34.7%, every body weight of silkworm larva comparison is according to increasing by 7.7% (Fig. 4), above presentation of results, method with injection MH, no matter the increase at unit volume phytase expression amount still all is being better than the food method of freshening of MH aspect the increase of sick body weight of silkworm larva, also lacks a procedure with injection than adding food MH simultaneously.The using method that MH is described with after recombinant virus mixes together injection for well.Embodiment three, different MH consumption are to the influence of silkworm phytase gene expression amount
With the JY1 kind is material, normally raises second day 5 age, injection 1.0 * 10
5The Bm-phy recombinant virus of pfu, and in Bm-phy, add the MH of 2 μ g, 4 μ g, 6 μ g/ heads respectively.With the close silkworm of 3 * 15 mean body weights as one group of experiment material, the experiment triplicate.Collect blood sample and be stored in-20 ℃, and measure the phytase vigor.The result is as follows: the vigor that every silkworm is injected the unit volume phytase of 2 μ g, 4 μ g, 6 μ g MH respectively all is significantly higher than contrast.Wherein best with the effect of injecting 4 μ g MH, the comparison of unit volume hemolymph phytase vigor is according to increasing by 52.2%; The effect that every silkworm is injected 6 μ g MH is taken second place, and comparison is according to increasing by 46.7%; And the effect of every injection 2 μ g MH is relatively poor, and only comparison is according to increasing by 36.6%.The sick body weight of silkworm larva of injection different amount MH (2 μ g, 4 μ g, 6 μ g) is compared respectively according to increasing by 4.71%, 3.50% and 1.74% (Fig. 5).From the The above results analysis, the effect of injecting 4 μ g MH with every silkworm is the best.Embodiment four, different MH duration of service is to the influence of silkworm phytase gene expression amount and sick body weight of silkworm larva
Second day healthy silkworm (JY1 kind) after five ages, 3 * 15 that the mean body weight of counting is close is one group, every injection 1.0 * 10
5The Bm-phy of pfu, the consumption of MH are every 4 μ g.It is 0 hour that experiment establishes that MH and virus injects simultaneously, and 24 hours injection MH are 24 hours treatment group behind the virus infection, is followed successively by 48 hours treatment group, 72 hours treatment group, 96 hours treatment group later on and does not inject the control group of MH, tests triplicate.Blood sampling when the most of morbidity of extremely sick silkworm is extremely dying is stored in-20 ℃, measures the phytase vigor.From the test-results of table one as can be known, the duration of service of MH no matter from the expression efficiency of phytase should sick body weight of silkworm larva all in recombinant virus infection, to use MH, both compare respectively according to increasing by 57% and 3.66%, have also reduced the amount of labour used when handling simultaneously.
Table one, the duration of service of different MH is to the influence (4 μ g MH/ head) of phytase expression amount
Duration of service, phytase increased the sick body weight of silkworm larva increase of index (%) index (%)
0 hour 157.0 103.66
24 hours 154.6 100.78
72 hours 149.2 99.2
The consumption of 96 hours 127.5 97.1 embodiment five, different MH is to the influence of silkworm chrysalis phytase gene expression amount
With the JY1 kind is experiment material, and method for breeding is raised to putting on an arrow routinely, and choosing 100 healthy of silkworm chrysalises is one group, respectively 1 * 10
5The MH that adds 2 μ g, 4 μ g, 6 μ g in the Bm-phy viral dilution liquid of pfu, what do not add MH is control treatment.60h behind virus infection, every 12h sampling and be stored in-20 ℃, until virus infection till the pupa death, measure phytase vigor in the hemolymph, its result is as shown in Figure 6: every pupa inject phytase vigor in the silkworm chrysalis unit volume hemolymph of 2 μ g MH after infection during 108h comparison according to high by 50.2%, every pupa is injected the enzyme activity comparison of 4 μ g MH according to high by 33.3%, every pupa is injected the comparison of 6 μ g MH according to high by 31.0%, and MH its phytase vigor when 2 μ g consumptions that every pupa be described increases effect and is the best.The phase curve is as shown in Figure 6 during to the expression of silkworm chrysalis phytase gene in pupal cell for the consumption of different MH.Phase when embodiment six, MH handle the expression of back phytase gene in the silkworm body
With the JY1 kind is experiment material, and rearing method is raised and played silkworm second day to 5 ages routinely, and choosing 100 of the close silkworms of health and mean body weight is one group, injects 4 μ g MH+1.0 * 10
5The Bm-phy virus of pfu (MH treatment group), behind virus infection by the time phase blood sampling and take by weighing the disease body weight of silkworm larva and measure enzyme activity, triplicate of 60h, 72h, 84h, 96h, 108h, 120h, 132h.Experimental result as can be seen from Figure 7, has improved the translation and the expression efficiency of the external source phytase gene in the silkworm body and has prolonged the effectively expressing time behind the use MH as shown in Figure 7.During 132h, its phytase vigor and sick body weight of silkworm larva increase by 35.1% and 6.7% respectively behind virus infection.Embodiment seven, different MH duration of service is to the influence of the silkworm half lethal time that infects recombinant phytase virus
Play behind the silkworm two days healthy silkworm five ages of JY1 kind, counting 100 is one group, every injection 1.0 * 10
5The Bm-phy of pfu, the consumption of MH are every 4 μ g.It is 0 hour that experiment is established MH simultaneously injected with virus, what 24 hours injected MH behind the virus infection is 24 hours treatment group, be followed successively by 48 hours treatment group, 72 hours treatment group, 96 hours treatment group later on and do not inject the control group that is of MH, when beginning death to the infection back sick silkworm of 112h, every mortality ratio of investigation in 4 hours, and calculate LT
50, experimental result as shown in Table 2, behind virus infection the different time use MH all can postpone effectively the morbidity death time, prolonged LT to some extent
50Time, wherein use the best results of MH, LT with 24h behind the virus infection
50Prolonged more than the 8h.
Table two, MH duration of service is to infecting the LT of recombinant phytase virus silkworm
50Influence
MH time of application CK 0h 24h 48h 72h 96h
LT
50(h)??????115.59????120.4????124.5????119.0????115.6????118.36
MH handle with the infection recombinant phytase of handling without MH after death rate of the onset the time mutually curve more as shown in Figure 8, use the time phase curve of MH from 24h behind virus infection, delayed the death time of sick silkworm after 4 μ g MH handle, prolonged foreign gene in the intravital effective expression of the silkworm time.Embodiment eight, MH handle behind the wild-type bombyx mori nuclear polyhedrosis virus infected silkworm of back the influence to polyhedrosis gene expression amount and morbidity process
Play behind the silkworm two days healthy silkworm five ages of JY1 kind, counting 100 is one group, every injection 1.0 * 10
5The wild-type Zhenjiang strain nuclear polyhedrosis virus BmNPVZJ-8 strain of pfu, MH is treated to: MH is woven into (4 μ g MH+1.0 * 10 in the diluent of BmNPVZJ-8
5The pfu/ head) injection together, what MH did not handle is contrast.60 hours beginning blood samplings behind the virus infection, later on every 12 or 6 hours respectively sampling once, and the sick body weight of silkworm larva of instrumentation, sample are behind the ultrasonic disruption cell, with the polyhedron number in every milliliter of the blood counting chamber counting, till the basic death of silkworm.
After MH handled, the polyhedron number of every milliliter of hemolymph compare when 96h according to increasing by 39.0%, and the comparison of sick body weight of silkworm larva is according to increasing by 2%, the MH processing is described after, also can increase the polyhedron number in the unit volume hemolymph effectively, promoted the expression of polyhedrosis gene.Handle about MH polyhedron number and the variation of sick body weight of silkworm larva in the silkworm body hemolymph of back and contrast the time curve is as shown in Figure 9 mutually.
Same another batch silkworm of handling, LT between investigation MH handles and do not handle
50Changing conditions, its result is as shown in Table 3.
Table three, MH are handled infecting the silkworm LT of wt BmNPVZJ-8
50Influence
| Handle | Infect the sick mortality ratio (%) of back 72h | Infect the sick mortality ratio (%) of back 84h | Infect the sick mortality ratio (%) of back 90h | ????LT 50 |
| ????CK ?MH(4μg) | ????6 ????2 | ????44.09 ????35.5 | ????86.6 ????65.0 | ????85.67 ????89.90 |
After further specifying MH and handle from The above results, when having increased the protein expression amount effectively and having prolonged the protein effective expression mutually.Embodiment nine, the bombyx mori cell that infects through MH treatments B mNPV have promoted duplicating of virus
Although raise under the condition the identical of same experiment, MH can significantly improve the expression amount of virogene in silkworm larva and pupal cell hemolymph by the utmost point, but between different experiments, owing to be subjected to the nutritional condition of mulberry silkworm rearing and the influence of envrionment conditions, the mobility scale that MH improves effect to phytase gene and polyhedron gene expression amount is big (30%-60%).By above experimental result, can promote recombinant virus phytase gene and the wild-type virus polyhedron gene expression amount in cellulous silkworm larva and pupal cell hemolymph, MH also should have similar facilitation effect in single silkworm cultured cell.For further proving the reliability of MH experiment conclusion in silkworm body and pupal cell, studied with MH and handled the influence that wild-type bombyx mori nuclear polyhedrosis virus is bred in the BmN cell of cultivating, its result is as shown in figure 10.
At 15cm
2Tissue Culture Flask in, the inoculation Suitable Density the BmN cell, treat cell attachment after, remove substratum, adding 1ml contains the serum free medium (MOI=0.1) of an amount of virus particle, cultivate 1h down for 27 ℃, remove the cell upper strata then and infect liquid, the 1ml serum free medium is washed once, add the normal substratum that contains 3 μ g/ml MH again, cultivated 3 days down for 27 ℃, collect 100 μ l supernatants, survey its TCID with the terminal point dilution method
50From infect the time phase curve as seen: 24h behind the virus infection, TCID between MH treatment group and the CK
50Begin to occur difference, 60-72h behind virus infection, the difference maximum of the two, the TCID that this period, MH handled
50Be about 3 times of control treatment.Embodiment ten, MH handle the influence that grow prothoracic gland the back
The JY1 kind plays silkworm by normally raising to 5 ages, establishes 6 kinds of processing, wherein do not do the contrast (CK) of any processing, only inject 4 μ g MH CK-MH, the injection 1.0 * 10
5The wt of pfu wild-type virus BmNPV-ZJ8, inject 4 μ gMH and 1.0 * 10 simultaneously
5The wt-MH of pfu wild-type virus BmNPV-ZJ8, injection 1.0 * 10
5The DEGT of the Degt virus that pfu removes the egt gene, and inject the MH and 1.0 * 10 of 4 μ g simultaneously
5The processing DEGT-MH of the Degt virus of pfu.The average inoculation temperature is 24 ℃, during 108h, dissects prothoracic gland according to method in " experiment of silkworm true tumor " (three province's halls, 1970) of gloomy essence to virus infection.With the thinnest portion of branch behind the unified measurement of the micrometer prothoracic gland, 6 complete prothoracic glands are dissected in every kind of processing at least.Its result as shown in Table 4.
Experimental result shows that healthy silkworm is after MH handles, because intravital MH titre is higher, reverse feedback has delayed the growth and the hormone secretion of self prothoracic gland.And behind the wild-type BmNPV infected silkworm, because the egt genetic expression in the BmNPV genome makes intravital moulting hormone lose physiological function, thereby makes silkworm in order to keep hormonal equilibrium, prothoracic gland is in secretor state always, has promoted the growth of prothoracic gland.After using MH simultaneously with wild-type BmNPV virus infection silkworm, the MH of external source has compensated the MH because of egt genetic expression inactivation, so the intravital hormone of silkworm is in comparatively standard state, and the prothoracic gland form approaches normally.When silkworm infects the recombinant virus DEGT of egt genetically deficient, owing to do not have egt gene pairs silkworm hormone in vivo equilibrated to disturb, so its form roughly approaches normally.And when DEGT and MH acting in conjunction, too high MH has suppressed the secreting function of silkworm body self prothoracic gland in the body, and prothoracic gland is diminished.
Table four, MH handles the influence that virus infection silkworm prothoracic gland is grown
Handle the dissection prothoracic gland and count prothoracic gland size (μ m)
CK??????????????????6????????????????????????65
CK-MH???????????????6????????????????????????36.7
Wt??????????????????8????????????????????????98.8
Wt-MH???????????????8????????????????????????58.8
DEGT????????????????7????????????????????????55.7
DEGT-MH?????????????7????????????????????????28.6
Prove from above-mentioned prothoracic gland anatomy experiment, can compensate the destruction of the caused normal hormonal equilibrium state of MH inactivation that egt genetic expression causes in the virus gene group due to illness after MH uses, and can improve the expression of exogenous gene amount by the egt gene inactivation.Embodiment 11, JH handle the influence to phytase gene expression amount in silkworm
The expression amount height of phytase gene in silkworm five-age larva hemolymph is by the size decision of phytase vigor in every boss silkworm hemolymph content and the unit hemolymph, and the content of hemolymph generally accounts for about 25% of silkworm larva body weight.48h injection recombinant baculovirus after the silkworm five-age larva plays silkworm, body spray 50ppm behind the inoculation 24h; 100ppm; The neotonin of 150ppm is investigated the disease body weight of silkworm larva, collects blood sample, measures the vigor of phytase.
After using the exogenous insect hormone JH of different concns,, can significantly improve the silkworm five-age larva body weight that infects recombinant virus, and improve the expression amount of phytase gene in silkworm larva hemolymph unit volume through significance test of difference.Figure 11 with the contrast body weight and the unit hemolymph in the phytase vigor be index, compared of the influence of JH concentration to expression amount.In lower concentration JH district, along with the increase of JH concentration, the expression amount of sick body weight of silkworm larva and phytase all raises gradually, and when JH concentration was 100ppm, the two all reached maximum value, and after this, along with the increase of JH concentration, sick body weight of silkworm larva and expression amount reduce on the contrary gradually.Variance analysis and test of significance result show, no matter the JH of 100ppm handles sick body weight of silkworm larva, still the phytase expression amount of unit hemolymph volume all is significantly higher than contrast, as shown in Figure 11: after the JH of 100ppm handles, the weight increase of sick silkworm 12.4%, the unit of activity of phytase has increased by 16.4% in every milliliter of hemolymph, and the average expression amount comparison that can extrapolate every silkworm thus is according to exceeding 30.86%.Embodiment 12, different developmental phases are used the influence of JH to phytase gene expression amount and sick silkworm median lethal time in silkworm
Behind five age silkworms, 48h infects the silkworm of recombinant baculovirus after 24h is with the JH body spray first feeding of 100ppm, a check plot injecting virus, not being JH handles, the " Invest, Then Investigate " disease of fully falling ill body weight of silkworm larva, collect blood sample and measure the vigor of phytase, investigate of the influence of each JH treatment time simultaneously sick silkworm median lethal time.
Shown in the table five: 0-72h uses JH, and sick body weight of silkworm larva all is significantly higher than contrast; Use behind the 96h, then and between the contrast do not have notable difference.Wherein, 72h uses JH after playing silkworm and playing silkworm, and sick body weight of silkworm larva is bigger, and the utmost point is significantly higher than contrast.It is not remarkable that different JH duration of service influences difference to the expression amount of recombinant baculovirus external source phytase gene in five age silkworm larva hemolymphs, but all be significantly higher than the contrast of not using JH.
Table five, JH different duration of service are to infecting the influence of Bm-phy silkworm larva body weight and expression amount
Use the sick silkworm intensification factor of JH time average phytase intensification factor on average alive
(h) (%) power (U/ml) is (%) for body weight (g)
CK?????????3.85d????????100????????177.18a??????????100
0??????????4.26a???????110.65??????235.18b?????????132.74
24?????????4.06c???????105.45??????225.80b?????????127.44
48?????????4.08bc??????105.57??????225.16b?????????127.08
72?????????4.21ab??????109.35??????224.21b?????????126.54
96?????????3.98cd??????103.38??????218.65b?????????123.41
120????????3.88d???????100.78??????207.21b?????????116.55
Annotate: lowercase is represented 5% conspicuous level; Capitalization is represented 1% conspicuous level; Down together.
96h behind the recombinant virus infection investigates the sick silkworm mortality ratio of every kind of processing.Test-results shows (table six): the use of JH can obviously prolong the median lethal time of disease silkworm, and 24h and 72h use JH can make disease silkworm disease time more than the contrast prolongation 4h; Other times use JH that disease time is prolonged about 2-3h.
Table six, JH duration of service is to infecting the influence of Bm-phy silkworm larva median lethal time in five ages
| Use the JH time (h) | Different JH disease duration of service silkworm mortality ratio (%) | ????LT 50 | ||
| ????116h | ????120h | ????124h | ||
| ????0 ????24 ????48 ????72 ????96 ????120 ????CK | ????25.86 ????13.46 ????20.00 ????21.05 ????16.67 ????29.31 ????42.58 | ????51.72 ????40.38 ????70.9 ????47.37 ????60.00 ????65.51 ????78.25 | ????75.86 ????82.69 ????89.09 ????82.47 ????85.00 ????87.93 ????94 | ????119.77 ????120.91 ????118.60 ????120.30 ????119.08 ????118.29 ????116.83 |
Comprehensive above test-results, the different time of JH uses and the disease body weight of silkworm larva increased and the prolongation genetic expression time, all can improve the exogenous gene expression amount compared with the control, but difference is not remarkable each other, comprehensive above test-results thinks that 72h uses the best results of exogenous corpus allatum hormone to the raising of recombinant baculovirus exogenous gene expression amount after the silkworm five-age larva plays silkworm or plays silkworm, but require to consider from silkworm rearing, think and use to for well to play behind the silkworm 72h.Phase during phytase gene expression under embodiment 13, JH handle
48h injection recombinant phytase virus after the silkworm five-age larva plays silkworm, the JH of 24h body spray 100ppm behind the virus infection, 60h begins every 12h and investigates the disease body weight of silkworm larva behind the silkworm, and mensuration phytase vigor.As seen from Figure 12: before the 84h, the body weight that JH handles the disease silkworm is lower than contrast behind the virus infection; Afterwards, JH processing beginning to be higher than contrast, this moment, the check plot began morbidity, and sick body weight of silkworm larva descends, and contrasts the disease silkworm to infection back 108h and falls ill cmpletely, handles through JH and then postpones 6h.Sick body weight of silkworm larva JH processing contrasts high by about 7% when finally fully falling ill.As seen from Figure 13: the preceding 96h of virus infection, the expression amount difference of phytase gene was not remarkable between JH handled and contrasts, even the expression amount that JH handles is a little less than contrast, after the 96h, the expression amount that JH handles begins apparently higher than the check plot, to 108h contrast beginning the mass mortality.Can know by inference mutually during from above-mentioned expression, the expression amount raising of phytase gene is mainly worked by prolonging expression time.Embodiment 14, JH processing reaches the influence of median lethal time mutually when the wild-type virus polyhedron gene is expressed in silkworm
Utilize the polyhedron number that forms after the polyhedrin embedding virus particle of wild-type bombyx mori nuclear polyhedrosis virus what as a token of.Investigation JH handles the influence to the polyhedron gene expression amount.48h injected WtBmNPV after five ages, silkworm larva played silkworm, the JH of body spray 100ppm behind the silkworm wild-type virus infection 24h, and the check plot is used clear water mutually and is handled.60h begins to collect disease silkworm blood sample behind the virus infection, after ultrasonic disruption cell and dilution, and numeration polyhedron number; Begin the once sick silkworm mortality ratio of every 6h investigation from 72h, test-results is seen Figure 14 and Figure 15.
As seen from Figure 14: in the early stage (before the virus infection 90h) that JH handles, the expression amount and the contrast of polyhedron gene do not have notable difference.In the 6h afterwards, notable difference appears rapidly in the polyhedron number of the two.During to virus infection 96h, every milliliter of hemolymph polyhedron number comparison that JH handles exceeds more than 35% according to the polyhedron number.
As seen from Figure 15, JH handles and can prolong the silkworm five-age larva disease time that infects wtBmNPV equally, makes disease time postpone the above (JH:LT of 4h
50=89.2h; CK:LT
50=84.8h).Embodiment 15, MH and JH cooperate when handling phytase gene expression mutually the influence with sick body weight of silkworm larva
Play silkworm five ages and begin to add food MH (4 μ g/ head) after 24 hours, every silkworm is injected 5 μ l and contains about 1 * 10 after 48 hours
5The viral dilution liquid of pfu virus particle, the JH of body spray 100ppm again after 72 hours collects the sick silkworm hemolymph of different onset time and also weighs, hemolymph in-20 ℃ of preservations to be used for the phytase activity analysis.Following MH and JH cooperate processing all to be undertaken by this.
Bm-phy virus infection 60 hours beginning was collected once sick silkworm and is weighed and get hemolymph every 12 hours, when MH cooperates JH to handle phytase gene expression mutually and the influence of sick body weight of silkworm larva as 16 and shown in Figure 17.Between virus infection 60-96 hour, the phytase activity of treatment group and control group does not have considerable change, and is active in contrast after 96 hours, and 132 hours active raisings reach 34% to the virus infection; Sick body weight of silkworm larva differed not obvious in 60-72 hour, then slightly descended after 72 hours, and maximum decline reaches 5.6% (132h behind the virus infection).Mutually, HORMONE TREATMENT has obviously prolonged the effective expression time of foreign gene during from the expression of enzyme activity.Embodiment 16, MH and JH cooperate the influence of handling when polyhedron gene expressed mutually with sick body weight of silkworm larva
Utilize the polyhedron number that forms after the wild-type bombyx mori nuclear polyhedrosis virus polyhedrin embedding virus particle in the unit volume hemolymph to be reference, the influence that observation MH and JH combination treatment are expressed polyhedron gene, result such as Figure 18 and shown in Figure 19.Virus infection began to collect the disease silkworm in 60 hours, weighed and got hemolymph, counted polyhedron through ultrasonic disruption cell, dilution back with blood counting chamber, after 72 hours, took a sample once every 6 hours.Between whole period of disease, the polyhedron number average is higher than contrast, and is particularly more obvious after 84 hours, and the maximum comparison is according to exceeding nearly 25% in the time of 90 hours.Yet number causes the silkworm morbidity to increase the weight of owing to polyhedron in the silkworm body after the HORMONE TREATMENT forms, so that sick silkworm mean body weight comparison of the later stage of falling ill is according to obvious reduction (reaching about 15%) is arranged.Test 17, MH and JH cooperate the influence of handling double lethal time
Table seven, MH and JH cooperate to be handled infecting the influence of Wt BmNPV silkworm larva in five ages
| Handle | Mortality ratio (%) | ????LT 50 | |
| Infect back 84h | Infect back 90h | ||
| ????CK ??MH+JH | ????44.09 ????19.7 | ????86.6 ????56.8 | ????84.83 ????88.9 |
From table seven as can be known, MH and JH combination treatment make the five-age larva morbidity lat theta-dead rate that infects wild-type bombyx mori nuclear polyhedrosis virus reduce LT
50Postpone more than 4 hours.This all is favourable to production and the virus multiplication that improves foreign protein.
Range of application of the present invention is not limited to above-mentioned example, says from the mechanism of action and can improve all foreign genes at the expression efficiency of insect baculovirus expression system and the duplicating efficiency of baculovirus.
Claims (10)
1. use the method that insect hormone improves insect baculovirus expression system expression of exogenous gene amount for one kind, it is characterized in that insect moulting hormones (MH), corpus allatum hormone (JH) and moulting hormone (MH) and neotonin (JH) are united use to improve foreign gene and the expression efficiency of polyhedron gene in insect baculovirus expression system.
2. improve insect baculovirus expression system expression of exogenous gene metering method according to the described application insect hormone of claim 1, it is characterized in that described baculovirus is: BmNPV, AcMNPV, ApNPV, BsSNPV, CfMNPV, EoSNPV, HaNPV, HzNPV, LdMNPV, MbMNPV, OpMNPV, SIMNPV, SeMNPV, and TnNPV.
3. improve insect baculovirus expression system expression of exogenous gene metering method according to the described application insect hormone of claim 1, it is characterized in that described insect host is: silkworm (Bombyx mori), wild silkworm (Bombyx mandarina), Semen Ricini silkworm (Philosomia cynthia ricini), wild silkworm (Dictyoplocajaponica), Philosamia cynthia (Philosamia cynthia pernyi), tussah (Antheraea pernyi), Japan giant silkworm (Antheraea yamamai), wild giant silkworm (Antheraea polyphymus), autographa california (Autographa californica), tea geometrid (Ectropis obliqua), cabbage army worm (Mamestrabrassicae), prodenia litura (Spodoptera littoralis), autumn mythimna separata (Spodoptera frugiperda), cabbage looper (Trichoplusia ni), armyworm (Thaumetopoea wilkinsoni), bollworm (Heliothis armigera), U.S. bollworm (Heliothis zea), oriental tobacco budworm (Heliothis assulfa), cigarette beetle (Heliothis virescens), oriental armyworm (Pseudaletia separata), and gypsymoth (Lymantria dispar).
4. improve insect baculovirus expression system expression of exogenous gene metering method according to the described application insect hormone of claim 1, it is characterized in that described insect is a silkworm, described baculovirus is a silkworm baculovirus.
5. improve insect baculovirus expression system expression of exogenous gene metering method according to the described application insect hormone of claim 1, it is characterized in that, described insect moulting hormones is: α-ecdysone, β-ecdysone and 20,26-dihydroxyl ecdyson, plant the ecdysteroids compound of source property, synthetic ecdysteroids compound.Described corpus allatum hormone is: terpenoid compound JH-0 (C19 neotonin), JH-I (C18 neotonin), JH-II (C17 neotonin); JH-III (C16 neotonin) and derivative 738 thereof, ZR515, ZR512, ZR619, ZR777.
6. improve insect baculovirus expression system expression of exogenous gene metering method according to right 1 described application insect hormone, it is characterized in that, described insect hormone using method is that puncture injection, mouthful food and body surface spray use.
7. improve insect baculovirus expression system expression of exogenous gene metering method according to the described application insect hormone of claim 6, it is characterized in that, described insect hormone is five length of times and the pupa time at silkworm larva duration of service.
8. improve insect baculovirus expression system expression of exogenous gene metering method according to the described application insect hormone of claim 1, it is characterized in that the amount ranges that described insect moulting hormones MH is used in silkworm larva is every silkworm of 1～8 μ g/; The amount ranges that is used in silkworm pupa is every pupa of 1～6 μ g/; The concentration range that described corpus allatum hormone JH is used in silkworm larva is the spray of 25～150ppm body; The amount ranges that described insect moulting hormones MH and neotonin JH unite use is 1～8 every of μ g MH/ and the spray of 25～150ppm JH body.
9. improve insect baculovirus expression system expression of exogenous gene metering method according to the described application insect hormone of claim 1, it is characterized in that the optimum consumption that described insect moulting hormones MH is used in silkworm larva is every silkworms of 4 μ g/; The optimum consumption that is used in silkworm pupa is every pupas of 2 μ g/; It is 100ppm that described corpus allatum hormone JH is used in the silkworm larva optimal concentration; Described insect moulting hormones MH and neotonin JH unite use, and optimum consumption is the JH body spray of 4 every of μ g MH/ and 100ppm.
10. improve insect baculovirus expression system expression of exogenous gene metering method according to claim 1,3 described application insect hormones, it is characterized in that, described insect moulting hormones MH is used in bombyx mori cell, and its concentration range that makes an addition in the cell culture medium is 1～5 μ g/ml substratum.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103898111A (en) * | 2012-12-26 | 2014-07-02 | 中国科学院遗传与发育生物学研究所 | Recombinant baculovirus expressing dsRNA inhibiting insect juvenile hormone binding protein gene |
| CN108220250A (en) * | 2017-12-18 | 2018-06-29 | 蔡国祥 | A kind of method for expanding numerous bombyx mori nuclear polyhydrosis virus |
| CN111727980A (en) * | 2020-06-12 | 2020-10-02 | 河南大学 | Pesticide for preventing and treating cotton bollworm by using nuclear polyhedrosis virus and preparation method and application thereof |
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2001
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103898111A (en) * | 2012-12-26 | 2014-07-02 | 中国科学院遗传与发育生物学研究所 | Recombinant baculovirus expressing dsRNA inhibiting insect juvenile hormone binding protein gene |
| CN103898111B (en) * | 2012-12-26 | 2016-12-28 | 中国科学院遗传与发育生物学研究所 | The recombinant baculovirus of the dsRNA of expression inhibiting corpus allatum hormone binding-protein gene |
| CN108220250A (en) * | 2017-12-18 | 2018-06-29 | 蔡国祥 | A kind of method for expanding numerous bombyx mori nuclear polyhydrosis virus |
| CN111727980A (en) * | 2020-06-12 | 2020-10-02 | 河南大学 | Pesticide for preventing and treating cotton bollworm by using nuclear polyhedrosis virus and preparation method and application thereof |
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