CN103060258B - Carcinogenic agent-induced high-yield baculovirus cell line and its preparation method and application - Google Patents
Carcinogenic agent-induced high-yield baculovirus cell line and its preparation method and application Download PDFInfo
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Abstract
The invention provides a high-yield baculovirus ovary cells line induced by a carcinogen, a preparation method and an application, a name of the cell line is IOZCAS-Spex12, and a preservation number is CGMCC No. 4804, the preparation method comprises the following steps: culturing beet armyworm ovary cells; and using the carcinogen to induce and converse the beet armyworm ovary cells obtained in the step 1 to obtain the high-yield baculovirus ovary cells line induced and conversed by the carcinogen; and the invention also relates to the application of high-yield baculovirus ovary cells line in baculovirus production.
Description
Technical field
The present invention relates to a kind of insect cell line and its preparation method and application, especially one derives from insect pupa ovary tissue, by the clone of carcinogen N-methyl-N'-nitro-N-nitroso-guani dine (MNNG) Induction Transformation, and by the method for outer being immortalized of the Induction Transformation clone of prosthesis, belong to transgenic animal technical field.
Background technology
Insect is the biotic population of most species in the world, has abundant cytogenetics system, no matter scientific research or practical application, and insect cell line comes into one's own day by day.Insect cell line is the important tool of the scientific researches such as physiology, developmental biology, cytobiology, molecular biology and biological chemistry always; It is the chief component composition of the rhabdovirus expression vector system of one of four large expression systems, can express the exogenous protein with great economy value and scientific meaning; As bio-reactor amplification insect baculovirus, particularly contain the recombinant baculovirus of foreign gene, for the R&D and production of viral organism sterilant.From Grace within 1962, setting up first can tussah (Autheraea pernyi) ovary cell line of cultured continuously since, in the time of 40 years, the insect cell line of having set up exceedes 500 kinds, they derive from respectively 170 various insects, comprising lepidopteran, Diptera, Homoptera, Hymenoptera, Orthoptera and Coleoptera etc.Although insect cell is cultivated the favor that has been subject to numerous scientists, also obtained in recent years deepening constantly and developing, but the foundation of a strain insect cell line often needs to consume a large amount of time and efforts, especially compared with the increasingly mature various culture technique of mammalian cell, the cultivation means of insect cell are aging, single, lack innovation.
Up to the present, have 6 strains from the clone of beet armyworm (Spodoptera exigua), conventionally derive from newly hatched larvae, hemocyte and fatty body.First clone of beet armyworm UCR-SE-1 is by Gelernter and Federici (Gelernter, W.D. with B.A.Federici J.Invertebr.Pathol.1986,48:199～207) set up, the second strain clone Se3FH is the (Hara such as Hara, K., K.Tsuda, M.Funakoshi and T.Kawarabata In Vitro Cell.Dev.Biol.1993,29A (12): 904～907) set up, two strain clones all derive from beet armyworm newly hatched larvae tissue.The 3rd strain Le-H-HNU7 clone and the 4th strain SeHe920-1a clone derive from beet armyworm hemocyte, and this clone is to Spodoptera litura nucleopolyhedrosis virus sensitivity.Other two strains are the clone that derives from beet exigua larvae fatty body of being set up by this laboratory, and this two strains clone is all very responsive to laphygma exigua nuclear polyhedrosis virus and autographa california nuclear polyhedrosis virus.Although the relevant report of the existing beet armyworm clone of aforementioned documents, but up to the present, also do not derive from the clone of beet armyworm ovary.The ovary of insect is normally paired, is the place of ovum initiation and development.Ovary is made up of ovariole (ovariole), and in all kinds of insects, the number of ovariole is widely different, and lepidopterous insects is generally 4,6 or 8.Since Grace utilizes ovary tissue to set up first insect cell line, ovary is the vital tissue source of Establishment of Cell Line always.The ovary tissue that the pupa that dissection will be sprouted wings obtains is easier to operation, reduces opportunities for contamination.Still there is very large demand in a greater variety of insect cell lines, sets up the new clone that derives from insect ovary, and the rhabdovirus expression vector system more efficiently that builds is very significant.
Traditional insect cell line establishment method is normally waited for cell spontaneous transformation, and because condition is difficult to control, usually due to pollution, all that has been achieved is spoiled.After cultured cell in vitro successfully goes down to posterity, in the time cultivating certain algebraically, can enter growth-inhibiting state, vitality obviously weakens, and multiplication capacity declines, and cessation of growth cessation is also final dead.It is the important means that obtains cellular immortalization that artificial induction's cultured cell in vitro transforms.Under the mutagen of artificial design, cell is transformed, transformation efficiency is higher, and passage cell growth cycle is long, proterties is stable.In cell cultivation process, manual-induced conversion comprises the various mutagen impacts such as chemistry, physics, biology (virus) in vitro.Under mutagenic compound exist, the cell DNA of vitro culture damages, but now cell is still survived, and cell carries out the self-regeneration of DNA for the existence of oneself, because just having the enzyme of many self-regeneration DNA, cell itself exists, its repair process is complicated, but in this complicated DNA repair process, mis repair also can occur unavoidably, the reparation of mistake can cause the change of DNA structure, repair and finish, this altered DNA structure is just fixed, and irreversible.This just indicates that hereditary change has occurred cell DNA.Under suitable condition, this cell just can continuous growth and breeding.
Transform with chemical carcinogen induced animal normal cell system separately, all have report (Nes now S, Garland H, Curt is G.Cancer let t, 1989 both at home and abroad; 47 (1-2): 91～97.; Tsuch iya T, Umeda M.Carcinogenes is.1995; 16 (8): 1887～1894.).N-methyl-N'-nitro-N-nitroso-guani dine (MNNG) is the nitrous acid amides representative of synthetic, for conventional chemical carcinogens, can there is covalent attachment with the 0-6 position of DNA guanine in its alkanisation group, form adducts, cause base mispairing, thereby bring out cell transformation (model rainbow, Dong Huifen, Jiang Mingsen, Ming Zhenping, Zhong Qinping, the research of effect of MNNG on cytoskeleton of cultured cells from adult Schistosoma japonicum, sick magazine, 2001,20 (6): 401-403 learned of place of china).According to domestic, N-methyl-N'-nitro-N-nitroso-guani dine is used for studying the propagation of mammalian cell.For example, the impact of the N-methyl-N'-nitro-N-nitroso-guani dine that recklessly waits report for 1994 quietly on vascular smooth muscle cell proliferation, the DNA that has obviously strengthened cell is synthetic, has significantly promoted division growth (Hu Jing, the Wen Jinkun of cell, Wei Suzhen, Zuo Lianfu, the impact of N-methyl-N'-nitro-N-nitroso-guani dine on vascular smooth muscle cell proliferation, Chinese physical medicine magazine, 1994,16 (4): 211-213).Zhang Wengeng etc. utilize MNNG Induction Transformation and have set up human neofetus dermal fibroblasts system (Zhang Wengeng, Xu Gang, Wang Yanping, Chen Xiaobing, the foundation of the human neofetus dermal fibroblasts system of MNNG Induction Transformation, 1994,16 (3): 125-128).Aspect invertebrates, MNNG focuses mostly in the research to Cells of Schistosoma japonicum inducing action, to the effect of Cultured Cells From Adult Schistosoma Japonicum skeleton, Phosphoric acid esterase and bird ammonia enzyme decarboxylase; The impact of nucleolus organizer region's organizer-region-associated Proteins; Research to Cultured Cells From Adult Schistosoma Japonicum propagation etc. all has report (model rainbow, Dong Huifen, Jiang Mingsen, Ming Zhenping, Zhong Qinping, the research of effect of MNNG on cytoskeleton of cultured cells from adult Schistosoma japonicum, the sick magazine of learning of place of china, 2001,20 (6): 401-403; Liu Qing, Dong Huifen, Jiang Mingsen, Ming Zhenping, Zhong Qinping, the research of Mnng On Phosphatase In The Cultured Cells From Adult Schistosoma Japonicum impact, Chinese prevention and cure of snail fever magazine, 2002,14 (1): 14-16; Model rainbow, Huang Min, Wang Qian, Li Manjun, the preliminary study of MNNG to the effect of chistosoma japonicum cell of adult ornithine decarboxylase activity, Chinese parasitology and parasitic disease magazine, 2002,20 (1): 35-36; Zhong Qinping, Ming Zhenping, Jiang Mingsen, Dong Huifen, MNNG induces the impact on Cultured Cells From Adult Schistosoma Japonicum nucleolus organizer region organizer-region-associated Proteins, Chinese prevention and cure of snail fever magazine, 2009,21 volumes (5): 378-381; Dong Huifen, Jiang Mingsen, Ming Zhenping, Zhong Qinping, Yang Mingyi, the research of N-methyl-N'-nitro-N-nitroso-guani dine induction Cultured Cells From Adult Schistosoma Japonicum propagation, Chinese public health, 2000,16 (10): 883-884).
Have no at present the report of N-methyl-N'-nitro-N-nitroso-guani dine (MNNG) for lepidopteran insect cell Induction Transformation.External evoked and set up clone to beet armyworm pupa ovary primary cell by N-methyl-N'-nitro-N-nitroso-guani dine (MNNG), for the foundation of insect cell line provides new method and approach.
Summary of the invention
Therefore, the object of the invention is for there is no at present the deficiency of N-methyl-N'-nitro-N-nitroso-guani dine (MNNG) for lepidopteran insect cell Induction Transformation, it is and its preparation method and application to think that the foundation of insect cell line provides new method and approach that a kind of high virus producing cell of carcinogen induction is provided.
On the one hand, the invention provides a kind of high yield virus ovary cell line of carcinogen induction, the name of this clone is called IOZCAS-Spex12, and its preserving number is CGMCC No.4808 (Classification And Nomenclature: the strain of beet armyworm pupa gonad cell, preservation date, on 05 03rd, 2011, depositary institution: China Committee for Culture Collection of Microorganisms's common micro-organisms center, address, No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, Institute of Microorganism, Academia Sinica, postcode: 100101).
Preferably, described carcinogen is N-methyl-N'-nitro-N-nitroso-guani dine.
Preferably, described gonad cell is Beet Armyworm Eggs nest clone.
On the other hand, the invention provides a kind of preparation method of high yield virus ovary cell line of carcinogen induction, under aseptic condition, carry out, specifically comprise the following steps:
Step 1: cultivate beet armyworm ovary cell;
Step 2: with the beet armyworm ovary cell obtaining in carcinogen Induction Transformation step 1; With
Step 3: the high yield baculovirus ovary cell line that obtains carcinogen Induction Transformation.
Preferably, in step 1, described cultivation beet armyworm ovary cell, comprises the following steps:
Step 1.1: female beet armyworm pupa is immersed in 3% hypochlorous acid solution to 5 minutes, and 10-20 minute in 75% ethanolic soln, carries out surface sterilization, then cleans insect with sterile distilled water, then blots pupa surface-moisture;
Step 1.2: dissecting insects, takes out beet armyworm ovary tissue;
Step 1.3: with organizing 2-3 time of obtaining in physiological saline cleaning step 1.2, clean with cell culture fluid again, after cleaning, this tissue is put into the Tissue Culture Flask that contains 1mL cell culture fluid I rinse, cover tightly bottle cap, put into the cell culture incubator of 27 ℃ of unglazed photographs and cultivate 24 hours;
Step 1.4: add appropriate cell culture fluid II, make tissue completely or major part is immersed in this nutrient solution, with step 1.3 similarity condition under cultivate;
Step 1.5: the nutrient solution of every 7-10 days sucking-off half amount, and the new cell culture fluid II of half amount that simultaneously changes to, until observe continuous expansion and start propagation cells fill whole culturing bottle.
Preferably, in step 2, described carcinogen is N-methyl-N'-nitro-N-nitroso-guani dine.
Preferably, the concrete reactions steps of described step 2 is as follows: use the cell culture fluid III that contains 3 μ g/mL N-methyl-N'-nitro-N-nitroso-guani dine (MNNG) instead, continue with step 1.3 similarity condition under cultivate.
Preferably, as follows in the concrete reactions steps of step 3:
Step 3.1: use containing the cell culture fluid III of N-methyl-N'-nitro-N-nitroso-guani dine (MNNG) and cultivate beet armyworm ovary tissue after 72 hours, phase microscope observation of cell state, change cell culture fluid II, the cell that success transforms continues and step 1.3: under similarity condition, cultivate;
Step 3.2: culturing cell under the condition identical with step 1.5 again, until obtain the high yield virus ovary cell line of carcinogen induction.
Again on the one hand, the high yield virus gonad cell that the invention provides a kind of carcinogen induction ties up to the application in baculovirus production, and can use rhabdovirus expression vector to express recombinant protein.
Preferably, described baculovirus is laphygma exigua nuclear polyhedrosis virus (SeNPV) and autographa california nuclear polyhedrosis virus (AcMNPV).
The explanation of cell culture fluid that the inventive method is used: cell culture fluid used in the present invention is the conventional insect cell nutrient solution adopting and the mixture of penicillin, Streptomycin sulphate and foetal calf serum, and the cell culture fluid pH being made into is between 6.0-6.8.Insect cell nutrient solution can be the insect cell nutrient solution of extensive stock, as TNM-FH, and Grace ' s, Sf 900, TC-100, IPL-41, Ex-Cell400 etc.
Wherein cell culture fluid I is the mixture of insect cell nutrient solution and penicillin, Streptomycin sulphate, phenylthiourea and foetal calf serum; Cell culture fluid II is the mixture of insect cell nutrient solution and penicillin, Streptomycin sulphate and foetal calf serum; Cell culture fluid III is the mixture of insect cell nutrient solution and penicillin, Streptomycin sulphate, N-methyl-N'-nitro-N-nitroso-guani dine (MNNG) and foetal calf serum.
Conventionally, in cell culture fluid, Penicillin Content is that 100U/mL, content of streptomycin are 100U/mL; Induction Transformation nutrient solution is that above-mentioned nutrient solution is containing N-methyl-N'-nitro-N-nitroso-guani dine (MNNG) 3 μ g/mL; Animal serum content is 10% (volume ratio) of cell culture fluid.
It is experiment material that the present invention utilizes beet armyworm pupa gonad cell, by the method for carcinogen N-methyl-N'-nitro-N-nitroso-guani dine (MNNG) Induction Transformation, built up a strain beet armyworm pupa ovary cell line, this clone is extremely sensitive to laphygma exigua nuclear polyhedrosis virus and autographa california nuclear polyhedrosis virus.
In the present invention, the biological property of the beet armyworm pupa ovary cell line of Induction Transformation is observed and is measured:
1. through experimental observation, this clone major part is attached cell, also has partial suspended in nutrient solution.The shape of cell has 3 types: most cells circle, small part fusiformis, less ellipse, easily assembles and form cell mass.(Fig. 1);
2.IOZCAS-Spex12 is to laphygma exigua nuclear polyhedrosis virus and autographa california nuclear polyhedrosis virus sensitivity, can observe typical cell pathology feature, be that nucleus increases, include a large amount of polyhedron particles, infection rate is respectively 91.4% and 65.5%; And at least can 3 generations of continuous passage.(Fig. 2-3)
3. according to McIntosh and Ignoffo, the method for 1989 (McIntosh, A.H. and C.M.Ignoffo J.Invertebr.Pathol.1989,54:97～102), measure the 11st generation cell growth curve and population doubling time.With 1.54 × 10
5the concentration of cell/mL is inoculated in 24 well culture plates, 27 ℃ of cultivations, every 48h measures cell concn, draws growth curve, and presses formula T=tlg2/[lg (N/N0)] calculate, the cell colony doubling time in the 11st generation is 71.06 hours.
Wherein T=is in one times of required time of logarithmic phase average increment
T=is inoculated into the time of measuring cell count
Cell count when N0=inoculation
The total cellular score that N=moment t measures
4. according to Takahashi etc. the method for (Takahashi, Mitsuhashi and Ohtaki (1980) Develop.Growth and Differ.22:11-19), measured the 12nd generation cell caryogram.Due to the chromosome number n=31 (Hara of beet armyworm, Tsuda, Funakoshi and Kawarabata In Vitro Cell.Dev.Biol.1993,29A (12): 904～907), and the chromosome number of IOZCAS-Spex12 is between 116-131, therefore, newly-established clone IOZCAS-Spex12 forms (Fig. 2) by 4 times of somatocyte.
5. use the method (McIntosh of DAF-PCR, Grasela and Matteri (1996), Insect Mol.Biol.5:187～195) identify that clone IOZCAS-Spex12 derives from beet armyworm really, but not the pollution of other clone (Fig. 4).The DNA being extracted by IOZCAS-Spex12 and beet armyworm pupa DNA, beet armyworm ovary DNA, to derive from banding pattern main after the DNA cloning of IOZCAS-SpexII-A of beet exigua larvae fatty body identical; And with contrast, the cell banding pattern of BCIRL-Hz-AM1 that derives from Sf9, the Heliothis zea pupa ovary of the greedy noctuid in meadow is obviously different.
6. use the frozen method of conventional cell to carry out frozen processing to the part cell of certain generation, preserve the kind money of cell, and successfully recovery.
The differential genes expression analysis of the beet armyworm pupa ovary cell line of Induction Transformation in the present invention:
IOZCAS-Spex12 has 10 genes on expressing, to have significantly or utmost point significant difference with IOZCAS-Spex11 (contrast, derive from equally beet armyworm pupa ovary tissue, process without MNNG).
IOZCAS-Spex11 sets up for this laboratory, derives from equally beet armyworm pupa ovary tissue, the clone of not processing through MNNG.Its preserving number is CGMCC No.4807 (Classification And Nomenclature: beet armyworm pupa gonad cell strain, preservation date, on 05 03rd, 2011, depositary institution: China Committee for Culture Collection of Microorganisms's common micro-organisms center, address, No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, Institute of Microorganism, Academia Sinica, postcode: 100101).
Accompanying drawing explanation
Below, describe by reference to the accompanying drawings embodiment of the present invention in detail, wherein:
Fig. 1 is the cultivation aspect graph of IOZCAS-Spex12 of the present invention, and in figure, scale is depicted as 400 μ m;
Fig. 2 obtains the polyhedrosis aspect graph of a large amount of virus after IOZCAS-Spex12 of the present invention infects laphygma exigua nuclear polyhedrosis virus, and in figure, scale is depicted as 200 μ m;
Fig. 3 is that many embedding nuclear polyhedrosis virus of IOZCAS-Spex12 infection autographa california of the present invention obtain the polyhedrosis aspect graph of a large amount of virus, and in figure, scale is depicted as 200 μ m;
Fig. 4 is the growth curve of IOZCAS-Spex12 of the present invention;
Fig. 5 is the caryogram of IOZCAS-Spex12 of the present invention;
Fig. 6 is the DNA fingerprint amplification collection of illustrative plates that DAF-PCR identifies IOZCAS-Spex12, in figure, 1 is the collection of illustrative plates of beet armyworm ovary (Spodoptera exigua ovary), 2 is the collection of illustrative plates of beet armyworm pupa (Spodoptera exigua pupa), 3 is the collection of illustrative plates of the IOZCAS-Spex II-A of fatty body, the collection of illustrative plates of 4 Sf9 for the greedy noctuid in meadow, 5 is the collection of illustrative plates of the BCIRL-Hz-AM1 of Heliothis zea pupa ovary, 6 is the collection of illustrative plates of IOZCAS-Spex12, and 7 is the collection of illustrative plates of DNA marker (DNA Marker).
Fig. 7 is for using 'beta '-tubulin (tubulin) to analyze ten genetic expressions as internal reference by Real-Time PCR method: in figure, ordinate zou is gene relative expression quantity, X-coordinate is followed successively by SUMO-1 activating enzyme (activating enzyme), BCCIP-proteinoid (like protein), little HSP (small HSP), 10kDa HSP, GST, CypA, the acceptor (receptor for activated PKC) of the PKC activating, PDI proteinoid ERp57 (PDI like protein ERp57), ALDH, dependent DBPA-the proteinoid of ATP DEAD box (DEAD box ATP-dependent RNA helicase-like protein).
the preservation information of biomaterial
The preserving number that name of the present invention is called the clone of IOZCAS-Spex12 is CGMCC No.4808, Classification And Nomenclature: beet armyworm pupa gonad cell strain, preservation date, on 05 03rd, 2011, depositary institution: China Committee for Culture Collection of Microorganisms's common micro-organisms center.
The preserving number that name is called the clone of IOZCAS-Spex11 is CGMCC No.4807, Classification And Nomenclature: beet armyworm pupa gonad cell strain, preservation date, on 05 03rd, 2011, depositary institution: China Committee for Culture Collection of Microorganisms's common micro-organisms center.
Embodiment
the foundation of embodiment 1 MNNG induction beet armyworm pupa ovary cell line
5 days female pupas of beet armyworm of getting last age (derive from plague of rats comprehensive regulation research National Key Laboratory of agricultural insect pest of Institute of Zoology, Academia Sinica, beet exigua larvae uses artificial diet at 26 ± 1.5% ℃, under the condition of relative humidity 65%-75%, raises.) being immersed in 3% hypochlorous acid solution 5 minutes, 10-20 minute in 75% ethanolic soln, carries out surface sterilization.Dissect this pupa and take out ovary tissue, when operation, keep it complete as far as possible.Clean this tissue 2-3 time with physiological saline, use again cell culture fluid I (take TNM-FH as main, containing the penicillin of 100U/mL, the foetal calf serum of the Streptomycin sulphate of 100U/mL and 10% (v/v), pH=6.2) clean 2-3 time, put into the 25cm that uses the rinse of 1mL cell culture fluid
2tissue Culture Flask in, put into the cell culture incubator of 27 ℃ of unglazed photographs Celsius and cultivate 24 hours.Then add 3mL cell culture fluid II, put under similarity condition and cultivate.Note key that the method is successfully set up clone be to make tissue be close to Tissue Culture Flask at the bottom of, do not make tissue suspension in cell culture fluid.Left and right sucking-off in later every 7-10 days is the nutrient solution of amount partly, and changes to the new nutrient solution II of half amount simultaneously.A large amount of single cells expansion to the periphery gradually dissociate this operation can be observed tissue after the 3rd day around.In the time that primary cell is bred to 9 days, use the cell culture fluid III containing 3 μ g/mL N-methyl-N'-nitro-N-nitroso-guani dine (MNNG) instead, after 72 hours, use normal cell nutrient solution II instead, in the cell culture incubator of 27 ℃ of unglazed photographs, cultivate.At the induction initial stage, not all cells sustains damage, and only has sub-fraction to enter conversion developmental stage, is called precancerous cell, is scattered in intac cell.Cell continues in culturing process normal cell because contact inhibition stops division until dead, and those precancerous cells are owing to losing the restriction of contact inhibition and accelerated the speed of breeding growth, and then division growth continuously.The cells contacting transforming suppresses to disappear, and has the trend of accumulated growth.After 60 days, in the time that cell proliferation extremely will be paved with whole culturing bottle, cell is put into new culturing bottle together with whole nutrient solution sucking-offs, and adds the cell culture fluid II that 2mL is new.Establishment of Cell Line initial success.After start to go down to posterity at cell the 8th day, start partly to measure for the second time sub-bottle and go down to posterity, the later generation time maintains 7 days.While passing to for the 11st generation, go down to posterity than 1: 3, the generation time maintains 6 days, and Growth of Cells starts to stablize, and while reaching for the 12nd generation, the generation time has foreshortened to 4-5 days.Final this clone is named as IOZCAS-Spex12.
observation of biological characteristics and the mensuration of embodiment 2 IOZCAS-Spex12
(1) morphological specificity: as shown in Figure 1, through microscopic examination, this cell line cell is easy to form cell mass and can stands high-density growth environment, has broken through contact inhibition.The shape of cell has 3 types: circle, fusiformis and ellipse.Most cells is adherent.
(2) growth of cell: at 27 ℃, in the 11st generation of clone,, population doubling time was 71.06h in the TNM-FH nutrient solution of the Streptomycin sulphate of the penicillin containing 10% foetal calf serum, 100U/mL, 100U/mL, 3 μ g/mL N-methyl-N'-nitro-N-nitroso-guani dine (MNNG).As shown in Figure 4, the high-density Yue Keda 1.6 × 10 of cell
6individual cell/mL.
(3) karyotyping: IOZCAS-Spex12 the 12nd generation cell is 4 times of somatocyte, and chromosome number scope 116-131 (2n=62), is shown in Fig. 5.
(4) DAF-PCR identifies: as shown in Figure 6, clone IOZCAS-Spex12 derives from beet armyworm really, but not the pollution of other clone.The DNA being extracted by IOZCAS-Spex12 is same as beet armyworm pupa, beet armyworm ovary (all derives from plague of rats comprehensive regulation research National Key Laboratory of agricultural insect pest of Institute of Zoology, Academia Sinica, beet exigua larvae uses artificial diet at 26 ± 1.5% ℃, under the condition of relative humidity 65%-75%, raises.) and fatty body (derive from agricultural insect pest of Institute of Zoology, Academia Sinica plague of rats comprehensive regulation research National Key Laboratory, beet exigua larvae uses artificial diet at 26 ± 1.5% ℃, under the condition of relative humidity 65%-75%, raises.Its source please be described) DNA fingerprint amplification collection of illustrative plates, and be different from the collection of illustrative plates of the BCIRL-Hz-AM1 of Sf9, the Heliothis zea pupa ovary (deriving from biotechnology research institute of Scientia Agricultura Sinica research institute) of the greedy noctuid in meadow (deriving from plague of rats comprehensive regulation research National Key Laboratory of agricultural insect pest of Institute of Zoology, Academia Sinica).
(5) freezing and thawing: use the frozen method of conventional cell to carry out frozen processing to the part cell of certain generation, preserve the kind money of cell, and can successfully recover.
the viral susceptibility of embodiment 3.
IOZCAS-Spex12 is to multiple nuclear polyhedrosis virus sensitivity: SeNPV, AcMNPV and SpltNPV budding pattern virus particle BV (all deriving from plague of rats comprehensive regulation research National Key Laboratory of agricultural insect pest of Institute of Zoology, Academia Sinica) are inoculated to IOZCAS-Spex12 with the concentration of 0.001 larva equivalent/milliliter, cultivate after 7 days, can observe typical cell pathology feature by inverted microscope, as Fig. 2-3, be that nucleus increases, include a large amount of polyhedron particles.And at least can 3 generations of continuous passage.Viral infection rate is respectively 91.4%, 81.4% and 31.9%.
the differential genes expression analysis of embodiment 4.IOZCAS-Spex12 and IOZCAS-Spex11
Using Invitrogen company's T rizol to extract respectively IOZCAS-Spex12 (contrasts with IOZCAS-Spex11, derive from equally beet armyworm pupa ovary tissue, process without MNNG) RNA of cell, then use DNase I to process and remove genomic dna.Use TakaRa company
rT reagent Kit Perfect Real Time test kit, RNA reverse transcription is become after complete cDNA, carry out real-time quantitative PCR reaction, as shown in Figure 7, show that IOZCAS-Spex12 has 10 genes on expressing, to have significantly or utmost point significant difference compared with IOZCAS-Spex11.The reverse transcription reaction primer of these 10 genes and internal reference is in table 1.
Table 1. real-time quantitative PCR reaction primer sequence.
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