CN102807968B - Transgenic fat body cell line of high-yield baculovirus and preparation method as well as application thereof - Google Patents
Transgenic fat body cell line of high-yield baculovirus and preparation method as well as application thereof Download PDFInfo
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Abstract
The invention provides a transgenic fat body cell line of high-yield baculovirus and a preparation method as well as application thereof. The cell line which is named as IOZCAS-Spex X has a preservation number of CGMCC No. 4505; and the preparation method of the transgenic fat body cell line comprises following steps of: culturing asparagus caterpillar fat body cells; transforming genes containing human telomerase reverse transeriptase into a known expression vector to obtain a recombinant expression vector; leading the recombinant expression vector into the asparagus caterpillar fat body cells; and enabling the asparagus caterpillar fat body cells to express human telomerase reverse transeriptase so as to obtain the transgenic fat body cell line. In addition, the invention discloses application of the transgenic fat body cell line to production of baculovirus.
Description
Technical field
The present invention relates to a kind of transgenic insect clone, relate in particular to transgenosis fatty body clone of a kind of high yield baculovirus and its preparation method and application, belong to transgenic animal technical field.
Background technology
Insect is the biotic population of most species in the world, has abundant cytogenetics system.The main application of insect cell line shows: as research material, be the important tool of the scientific researches such as physiology, developmental biology, cytobiology, molecular biology and biological chemistry always; As the important component part of rhabdovirus expression vector system, can express the exogenous protein with great economy value and scientific meaning; As bio-reactor, amplification insect baculovirus, particularly contains the recombinant baculovirus of foreign gene, produces biotic pesticide.It is reported, nineteen sixty-five first strain insect cell line after successfully setting up so far, in the time of nearly 40 years, the insect cell line of having set up exceedes 500 kinds.They derive from respectively 170 various insects such as lepidopteran, Diptera, Homoptera, Hymenoptera, Orthoptera and Coleoptera, and wherein major part derives from lepidopteran and dipteral insect.Lepidopterous clone derives from Various Tissues, comprises ovary, embryo, hemocyte, fatty body etc.The fatty body of insect has similar function with mammiferous liver, is important physiological metabolism tissue, be also baculovirus main infect, copy one of position, there is important using value.The foundation of insect cell line often needs to consume a large amount of time and efforts.In culturing process, owing to being subject to the impact of various environmental factors, there is spontaneous transformation in cell, by original diploid caryogram, is transformed into polyploid/heteroploid caryogram in vitro sometimes, and the growth characteristics of cell change and being immortalized.The cell of immortalization loses contact inhibition in process of growth, can infinitely breed and go down to posterity.Such conversion not only the time long, and transformation efficiency is extremely low.Setting up a strain clone often needs to cultivate a large amount of primary cells, wastes time and energy, and often trusts to chance and strokes of luck formula and waits for cell spontaneous transformation, and condition is difficult to control, and usually because of pollution, all that has been achieved is spoiled.And after cultured cell in vitro successfully goes down to posterity, when cultivating certain algebraically, conventionally can enter growth-inhibiting state, and vitality obviously weakens, and multiplication capacity declines, and cessation of growth cessation is also final dead.It is the important means of being immortalized cell that artificial induction's cultured cell in vitro transforms.Under the mutagen of artificial design, cell is transformed, conventionally only need within 1-3 month, just can realize, and transformation efficiency is higher, passage cell growth cycle is long, proterties is stable.
Find at present, cellular immortalization and telomere and Telomerase are in close relations.Telomere and Telomerase are a kind of special constructions of eukaryotic cell end of chromosome, telomeric dna and telomere protein, consist of.Telomeric dna is the repeated nucleotide sequences that is rich in the high conservative of G, participates in DNA replication dna, and stablizes and completely copy and play an important role remaining chromosomal.Human cell's telomere is comprised of tumor-necrosis factor glycoproteins (TTAGGG) n of 10-15kb; In plant, telomere occurs with (TTTAGGG) n tumor-necrosis factor glycoproteins; Insect cell telomere is generally comprised of the tumor-necrosis factor glycoproteins (TTAGG) of 6-8kb.Known at present, in insect, there are three kinds of telomeric dna types: the telomeric dna of silkworm (Bombyx mori) forms (Okazaki et al.1993) by tumor-necrosis factor glycoproteins (TTAGG) n; Fruit bat (Drosophila melanogaster) telomeric dna forms (Biessmann et al.1990, Levis et al.1993) by transposable element HeT-A and TART; Telomeric dna in midge forms (Nielsen & by complicated gene tandem repetitive sequence
1993, Zhang et al.1994).Once, telomere shortens 50-200bp in the every division of cell of vitro culture, and when foreshortening to a threshold value, cell will stop division, moves towards old and feeble and dead.Telomerase is a kind of ribonucleoprotein enzyme, RNA and protein, consists of, and has the function of reversed transcriptive enzyme, take self RNA as the synthetic telomeric dna of template, to maintain the length of telomere.Cellular immortalization refers to that the cell of vitro culture, due to the change of self or the impact of external conditions, escapes cellifugal senescence phase and climacteric, and it is stable that telomere length maintains all the time, thereby obtains the process of unlimited multiplication capacity.Cellular immortalization and Activation of Telomerase are closely related.Telomere maintain the activation that depends on Telomerase.(the human telomerase catalytic subunit of stably express human telomerase reverse transcriptase gene in primary cultured cells, hTERT) can stablize the length of telomere, make cell be easy to immortalization (Cemi C.Telomeres, telomerase, and myc.An update[J] .Mutat Res.2000,462 (1): 31-47.).Although a lot of about telomerase gene being applied to the report of mammalian cell immortalization, have no and relate to the report that utilizes transfected with human telomerase gene to obtain insect immortalized cell line.By artificial induction's vitro conversion method, set up and derive from insect fatty body and express the clone of human telomerase reverse transcriptase's gene, for being immortalized clone provides important channel.
Summary of the invention
Therefore, the object of the invention is the deficiency that contains human telomerase insect cell line for there is no at present, the transgenosis fatty body clone that a kind of high yield baculovirus is provided, it contains human telomerase reverse transcriptase's gene, for being immortalized insect cell line provides important channel.
Another object of the present invention is to provide the preparation method of the transgenosis fatty body clone of a kind of high yield baculovirus.
A further object of the present invention is to provide the application of the transgenosis fatty body clone of a kind of high yield baculovirus.
For above-mentioned purpose, technical scheme of the present invention is as follows:
One aspect of the present invention provides the transgenosis fatty body clone of a kind of high yield baculovirus, and the name of this clone is called IOZCAS-SpexX, and the preserving number of this clone is CGMCC No.4505.
Preferably, described transgenosis fatty body clone contains human telomerase reverse transcriptase's gene (hTERT).
Preferably, the nucleotide sequence of described human telomerase reverse transcriptase's gene is as shown in SEQ ID NO 1.
Another aspect of the present invention provides the preparation method of the transgenosis fatty body clone of a kind of high yield baculovirus, comprises the following steps:
Step 1: cultivate beet armyworm fatty body cell;
Step 2: will enter in known expression vector containing human telomerase reverse transcriptase's gene transformation, and obtain recombinant expression vector;
Step 3: described recombinant expression vector is imported in described beet armyworm fatty body cell; With
Step 4: make described beet armyworm fatty body cell can express human telomerase reverse transcriptase, obtain transgenosis fatty body clone.
Preferably, in step 1) in, described cultivation beet armyworm fatty body cell, comprises the following steps:
Step 1.1: beet armyworm end instar larvae 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 fatty body tissue;
Step 1.3: with organizing 2-3 time of obtaining in physiological saline cleaning step 2, with cell culture fluid I, clean 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 I, 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 known expression vector is pIZT-V5-His or pIB-V5-His.
Preferably, in step 3) in, described recombinant expression vector imports in described beet armyworm fatty body cell by liposome.
Preferably, in step 4) in, specifically by following steps, obtain transgenic insect clone:
Step 4.1: put into step 1.3 similarity condition under cultivate at least 4 hours, and frequently rock.After transfection, change appropriate cell culture fluid II continue with step 1.3 similarity condition under cultivate;
After step 4.2:72 hour, fluorescence inverted microscope is observed transfection effect, change contain eukaryotic cell screen antibiotic cell culture fluid III continue with step 1.3 similarity condition under cultivate;
Step 4.3: by identical with step 1.5 this cell culturing cell, until obtain transgenic insect clone.
Above-mentioned whole process is all carried out under aseptic condition; Four kinds of wherein said cell culture fluids are respectively that 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, bleomycin (zeocin) and foetal calf serum, and the cell culture fluid pH being made into is 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-Cell 400 etc.
Conventionally, in cell culture fluid, Penicillin Content is that 100U/mL, content of streptomycin are 100U/mL; The nutrient solution of screening transgenic cell is that above-mentioned nutrient solution is containing bleomycin (zeocin) 400 μ g/mL; Transgenic cell line nutrient solution is that above-mentioned nutrient solution is containing bleomycin (zeocin) 50 μ g/mL; Animal serum content is 10% (volume ratio) of cell culture fluid.
The application that the transgenic insect clone of a kind of high yield baculovirus is provided on the one hand again of the present invention, the application of described transgenic insect clone in baculovirus produces.
Preferably, described baculovirus is laphygma exigua nuclear polyhedrosis virus (SeNPV) or autographa california nuclear polyhedrosis virus (AcMNPV).
In the present invention, having expressed the biological property of the beet armyworm fatty body clone of human telomerase reverse transcriptase (hTERT) gene observes and measures:
1. through experimental observation, this clone major part is attached cell, also has and is suspended on a small quantity in nutrient solution.The shape of cell has 3 types: most cells circle, small part fusiformis, and less like macrophage-shaped.
2.IOZCAS-SpexX, to beet armyworm and autographa california nuclear polyhedrosis virus sensitivity, can observe typical cell pathology feature, and nucleus increases, and includes a large amount of polyhedron particles; And at least can 3 generations of continuous passage.
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 12nd generation cell growth curve and population doubling time.With the concentration of 1 × 105 cell/mL, be 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 12nd generation is 56.98 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 during N0=inoculation
The total cellular score that N=moment t measures
4. according to the method for Takahashi et al. (Takahashi, Mitsuhashi and Ohtaki (1980) Develop.Growth and Differ.22:11-19), measured the 22nd 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-SpexX is between 116-131, therefore, newly-established clone IOZCAS-Spex X is comprised of 4 times of somatocyte.
5. use the method (McIntosh of DAF-PCR, Grasela and Matteri (1996), Insect Mol.Biol.5:187～195) identified that clone IOZCAS-Spex X derives from beet armyworm really, but not the pollution of other clone.The DNA being extracted by IOZCAS-Spex X and beet armyworm pupa 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 IOZCAS-Ha-I, the Heliothis zea pupa ovary of Sf9, the bollworm fatty body 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.
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-Spex X of the present invention, and in figure, scale is depicted as 400 μ m;
Fig. 2 is transfection fluorescence (the green fluorescence GFP mark) figure of IOZCAS-Spex X of the present invention, and in figure, scale is depicted as 400 μ m;
Fig. 3 obtains a large amount of viral polyhedron test-results figure after IOZCAS-Spex X of the present invention infects beet armyworm, and in figure, scale is depicted as 200 μ m;
Fig. 4 obtains a large amount of viral polyhedron test-results figure after IOZCAS-SpexX of the present invention infects autographa california nuclear polyhedrosis virus, and in figure, scale is depicted as 200 μ m;
Fig. 5 is the growth curve of IOZCAS-SpexX of the present invention;
Fig. 6 is the caryogram of IOZCAS-SpexX of the present invention;
Fig. 7 is the DNA fingerprint amplification agarose electrophoresis figure that DAF-PCR identifies IOZCAS-SpexX, in figure, 1 is beet armyworm pupa (Spodoptera exigua pupa), 2 is the IOZCAS-Spex II-A of fatty body, 3 is the IOZCAS-Ha-I of bollworm fatty body, 4 is the BCIRL-Hz-AM1 of Heliothis zea pupa ovary, 5 Sf9 for the greedy noctuid in meadow, 6 is DNA marker (DNA Marker), 7 is IOZCAS-Spex X;
Fig. 8 is the expression vector plasmid pIZT-V5-His that contains hTERT gene, has multiple clone site, baculovirus getting up early promotor, V5 epi-position and anti-Zeocin gene;
Fig. 9 is the expression vector plasmid pIB-V5-His that contains hTERT gene, has multiple clone site, baculovirus getting up early promotor, V5 epi-position and anti-Blasticidin gene;
Figure 10 is the agarose electrophoresis result schematic diagram of transgenic insect clone pcr amplification, in figure, 1 is DNA marker (DNA Marker), 2 is the Sf9 of the fall army worm of transfection hTERT gene, 3 is the Sf9 of fall army worm, 4 is IOZCAS-Spex X cell system, the 5 IOZCAS-Spex II-A that are fatty body.
Embodiment
The technology of using in following examples, comprises gene amplification, gene clone, and cell transfecting, and cell cultures, detection technique, unless stated otherwise, be routine techniques known to those skilled in the art; The plant and instrument that uses, reagent, cell etc., only specify in specification sheets, is that those skilled in the art can obtain by public approach.
the foundation of embodiment 1. transgenic beet exigua larvae fatty body clones
The beet exigua larvae of getting end age is immersed in 3% hypochlorous acid solution 5 minutes, and 10-20 minute in 75% ethanolic soln, carries out surface sterilization.Dissect this insect and take out fatty body tissue, during 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 1-2 time, put into the 25cm that uses the rinse of 1mL nutrient solution
2tissue Culture Flask in, put into the cell culture incubator of 27 ℃ of unglazed photographs Celsius and cultivate 24 hours.Then add the above-mentioned cell culture fluid I of 3mL, put under similarity condition and cultivate.Note key that the method is successfully set up clone be to make tissue block be close to Tissue Culture Flask at the bottom of, do not make tissue block be suspended 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 cell culture fluid II of half amount simultaneously.After operating 7-10 days, this can observe dissociate around tissue block a large amount of single cells expansion to the periphery gradually.When primary cell is bred to 20 days, according to the Cellfectin II Reagent (Cat.no.10362-100 of invitrogen company, human telomerase reverse transcriptase's Transfected Recombinant Plasmid that 10362-125) method builds this laboratory, in primary cell, is cultivated in the cell culture incubator of 27 ℃ of unglazed photographs.Within after transfection the 3rd day, can observe transfection effect by fluorescence inverted microscope.The transfection initial stage, the floating death of part cell, within every 7 days, half amount is changed cell culture fluid II, observation of cell propagation situation.When cell has agglomerate propagation trend, add 400 μ g/mL bleomycin (zeocin) screenings, successful transfection the cell attachment growth of recombinant plasmid gene normal, the cell of untransfected is floating death.After 49 days, when 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 III that 2mL is new (containing 50 μ g/mL bleomycin (zeocin)).Establishment of Cell Line initial success.After start to go down to posterity at cell the 20th day, start sub-bottle for the second time and go down to posterity, the later generation time shortens gradually, and while passing to for the 5th generation, the generation time foreshortens to 12 days, and Growth of Cells starts to stablize, and while reaching for the 22nd generation, the generation time has foreshortened to 3-4 days.Final this clone is named as IOZCAS-Spex X.IOZCAS-Spex X is deposited in China Committee for Culture Collection of Microorganisms's common micro-organisms center on December 28th, 2010, and preserving number is CGMCC No.4505.
Utilize vector plasmid pIZT-V5-His construction recombination plasmid pIZT-hTERT.As shown in Figure 8, vector plasmid pIZT-V5-His has multiple clone site, baculovirus getting up early promotor, V5 epi-position and anti-Zeocin gene.Vector plasmid pIZT-V5-His is carried out to EcoR I and EcoR V restriction enzyme digestion, hTERT gene (its sequence is as shown in SEQ ID NO 1) (from plasmid pBABE-puro-hTERT) is carried out to SalI restriction enzyme digestion, after dNTP fills, EcoRI enzyme cuts, be cloned into and in vector plasmid, obtain hTERT recombinant plasmid pIZT-hTERT.
Utilize vector plasmid pIB-V5-His construction recombination plasmid pIB-hTERT.As shown in Figure 9, vector plasmid pIB-V5-His has multiple clone site, baculovirus getting up early promotor, V5 epi-position and anti-Blasticidin gene.Vector plasmid pIB-V5-His is carried out to EcoR I and EcoR V restriction enzyme digestion, hTERT gene (from plasmid pBABE-puro-hTERT) is carried out to SalI restriction enzyme digestion, after dNTP fills, EcoRI enzyme cuts, be cloned into and in vector plasmid, obtain hTERT recombinant plasmid pIB-hTERT.
the Observation of biological characteristics of embodiment 3.IOZCAS-SpexX and mensuration
(1) morphological specificity: 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.As shown in Figure 1-2, the shape of cell has 3 types: circle, fusiformis and ellipse.Most cells is adherent.
(2) growth of cell: at 27 ℃, the 12nd generation of clone, population doubling time was 56.98h in the TNM-FH nutrient solution of the Streptomycin sulphate of the penicillin containing 10% foetal calf serum, 1OOU/mL, 100U/mL, 50 μ g/mL bleomycin.As shown in Figure 5,1.3 × 106 cell/mL of high-density Yue Keda of cell.
(3) karyotyping: as shown in Figure 6, IOZCAS-Spex X the 22nd generation cell is 4 times of somatocyte, chromosome number scope 116-131 (2n=62).
(4) DAF-PCR identifies: as shown in Figure 7, clone IOZCAS-Spex X derives from beet armyworm really, but not the pollution of other clone.The DNA being extracted by IOZCAS-SpexX is same as the DNA fingerprint amplification collection of illustrative plates of beet armyworm pupa and fatty body, and is different from the collection of illustrative plates of the BCIRL-Hz-AM1 of IOZCAS-Ha-I, the Heliothis zea pupa ovary of Sf9, the bollworm fatty body of the greedy noctuid in meadow.
(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 4.
IOZCAS-Spex X is to laphygma exigua nuclear polyhedrosis virus sensitivity: the concentration inoculation IOZCAS-Spex X by SeNPV and AcMNPV budding pattern virus particle BV with 0.001 larva equivalent/milliliter, as shown in Figure 3-4, cultivate after 7 days, by inverted microscope, can observe typical cell pathology feature, 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 91.2% and 85.4%.
Utilize round pcr, measure the existence of hTERT gene in the clone IOZCAS-SpexX that turns hTERT gene.
Primer:
hTERT?up：AGC?TGC?GGC?CCT?CCT?TCC?TAC?TCA
hTERT?down：GAC?GCT?CGG?CCC?TCT?TTT?CTC?TGC
Reaction conditions:
95℃，2min；
95 ℃/30S, 57 ℃/30S, 72 ℃/60S; 30 circulations
72℃，5min。
As shown in figure 10, PCR detected result proves, has hTERT gene in genetically modified cell strain.
Claims (2)
1. a transgenosis fatty body clone for high yield baculovirus, the name of this clone is called IOZCAS-Spex X, and preserving number is CGMCC No.4505.
2. the application of transgenosis fatty body clone according to claim 1, the application of described transgenosis fatty body clone in baculovirus produces, described baculovirus is laphygma exigua nuclear polyhedrosis virus or autographa california nuclear polyhedrosis virus.
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