CN102807969A - Transgenic insect cell line for high-yield baculovirus, and preparation method and application thereof - Google Patents
Transgenic insect cell line for high-yield baculovirus, and preparation method and application thereof Download PDFInfo
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Abstract
The invention provides a transgenic insect cell line for high-yield baculovirus, and a preparation method and the application thereof. The name of the transgenic insect cell line is IOZCAS-Spex IX and the preservation number of the cell line is CGMCC No.4506. The preparation method of the transgenic insect cell line comprises the steps as follows: spodoptera exigua ovary cells are cultured; human telomerase reverse transcriptase genes are transformed into known expression vectors to obtain recombinant vectors; the recombinant vectors are introduced into the spodoptera exigua ovary cells; and the spodoptera exigua ovary cells are enabled to express human telomerase reverse transcriptase, so as to obtain the transgenic insect cell line. The transgenic insect cell line is applied to the production of the baculovirus.
Description
Technical field
The present invention relates to a kind of transgenic insect clone, relate in particular to the transgenic ovary cell line of a kind of high yield baculovirus, belong to the transgenic animal technical field.
Background technology
The main application of insect cell line shows: as research material, be the important tool of 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 exogenous protein with great economy value and scientific meaning; As bio-reactor, the amplification insect baculovirus particularly contains the recombinant baculovirus of foreign gene, produces biotic pesticide.Insect cell line has important economy and using value, but its foundation often need consume great amount of time and energy.It is reported that so far, in nearly 40 years time, the insect cell line of having set up is above 500 kinds after nineteen sixty-five first, the strain insect cell line was successfully set up.They derive from 170 various insects such as lepidopteran, Diptera, Homoptera, Hymenoptera, Orthoptera and Coleoptera respectively, and wherein major part derives from lepidopteran and dipteral insect.Lepidopteran (Lepidoptera) is second largest order of Insecta, and the known kind in the whole world reaches more than 100,000 kinds, and relevant with agricultural have two suborders.Lepidopterous insects has the great economy meaning, and except that only a few was got the food polycarpeae, many was agricultural important pests.Set up lepidopteran insect cell system and valuable biologic material is provided for scientific research and biological control.Lepidopterous clone derives from multiple tissue, comprises ovary, embryo, hemocyte, fatty body etc.
Up to the present, the clone from beet armyworm (Spodoptera exigua) has 6 strains.Gelernter and Federici (Gelernter, W.D.and B.A.Federici J.Invertebr.Pathol.1986,48:199～207) have set up first clone of beet armyworm UCR-SE-1 from the beet armyworm newly hatched larvae tissue that shreds.Through clone forming method, they have separated SE-UCR-1A clone again from UCR-SE-1 clone.On the basis of SE-UCR-1A clone, (1990,26 (8): 824～828) screening obtains a clone that lacks nucleoside kinase for Mccarthy W J, Mckedy D.Dev.Biol. for Mccarthy and Mckedy.(Hara, K., K.Tsuda, M. Funakoshi and T.Kawarabata In Vitro Cell.Dev.Biol.1993,29A (12): 904～907) also from beet armyworm newly hatched larvae tissue, set up the second strain clone Se3FH such as Hara.Se3FH is responsive to SeNPV, and the speed of growth is faster than UCR-SE-1.Can be infected by SeNPV through the Se301 of clone and separate clone 100%, the speed of growth is faster than Se3FH, and the plaque of generation is bigger than Se3FH.The 3rd strain Le-H-HNU7 clone is set up through the beet armyworm hemocyte, and this clone is responsive to Spodoptera litura nucleopolyhedrosis virus.The 4th strain SeHe920-1a clone also is to derive from the beet armyworm hemocyte, behind the spore of access microsporidium (Vairimorpha sp.), has higher cells infected ability than other clone that derives from the non-hemocyte of lepidopterous insects.The ability of SeHe920Y7 infection microsporidium is the strongest among its 12 clone strain SeHe920Y1 to SeHe920Y12.Other two strains are the clone of being set up by this laboratory that derives from the beet exigua larvae fatty body, 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, yet 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 that ovum takes place and grows.Ovary is made up of ovariole (ovariole), and the number of ovariole is widely different in all kinds of insects, and lepidopterous insects is generally 4,6 or 8.Since Grace utilized ovary tissue to set up first insect cell line, ovary was the vital tissue source that clone is set up 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 big demand in a greater variety of insect cell lines, set up the new clone that derives from the insect ovary, and making up more efficiently, the rhabdovirus expression vector system is very significant.
Compare with the sophisticated culture technique of mammalian cell, the insect cell culture technique is demanded urgently setting up with perfect.Insect is the biotic population of most species in the world, and abundant cytogenetics system is arranged.Spontaneous transformation owing to receive the influence of various environmental factors, takes place, i.e. immortalization in cell sometimes in the vitro culture process.The cell of immortalization loses contact inhibition in process of growth, can infinitely breed to go down to posterity, and such conversion not only required time is long, and transformation efficiency is extremely low.Set up a strain clone and often need cultivate a large amount of primary cells, waste time and energy, wait for the cell spontaneous transformation with often trusting to chance and strokes of luck formula, condition is difficult to control, and usually all that has been achieved is spoiled because of pollution.After cultured cell in vitro successfully goes down to posterity, when cultivating certain algebraically, can get into the growth-inhibiting state usually, vitality obviously weakens, and multiplication capacity descends, and cessation of growth cessation is also final dead.It is the important means that obtains cell immortalityization that artificial induction's cultured cell in vitro transforms.Under the mutagen of manual work design, cell is transformed, only needed just can realize usually in 1-3 month, and transformation efficiency is higher, the passage cell growth cycle is long, proterties is stable.
Find that at present cell immortalityization is in close relations with telomere and Telomerase.Telomere and Telomerase are a kind of special constructions of eukaryotic cell end of chromosome, are made up of telomeric dna and telomere protein.Telomeric dna is the repeated nucleotide sequences that is rich in the high conservative of G, participates in dna replication dna, and plays an important role to keeping chromosomal stability and duplicating fully.The cell per minute of vitro culture splits once, and telomere shortens 50-200bp, and when foreshortening to a threshold value, cell will stop division, moves towards old and feeble and dead.Human cell's telomere is made up of Tumor-necrosis factor glycoproteins (TTAGGG) n of 10-15kb; Telomere occurs with (TTTAGGG) n Tumor-necrosis factor glycoproteins in plant; The insect cell telomere generally is made up of Tumor-necrosis factor glycoproteins (TTAGG) n of 6-8kb.Known at present, in insect, have three kinds of telomeric dna types: the telomeric dna of silkworm (Bombyx mori) constitutes (Okazaki et al.1993) by Tumor-necrosis factor glycoproteins (TTAGG) n; Fruit bat (Drosophila melanogaster) telomeric dna is formed (Biessmann et al.1990, Levis et al.1993) by transposable element HeT-A and TART; Telomeric dna in the midge is formed (Nielsen &
1993, Zhang et al.1994) by the gene tandem repetitive sequence of complicacy.Telomerase is a kind of ribonucleoprotein enzyme, is made up of RNA and protein, has the function of reversed transcriptive enzyme, is the synthetic telomeric dna of template with self RNA, to keep the length of telomere.Stably express hTRT gene (hTERT) can be stablized the length of telomere in former generation cultured cells; Make cell be easy to immortalization (Cemi C.Telomeres; Telomerase, and myc.An update [J] .Mutat Res.2000,462 (1): 31-47.).
Though relevant that telomerase gene is applied to the report of mammalian cell immortalization is a lot, does not see to relate to the report that utilizes transfection hTRT gene (hTERT) to obtain the insect immortalized cell line.Set up the clone that derives from insect pupa ovary and expressing human telomerase reverse transcriptase gene through artificial induction's vitro conversion method, for being immortalized insect cell line provides the important channel.
Summary of the invention
Therefore; The objective of the invention is to still there not being at present the deficiency that contains the hTERT insect cell line; The transgenic ovary cell line of a kind of high yield baculovirus is provided, and it contains hTRT's gene, for being immortalized insect cell line provides the important channel.
Another object of the present invention provides the preparation method of the transgenic ovary cell line of a kind of high yield baculovirus.
A purpose more of the present invention provides the application of the transgenic ovary cell line of a kind of high yield baculovirus.
To above-mentioned purpose, technical scheme of the present invention is following:
One aspect of the present invention provides the transgenic ovary cell line of a kind of high yield baculovirus, and the name of this clone is called the transgenic insect clone of IOZCAS-Spex IX, and its preserving number is CGMCC No.4506.
Preferably, said transgenic ovary cell line contains hTRT's gene (hTERT).
Preferably, the nucleotide sequence of said hTRT's gene is shown in SEQ ID NO 1.
Another aspect of the present invention provides the preparation method of the transgenic ovary cell line of a kind of high yield baculovirus, may further comprise the steps:
Step 1: cultivate the beet armyworm gonad cell;
Step 2: will contain hTRT's gene transformation and go in the known expression vector, and obtain recombinant expression vector;
Step 3: said recombinant expression vector is imported in the described beet armyworm gonad cell; With
Step 4: make the said beet armyworm gonad cell can the expressing human reverse transcriptase of telomere, obtain transgenic insect clone.
Preferably, in step 1), said cultivation beet armyworm gonad cell may further comprise the steps:
Step 1.1: the female pupa of beet armyworm is immersed in the 3% hypochlorous acid solution 5 minutes, in 75% ethanolic soln 10-20 minute, carry out surface sterilization, clean insect with sterile distilled water then, blot the pupa surface-moisture again;
Step 1.2: dissect insect, take out the beet armyworm ovary tissue;
Step 1.3: organize 2-3 time with what obtain in the saline water cleaning step 2; Clean with cell culture fluid I again; Clean the back and put into the Tissue Culture Flask that contains 1mL cell culture fluid I rinse to this tissue, cover tight bottle cap, put into the cell culture incubator of 27 ℃ of unglazed photographs and cultivated 24 hours;
Step 1.4: add an amount of cell culture fluid I, make tissue fully or major part be immersed in this nutrient solution, with the cultivation down of step 1.3 similarity condition;
Step 1.5: the nutrient solution of every 7-10 days sucking-off half amount, and change to the new cell culture fluid II of half amount simultaneously, be full of whole culturing bottle until observing continuous expansion and beginning proliferating cells.
Preferably, in step 2, said known expression vector is pIZT-V5-His or pIB-V5-His.
Preferably, in step 3), said recombinant expression vector imports in the described beet armyworm gonad cell through liposome.
Preferably, in step 4), specifically obtain the transgenic ovary cell line through following steps:
Step 4.1: put into step 1.3 similarity condition and cultivated at least 4 hours down, and rock frequently.Change after the transfection an amount of cell culture fluid II continue with the cultivation down of step 1.3 similarity condition;
The fluorescence inverted microscope is observed the transfection effect after step 4.2:72 hour, changes to contain eukaryotic cell and screen antibiotic cell culture fluid III or cell culture fluid IV screening, and the cell of successful expression hTERT continues to cultivate down with step 1.3 similarity condition;
Step 4.3: with the identical culturing cell of this cell, until obtaining transgenic insect clone with step 1.5.
Above-mentioned whole process is all carried out under aseptic condition; Wherein said four kinds of cell culture fluids are respectively that cell culture fluid I is the mixture of insect cell nutrient solution and penicillium mould, Streptomycin sulphate, phenylthiourea and foetal calf serum; Cell culture fluid II is the mixture of insect cell nutrient solution and penicillium mould, Streptomycin sulphate and foetal calf serum; Cell culture fluid III is the mixture of insect cell nutrient solution and penicillium mould, Streptomycin sulphate, bleomycin (zeocin) and foetal calf serum; Cell culture fluid IV is the mixture of insect cell nutrient solution and penicillium mould, Streptomycin sulphate, blasticidin (blasticidin) and foetal calf serum, and the cell culture fluid pH that is made into is 6.0-6.8.
The insect cell nutrient solution can be the insect cell nutrient solution of extensive stockization, like TNM-FH, and Grace ' s, Sf 900, TC-100, IPL-41, Ex-Cell 400 or the like.
Usually, penicillium mould content is that 100U/mL, content of streptomycin are 100U/mL in the cell culture fluid; The nutrient solution of screening transgenic cell is that above-mentioned nutrient solution contains bleomycin (zeocin) 400 μ g/mL; The transgenic cell line nutrient solution is that above-mentioned nutrient solution contains bleomycin (zeocin) 50 μ g/mL; Animal serum content is 10% (volume ratio) of cell culture fluid.
The application that the transgenic ovary cell line of a kind of high yield baculovirus is provided on the one hand again of the present invention, said transgenic ovary born of the same parents tie up to the application in the baculovirus production.
Preferably, said baculovirus is laphygma exigua nuclear polyhedrosis virus (SeNPV) or autographa california nuclear polyhedrosis virus (AcMNPV).
Having expressed the biological property of the beet armyworm pupa ovary cell line of hTRT (hTERT) gene among the present invention observes and measures:
1. through experimental observation, this clone major part is an attached cell, also has part to be suspended in the nutrient solution.The shape of cell has 3 types: circular, the few part fusiformis of most of cell, and less like scavenger cell shape, be prone to gathering and form cell mass; The fluorescent signal transfection initial stage is stronger, and transfection efficiency is high.
2.IOZCAS-Spex IX is responsive to laphygma exigua nuclear polyhedrosis virus and autographa california nuclear polyhedrosis virus; Can observe typical cell pathology characteristic; Be that nucleus increases, include a large amount of polyhedron particles, infection rate is respectively 92.41% and 83.15%; And at least can continuous passage 3 generations.
3. according to McIntosh and Ignoffo, the method for 1989 (McIntosh, A.H.and C.M.IgnoffoJ.Invertebr.Pathol.1989,54:97～102), measure the 10th generation cell growth curve and population doubling time.Concentration with 2.5 * 105 cells/mL is inoculated in 24 well culture plates, 27 ℃ of cultivations, and every 48h measures cell concn, draws growth curve, and by formula T=tlg2/ [lg (N/N0)] calculates, and the cell colony doubling time in the 10th generation is 70.09 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 the N0=inoculation
The N=TCS that t measured constantly
4. according to the method for Takahashi et al. (Takahashi, Mitsuhashi and Ohtaki (1980) Develop.Growth and Differ.22:11-19), measured the 12nd generation cell caryogram.Because 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-Spex IX is between 116-131; Therefore, newly-established clone IOZCAS-Spex IX is made up of 4 times of somatocyte.
5. use the method (McIntosh, Grasela and Matteri (1996), InsectMol.Biol.5:187～195) of DAF-PCR to identify that clone IOZCAS-SpexIX derives from beet armyworm really, but not the pollution of other clone.The DNA that is extracted by IOZCAS-SpexIX 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 of IOZCAS-Ha-I, Heliothis zea pupa ovary that derives from Sf9, the bollworm fatty body of the greedy noctuid in meadow is obviously different.
6. use the frozen method of conventional cell that the part cell of certain generation is carried out frozen processing, preserve the kind money of cell, and successfully recovery.
Description of drawings
Below, specify embodiment of the present invention in conjunction with accompanying drawing, wherein:
Fig. 1 is the cultivation aspect graph of IOZCAS-Spex IX of the present invention, and scale is depicted as 400 μ m among the figure;
Fig. 2 is transfection fluorescence (the green fluorescence GFP mark) figure of IOZCAS-Spex IX of the present invention, and scale is depicted as 400 μ m among the figure;
Fig. 3 obtains a large amount of viral polyhedron test-results figure after IOZCAS-Spex IX of the present invention infects beet armyworm, and scale is depicted as 200 μ m among the figure;
Fig. 4 obtains a large amount of viral polyhedron test-results figure after IOZCAS-Spex IX of the present invention infects autographa california nuclear polyhedrosis virus, and scale is depicted as 200 μ m among the figure;
Fig. 5 is the growth curve of IOZCAS-Spex IX of the present invention;
Fig. 6 is the caryogram of IOZCAS-Spex IX of the present invention;
Fig. 7 identifies the dna fingerprint amplification agarose electrophoresis figure of IOZCAS-Spex IX for DAF-PCR; 1 is IOZCAS-Spex IX among the figure, and 2 is beet armyworm pupa (Spodoptera exigua pupa), and 3 is the IOZCAS-Spex II-A of fatty body; 4 is the IOZCAS-Ha-I of bollworm fatty body; 5 is the BCIRL-Hz-AM1 of Heliothis zea pupa ovary, 6 Sf9 for the greedy noctuid in meadow, and 7 is dna marker (DNAMarker);
Fig. 8 is expression vector plasmid pIZT-V5-His, has MCS, baculovirus getting up early promotor, V5 epi-position and anti-Zeocin gene;
Fig. 9 is expression vector plasmid pIB-V5-His, has MCS, baculovirus getting up early promotor, V5 epi-position and anti-Blasticidin gene;
Figure 10 is the agarose electrophoresis synoptic diagram as a result of transgenic insect clone pcr amplification, and 1 is IOZCAS-Spex IX clone among the figure, and 2 is beet armyworm ovary (spodoptera exigua ovary), and 3 is dna marker (DNA Marker).
Embodiment
Employed technology comprises gene amplification in following examples, gene clone, and cell transfecting, and cell cultures, detection technique unless stated otherwise, are routine techniques known to those skilled in the art; Employed plant and instrument, reagent, cell etc. only specify in the specification sheets, are that those skilled in the art can obtain through public approach.
The foundation of embodiment 1. transgenic beet noctuid pupa ovary cell lines
5 days female pupas of beet armyworm of getting end age are immersed in the 3% hypochlorous acid solution 5 minutes, in 75% ethanolic soln 10-20 minute, carry out surface sterilization.Dissect this pupa and take out ovary tissue, keep it complete during operation as far as possible.Clean this tissue 2-3 time with saline water, (with TNM-FH is main, contains the penicillium mould of 100U/mL, and the foetal calf serum of the Streptomycin sulphate of 100U/mL and 10% (v/v) pH=6.2) cleans 1-2 time, puts into the 25cm that uses the rinse of 1mL nutrient solution to use cell culture fluid I again
2Tissue Culture Flask in, put into the cell culture incubator of 27 ℃ of unglazed photographs Celsius and cultivated 24 hours.Add 3mL cell culture fluid I then, put under the similarity condition and cultivate.Notice that it is that tissue is close at the bottom of the Tissue Culture Flask that this method is successfully set up the key of clone, does not make tissue suspension in cell culture fluid.Left and right sides 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 this operates the 3rd day, can observe and dissociate a large amount of one cells around the tissue, and expansion to the periphery gradually.When the primary cell of beet armyworm ovary is bred to 10 days; According to the Cellfectin II Reagent (Cat.no.10362-100 of invitrogen company; 10362-125) method in primary cell, is cultivated hTRT's recombinant plasmid transfection of this laboratory structure in the cell culture incubator of 27 ℃ of unglazed photographs.Can observe the transfection effect through the fluorescence inverted microscope on the 3rd day after the transfection.The transfection initial stage, the floating death of part cell, amount was changed cell culture fluid II in per 7 days half, observation of cell propagation situation.When cell has when gathering propagation trend, add the screening of 400 μ g/mL bleomycin (zeocin), successful transfection the cell attachment growth of recombinant plasmid gene normal, the then floating death of the cell of untransfected.When cell proliferation extremely will be paved with whole culturing bottle, cell was put into new culturing bottle together with whole nutrient solution sucking-offs after 11 days, and added new cell culture fluid III or the IV (containing 50 μ g/mL bleomycin (zeocin)) of 2mL.Clone is set up initial success.Begin to divide bottle to go down to posterity for the second time after begin to go down to posterity at cell the 17th day, the later generation time shortens gradually, and when passing to for the 5th generation, the generation time foreshortens to 10 days, and the cell growth begins to stablize, and when reaching for the 12nd generation, the generation time has foreshortened to 4-5 days.Final this clone is named as IOZCAS-Spex IX.IOZCAS-Spex IX has been deposited in China Committee for Culture Collection of Microorganisms common micro-organisms center, deposit number on December 28th, 2010: CGMCC No.4506, classification name: beet armyworm pupa ovary transgenosis cell strain.
The structure of embodiment 2. hTRTs (hTERT) gene recombination plasmid pIZT-hTERT and pIB-hTERT
Utilize vector plasmid pIZT-V5-His construction recombination plasmid pIZT-hTERT.As shown in Figure 8, vector plasmid pIZT-V5-His has MCS, baculovirus getting up early promotor, V5 epi-position and anti-Zeocin gene.Vector plasmid pIZT-V5-His is carried out EcoR I and EcoR V restriction enzyme digestion; (its sequence is shown in SEQ ID NO 1) (from plasmid pBABE-puro-hTERT) carries out Sal I restriction enzyme digestion with the hTERT gene; After that dNTP mends is flat, EcoR I enzyme cuts, is cloned into and obtains hTERT recombinant plasmid pIZT-hTERT in the vector plasmid.
Utilize vector plasmid pIB-V5-His construction recombination plasmid pIB-hTERT.As shown in Figure 9, vector plasmid pIB-V5-His has MCS, baculovirus getting up early promotor, V5 epi-position and anti-Blasticidin gene.Vector plasmid pIB-V5-His is carried out EcoR I and EcoR V restriction enzyme digestion; (from plasmid pBABE-puro-hTERT) carries out Sal I restriction enzyme digestion with the hTERT gene; After that dNTP mends is flat, EcoR I enzyme cuts, is cloned into and obtains hTERT recombinant plasmid pIB-hTERT in the vector plasmid.
The biological characteristics of embodiment 3.IOZCAS-Spex IX is observed and is measured
(1) morphological specificity: through microscopic examination, this cell line cell is easy to form cell mass and can stands the high-density growth environment, has broken through contact inhibition.Shown in Fig. 1-2, the shape of cell has 3 types: circle, fusiformis and ellipse.Most of cell attachment.
(2) growth of cell: under 27 ℃, in the 10th generation of clone,, population doubling time was 70.09h in the TNM-FH nutrient solution of the Streptomycin sulphate of the penicillium mould that contains 10% foetal calf serum, 100U/mL, 100U/mL, 50 μ g/mL bleomycin.As shown in Figure 5, the high-density Yue Keda 2.7 * 10 of cell
6Individual cell/mL.
(3) karyotyping: as shown in Figure 6, IOZCAS-Spex IX the 12nd 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 IX derives from beet armyworm really, but not the pollution of other clone.The DNA that is extracted by IOZCAS-Spex IX 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 that the BCIRL-Hz-AM1 of the IOZCAS-Ha-I of the Sf9 of noctuid, bollworm fatty body, Heliothis zea pupa ovary is coveted on the meadow.
(5) frozen and recovery: use the frozen method of conventional cell that the part cell of certain generation is carried out frozen processing, preserve the kind money of cell, and can successfully recover.
IOZCAS-Spex IX is responsive to laphygma exigua nuclear polyhedrosis virus: SeNPV and AcMNPV budding pattern virus particle BV are inoculated IOZCAS-SpexIX with the concentration of 0.001 larva equivalent/milliliter; Cultivate after 7 days; Shown in Fig. 3-4; Can observe typical cell pathology characteristic through inverted microscope, promptly nucleus increases, and includes a large amount of polyhedron particles.And at least can continuous passage 3 generations.Viral infection rate is respectively 92.41% and 83.15%.
The mensuration that embodiment 5. changes hTERT gene in the hTERT gene cell
Utilize round pcr, measure the existence of hTERT gene among the clone IOZCAS-Spex IX that changes the hTERT gene.
Primer:
HTERT upstream primer: AGC TGC GGC CCT CCT TCC TAC TCA
HTERT downstream primer: GAC GCT CGG CCC TCT TTT CTC TGC
Reaction conditions:
95℃，2min；
95 ℃/30S, 57 ℃/30S, 72 ℃/60S; 30 circulations
72℃，5min。
Shown in figure 10, the PCR detected result proves, has the hTERT gene in the genetically modified cell strain.
Claims (7)
1. the transgenic ovary cell line of a high yield baculovirus, the name of this clone is called the transgenic insect clone of IOZCAS-Spex IX, and its preserving number is CGMCC No.4506.
2. transgenic ovary cell line according to claim 1 is characterized in that, said transgenic ovary cell line contains hTRT's gene.
3. transgenic ovary cell line according to claim 2 is characterized in that, the nucleotide sequence of said hTRT's gene is shown in SEQ ID NO 1.
4. the preparation method who according to each described transgenic ovary born of the same parents of claim 1 to 3 is may further comprise the steps:
Step 1: cultivate the beet armyworm gonad cell;
Step 2: will contain hTRT's gene transformation and go in the known expression vector, and obtain recombinant expression vector;
Step 3: said recombinant expression vector is introduced in the described beet armyworm gonad cell; With
Step 4: make the said beet armyworm gonad cell can the expressing human reverse transcriptase of telomere, obtain transgenic insect clone.
5. the method for transgenic ovary cell line according to claim 4 is characterized in that, in step 2, said known expression vector is pIZT-V5-His or pIB-V5-His.
6. according to the application of each described transgenic ovary cell line of claim 1 to 3, said transgenic gonad cell ties up to the application in the baculovirus production.
7. application according to claim 6 is characterized in that, said baculovirus is laphygma exigua nuclear polyhedrosis virus or autographa california nuclear polyhedrosis virus.
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| CN112695010A (en) * | 2019-10-23 | 2021-04-23 | 中国科学院动物研究所 | Cotton bollworm pupa ovarian cell line for high yield of baculovirus and preparation method and application thereof |
| CN112760277A (en) * | 2019-10-21 | 2021-05-07 | 中国科学院动物研究所 | Oriental myxozoon pupa ovarian cell line for high yield of baculovirus and preparation method and application thereof |
| CN115851574A (en) * | 2022-12-27 | 2023-03-28 | 青岛农业大学 | Beet armyworm cell line |
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| CN112760277A (en) * | 2019-10-21 | 2021-05-07 | 中国科学院动物研究所 | Oriental myxozoon pupa ovarian cell line for high yield of baculovirus and preparation method and application thereof |
| CN112760277B (en) * | 2019-10-21 | 2022-05-17 | 中国科学院动物研究所 | Oriental myxozoon pupa ovarian cell line for high yield of baculovirus and preparation method and application thereof |
| CN112695010A (en) * | 2019-10-23 | 2021-04-23 | 中国科学院动物研究所 | Cotton bollworm pupa ovarian cell line for high yield of baculovirus and preparation method and application thereof |
| CN112695010B (en) * | 2019-10-23 | 2023-01-10 | 中国科学院动物研究所 | Cotton bollworm pupa ovarian cell line for high yield of baculovirus and preparation method and application thereof |
| CN115851574A (en) * | 2022-12-27 | 2023-03-28 | 青岛农业大学 | Beet armyworm cell line |
| CN115851574B (en) * | 2022-12-27 | 2024-04-19 | 青岛农业大学 | Beet armyworm cell line |
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