CN101070533B - American cotton bollworm yong-insect fatbody cell line of high yield stab-like virus, its constitution and use - Google Patents
American cotton bollworm yong-insect fatbody cell line of high yield stab-like virus, its constitution and use Download PDFInfo
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Abstract
This invention publicized one kind to originate from the insect fat body organization, and to the rod-shaped viral high sensitive cell line, this invention has also publicized this cell line establishment method, as well as this cell line the use which grew into the rod-shaped viral scale metaplasia. This cell line may use for to duplicate this kind of virus, uses in the rod-shaped viral pesticide the formalization production, as well as the construction rod-shaped virus the expression vector system, the expression has commercial or the scientific value protein. Therefore it has the extremely high trade and the scientific research value.
Description
Technical field
The present invention relates to a kind of insect cell line, especially a kind of insect fatty body tissue that derives from, and, the invention still further relates to the establishment method of this clone to the high responsive clone of baculovirus, and the purposes of this clone in baculovirus mass-producing growth.
Technical background
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.Insect cell line is as research material, it is the important tool of scientific researches such as physiology, developmental biology, cytobiology, molecular biology and biological chemistry always, and insect cell has been expressed a large amount of exogenous protein with great economy meaning or scientific meaning as the important component part of rhabdovirus expression vector system; As bio-reactor, the amplification insect baculovirus is as biotic pesticide simultaneously, and particularly amplification contains the recombinant baculovirus insecticides of foreign gene, and insect cell has also been brought into play important effect.
A large amount of commercial clones of lepidopterous insects that derive from have obtained people and have used widely, such as, derive from Sf-21 and its clone strain Sf-9 of the greedy noctuid (Spodoptera frugiperda) in meadow, derive from the Tn-368 of cabbage looper (Trichoplusia ni) and its clone strain Tn-5B1-4 (commodity be called High Five) etc., being applied to of success expressed and the production recombinant protein.
Lepidopterous clone derives from a lot of tissues, comprises ovary, embryo, hemocyte, fatty body etc.Yet, what deserves to be mentioned is that dedifferente because insect cell is incomplete, the insect cell line of having set up that has still keeps the feature of certain original structure and organ.Derive from the clone of different sorts insect or derive from and have nothing in common with each other with the clone amplicon virus or the Recombinant Protein Expression ability at a kind of insect different tissues position.Therefore setting up and screen new, more baculovirus sensitive cell line (strain) is a job that is necessary very much and has theoretical and practice significance.
Up to the present, from total about 10 strains of the clone of bollworm (Helicoverpa armigera (Hbn.)) (McIntosh, 1983; Yang and Xie, 1983; Su D.M., 1987; Kolokol`tsova, 1995; McIntosh, 1999; SudeepA.B., 2002A; Sudeep, 2002B) McIntosh, A.H. etc. (McIntosh, A.H., et al.In Vitro 1983,19:589～590) use the ovary of female pupa to be material, and at first having set up bolworm cell is BCIRL-HA-AM1.Find by inoculation test, this clone is reproducible Heliothis zea simple grain embedding nucleopolyhedrosis virus (HzSNPV), many embedding nucleopolyhedrosis viruss of bollworm (HaMNPV) and many embedding nucleopolyhedrosis viruss of autographa california (AcMNPV) not, both do not had polyhedron to occur, the concentration of virus does not increase yet.(L-7, Lenz, C.J., et al., J.Invertebr.Pathol.57:227～233 such as Lenz; 1991) cloned this clone in 1991, obtained cell strain CSIRO-BCIRL-HA1, this cell strain is very responsive to HaMNPV.(Yang Shuyan such as Yang Shuyan, thank to Rong Dong .In: Shanghai Inst of Insectology, Chinese Academy of Sciences, ed. entomology research collected papers (the 3rd collection). Shanghai: the .1983:129-136 of Shanghai science tech publishing house) also set up the clone (SIE-Ha-798 and SIE-Ha-806) of two strains from female pupa ovary.(Su D.M. such as Su D.M., et al.In " Biotechonology inInvertebrate Pathology and Cell Culture " (K.Maramorosh, ed.) New York:AcademicPress, 1987,375-383) delivered the clone (SFE-HA-831) of another strain from female pupa ovary.Nineteen ninety-five, (Kolokol`tsova T.D., et al., Vopr Virusol 1995,40:135～138) such as the scholar Kolokol`tsova T.D. of Russia also use the ovary of the female pupa of bollworm to set up a strain clone.McIntosh, (McIntosh, A.H. such as A.H., et al., In vitro Cell.Dev.Biol.1999,35 (2): 94～97) from the ovary tissue of bollworm female adult worm, set up three strain clone CSIRO-BCIRL-HA1 afterwards, CSIRO-BCIRL-HA2, CSIRO-BCIRL-HA3.Though it is many that three strain clones, are duplicated the amount that produces virus to the HzNPV sensitivity and are not so good as cell strain BCIRL-HA-AM1-A11.Clone NIV-HA-1195 (Sudeep A.B., et al., Indian J.Exptl.Biol.2002 40:69-73) sets up by the cotton bollworm larvae hemocyte, this clone is to AcNPV, Spodoptera litura nucleopolyhedrosis virus (SpltNPV) and HaNPV sensitivity.(Sudeep A.B.et al., In Vitro Cell Dev.Biol.-Animal 2002,38:262～264) such as Sudeep A.B. have set up clone NIV-HA-197, it is from the embryo of bollworm, to AcNPV, SpltNPV and HaSNPV sensitivity, 90% cell can infect HaSNPV.
Although the relevant report of the existing bolworm cell of aforementioned documents system, yet up to the present, also do not derive from the clone of bollworm fatty body.The fatty body of insect has similar function with mammiferous liver, is important physical metabolizing tissue, also be baculovirus main infect, duplicate one of position.Still there is very big demand in a greater variety of insect cell lines, set up the new clone that derives from the insect fatty body, and making up more efficiently, the rhabdovirus expression vector system is very significant.
Summary of the invention
The object of the present invention is to provide that a kind of new bollworm fatty body is tissue-derived and that have highly viral susceptibility, the cotton bollworm larvae fatty body clone of high yield baculovirus.
Another object of the present invention is to provide the construction process of the cotton bollworm larvae fatty body clone of a kind of described high yield baculovirus.
Another purpose of the present invention is to provide the purposes of the cotton bollworm larvae fatty body clone of described high yield baculovirus.
The name of the cotton bollworm larvae fatty body clone of high yield baculovirus provided by the invention is called IOZCAS-Ha-I, is deposited in China Committee for Culture Collection of Microorganisms common micro-organisms center on April 19th, 2006, and preserving number is CGMCC No.1690.
The invention provides the construction process of the cotton bollworm larvae fatty body clone of a kind of described high yield baculovirus, it is experiment material for utilizing cotton bollworm larvae fatty body cell, adopt a kind of method that can set up insect cell line fast, built up a strain to the high responsive bollworm fatty body clone of heliothis armigera nuclear polyhedrosis virus, called after IOZCAS-Ha-I.Preserving number is CGMCC No.1690.
It is as follows that the present invention sets up the concrete grammar of this clone:
(1) bollworm end instar larvae is immersed in the ethanolic soln 10～20 minutes, carries out surface sterilization, clean insect with sterile distilled water then, blot the polypide surface again;
(2) dissect insect, take out complete bollworm fatty body tissue;
(3) clean (2) middle tissue that obtains 2～3 times with physiological saline, clean with cell culture fluid again, clean the back this tissue is put into the Tissue Culture Flask that contains the rinse of 1mL cell culture fluid, cover tight bottle cap, put into the cell culture incubator of 27 ℃ of unglazed photographs and cultivated 24 hours;
(4) add an amount of cell culture fluid, make tissue block fully or major part be immersed in this nutrient solution, with the cultivation down of step (3) similarity condition;
(5) nutrient solution of sucking-off in per 7～10 days half amount, and change to the new cell culture fluid of half amount simultaneously, be full of whole culturing bottle until observing continuous expansion and beginning proliferating cells;
(6) containing the new individual cells that of breeding, put into new culturing bottle together with whole cell culture fluid sucking-offs, and add new cell culture fluid, and add again with the new cell culture fluid of sucking-off amount as much in the former culturing bottle that contains tissue block, two culturing bottles are put into incubator and are cultivated, cell begins to go down to posterity, and clone is set up successfully, obtains the cotton bollworm larvae fatty body clone of high yield baculovirus of the present invention;
Whole process is all carried out under aseptic condition.
The cell culture fluid that uses is the conventional insect cell nutrient solution that adopts and the mixture of penicillin, Streptomycin sulphate and foetal calf serum in the aforesaid method of the present invention, and the cell culture fluid pH that is made into is between 6.0-6.5.The insect cell nutrient solution can be the insect cell nutrient solution of extensive stockization, as TNM-FH, and Grace ' s, Sf900, TC-100, IPL-41, Ex-Cell 400 or the like.
Usually, penicillin content is 100U/mL in the cell culture fluid; Content of streptomycin is that preferred 100U/mL foetal calf serum content is 5～10% (volume ratios) of cell culture fluid.
The biological property of two strain bollworm fatty body clones is observed and is measured among the present invention:
1. through experimental observation, this clone major part is suspended in the nutrient solution, and the part attached cell is also arranged.The shape of cell has 3 types: most of cell circle, small part fusiformis, and less like scavenger cell shape (Fig. 1).
2. 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 10th generation cell growth curve and population doubling time.With 2 * 10
5The concentration of cell/mL is inoculated into T-12.5cm
2In the culturing bottle, 27 ℃ of cultivations, every 24h measures cell concn, draws growth curve, and T=tlg2/[lg (N/N by formula
0)] calculate, the cell colony doubling time in the 10th generation is 65.2 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
N
0Cell count during=inoculation
The N=total cellular score that t measured constantly
3. according to the method for Takahashi et al. (Takahashi, Mitsuhashi and Ohtaki (1980) Develop.Growth and Differ.22:11-19), measured the 7th generation cell caryogram.The chromosome number of IOZCAS-Ha-I between 58～239, nearly 52% cell chromosome number between 80～119,38% cell chromosome number between 160～199 (Fig. 2).
4. use the method (McIntosh, Grasela and Matteri (1996), Insect Mol.Biol.5:187～195) of DAF-PCR to identify that clone IOZCAS-Ha-I derives from bollworm really, but not the pollution of other clone (Fig. 3).Banding pattern main after the DNA that is extracted by IOZCAS-Ha-I and the cotton bollworm larvae DNA cloning is identical, and derives from the SL2 of fruit bat, the IOZCAS-Spex II of this self-built beet armyworm in laboratory with contrast, and IOZCAS-Spex III cell banding pattern is difference obviously.
5.IOZCAS-Ha-I to the heliothis armigera nuclear polyhedrosis virus sensitivity, can observe typical cell pathology feature, promptly nucleus increases, and includes a large amount of polyhedron particles (Fig. 4).And at least can continuous passage 3 generations.The viral polyhedron of using clone to produce is carried out biological assay, obtains LC50=5.88 * 10
5PIB/mL is similar to the result who measures with the viral polyhedron of polypide production.
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.
The cotton bollworm larvae fatty body clone of high yield baculovirus provided by the invention can be used for duplicating baculovirus, is used for the large-scale production of baculovirus or baculovirus insecticides; This clone also can be applicable to use the rhabdovirus expression vector express recombinant protein, and this recombinant protein has high commerce and scientific research is worth.
Description of drawings
Fig. 1 is the cultivation form of IOZCAS-Ha-I;
Fig. 2 is the caryogram of IOZCAS-Ha-I;
Fig. 3 is same as the dna fingerprint amplification collection of illustrative plates of bollworm H.armigera, and is different from clone SL2 of fruit bat and the collection of illustrative plates of IOZCAS-Spex II, IOZCAS-Spex III for the dna fingerprint amplification collection of illustrative plates that DAF-PCR identifies IOZCAS-Ha-I;
Fig. 4 obtains a large amount of viral polyhedroies for IOZCAS-Ha-I after infecting heliothis armigera nuclear polyhedrosis virus.
Embodiment
The foundation of embodiment 1. cotton bollworm larvae fatty body clones
The cotton bollworm larvae of getting end age is immersed in 70% the ethanolic soln 10 minutes, carries out surface sterilization.Dissect this insect and take out the fatty body tissue, keep it complete during operation as far as possible.Clean this tissue 2-3 time with physiological saline, (based on TNM-FH, contain the penicillin of 100U/mL, 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 again
2Tissue Culture Flask in, put into the cell culture incubator of 27 ℃ of unglazed photographs Celsius and cultivated 24 hours.Add the above-mentioned cell culture fluid of 3mL then, put under the similarity condition and cultivate.Notice that it is that tissue block 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 block be suspended in the cell culture fluid.The nutrient solution of left and right sides sucking-off in later every 7-10 days half amount, and change to the half new nutrient solution of measuring simultaneously.After this operates 7-10 days, can observe and dissociate a large amount of one cells around the tissue block, and expansion to the periphery gradually.See cell after the 28th day and constantly breed and be full of whole culturing bottle,, put into new culturing bottle together with whole cell culture fluid sucking-offs, and add the new cell culture fluid of 2mL containing the new cell that of breeding.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 7th day, the later generation time shortens gradually, and when passing to for the 8th generation, the cell growth begins to stablize, and finally this clone is named as IOZCAS-Ha-I.IOZCAS-Ha-I is deposited in China Committee for Culture Collection of Microorganisms common micro-organisms center on April 19th, 2006, and preserving number is CGMCC No.1690.
The biological characteristics of embodiment 2.IOZCAS-Ha-I is observed and is measured
(1) morphological specificity: through microscopic examination, this clone is suspended in the nutrient solution usually, and the shape of cell has 3 types: circular, fusiformis and like scavenger cell shape (Fig. 1).Round cell accounts for 85.6% among the IOZCAS-Ha-I, mean diameter 14.57 μ m; Spindle cell accounts for 9.5%, is about 16.6-42.0 μ m (average 23.95 μ m), wide about 8.0-15.2 μ m (average 11.53 μ m); About 4.9% cell is that diameter range is between 21.7-28.2 μ m like scavenger cell.
(2) growth of cell: under 27 ℃, in 2 the 10th generations of clone,, the 9th cell colony doubling time in generation was 65.2 hr in the TNM-FH of the Streptomycin sulphate of the penicillin that contains 10% foetal calf serum, 100U/mL, 100U/mL nutrient solution.The high-density Yue Keda 8 * 10 of cell
5Individual cell/mL.
(3) karyotyping: IOZCAS-Ha-I the 7th generation cell chromosome number is between 58-239, and nearly 52% cell chromosome number is between 80-119, and 38% cell chromosome number is seen Fig. 2 between 160-199.
(4) DAF-PCR identifies: clone IOZCAS-Ha-I derives from bollworm really, but not the pollution of other clone (Fig. 3).Banding pattern main after the DNA that is extracted by IOZCAS-Ha-I and the cotton bollworm larvae DNA cloning is identical, and derive from the SL2 (Schneider ' s Drosophila Line 2 of fruit bat with contrast, ATCC preserving number: CRL-1963), the IOZCAS-Spex II of this self-built beet armyworm in laboratory, the banding pattern of IOZCAS-Spex III (China Committee for Culture Collection of Microorganisms common micro-organisms center, preserving number CGMCC No.1690) cell is obviously different.
(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.
The mensuration of embodiment 3. viral susceptibility and output
IOZCAS-Ha-I is to the heliothis armigera nuclear polyhedrosis virus sensitivity: with the concentration inoculation IOZCAS-Ha-I of HaNPV budding pattern virus particle BV with 0.001 larva equivalent/milliliter, cultivate after 10 days, harvested cell and viral polyhedron, count viral polyhedron number with blood counting chamber at microscopically, the HaNPV of IOZCAS-Ha-I amplification as a result can reach every milliliter of nutrient solution and produce 7.3 * 10
7The output of PIB.Simultaneously can observe typical cell pathology feature, promptly nucleus increases, and includes a large amount of polyhedron particles (Fig. 4).And at least can continuous passage 3 generations.
The viral polyhedron of using clone to produce with aforesaid method is carried out biological assay to the cotton bollworm larvae in 2 latter stages in age.Its method is as follows: IOZCAS-Ha-I inoculation HaNPV virus is after 10 days, collecting cell and nutrient solution thereof, 10% sodium lauryl sulphate (SDS) that adds 1/10th volumes, concuss, centrifugal 5 minutes of 5000g, precipitation suspends with a small amount of 1%SDS, the polyhedrosis content of blood counting chamber counting HaNPV.With distilled water the HaNPV suspension that obtains is mixed with 1 * 10
7PIB/mL, 5 * 10
6PIB/mL, 1 * 10
6PIB/mL, 5 * 10
5PIB/mL, 1 * 10
5PIB/mL, 1 * 10
4The concentration gradient of six concentration of PIB/mL is made blank with distilled water.The feed of bollworm is immersed in the viral suspension of different concns 1 minute respectively, take out feed, blot the liquid on feed surface with thieving paper, put into 24 lattice insect box, every lattice insert the cotton bollworm larvae in 12 latter stage in age, put into illumination in 26 ℃, 14 hours: dark illumination box was cultivated in 10 hours.Change to the fresh feed that does not contain virus after 24 hours and continue to cultivate, 72 bollworms of each concentration experiment.The mortality ratio of five days " Invest, Then Investigate " bollworms of experiment is calculated LC
50The 5th day LC as a result
50Be 5.88 * 10
5PIB/mL carries out biological assay, its LC and use by the polyhedron of cotton bollworm larvae amplification
50Be 6.60 * 10
5PIB/mL.With the same HaNPV of the HaNPV of IOZCAS-Ha-I cell amplification, its LC with the polypide amplification
50On statistical significance, do not have significant difference, illustrate that the virulence of the heliothis armigera nuclear polyhedrosis virus of producing with IOZCAS-Ha-I (HaNPV) does not change, can be used for the production of HaNPV.
Claims (3)
1. the cotton bollworm larvae fatty body clone of a high yield baculovirus, its name is called IOZCAS-Ha-I, is deposited in China Committee for Culture Collection of Microorganisms common micro-organisms center on April 19th, 2006, and preserving number is CGMCC No.1690.
2. the purposes of cotton bollworm larvae fatty body clone in the scale operation of baculovirus of the described high yield baculovirus of claim 1.
3. purposes as claimed in claim 2, baculovirus wherein is a heliothis armigera nuclear polyhedrosis virus.
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| CN104735977B (en) * | 2012-06-12 | 2016-09-21 | 替代基因表达公司 | For expressing the baculovirus DNA element of recombiant protein in host cell |
| CN105861416B (en) * | 2016-06-06 | 2019-08-02 | 西北民族大学 | It is a kind of for the serum free medium and its preparation method and application for cultivating insect cell that suspends entirely |
| 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 |
| 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 |
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| CN1162545C (en) * | 2001-09-30 | 2004-08-18 | 山东大学 | Cathepsin B gene of bollworm and its clone, recombination and expression techniques |
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| 张晓飞.鳞翅目昆虫细胞株的建立与应用.植物保护27 4.2001,27(4),38-40. |
| 李艺丹 |
| 李艺丹;程罗根;张晓飞.鳞翅目昆虫细胞株的建立与应用.植物保护27 4.2001,27(4),38-40. * |
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Assignee: Beijing TransGen Biotech Co., Ltd. Assignor: Institute of Animals, Chinese Academy of Sciences Contract record no.: 2012990000427 Denomination of invention: American cotton bollworm yong-insect fatbody cell line of high yield stab-like virus, its constitution and use Granted publication date: 20110928 License type: Exclusive License Open date: 20071114 Record date: 20120618 |
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