CN100404667C - Young beet armyworms fat body cell system for producing baculiform virus with high yields - Google Patents
Young beet armyworms fat body cell system for producing baculiform virus with high yields Download PDFInfo
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Abstract
A high-yield rod-shaped virus beet noctuids larval fatty somatic system, its construction and use are disclosed. It can be used to copy this kind of virus, construct rod-shaped virus expression carrier system and for scaled production.
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, the clone from beet armyworm (Spodoptera exigua) has 4 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, (26 (8): 824～828) screening obtains a clone that lacks nucleoside kinase for Mccarthy W J, Mckedy D.Dev.Biol.1990 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 to the SeNPV sensitivity, 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-HNU
7Clone is set up by the beet armyworm hemocyte, and this clone is to the Spodoptera litura nucleopolyhedrosis virus sensitivity.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.
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 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 purpose of this invention is to provide a kind of new beet armyworm fatty body clone tissue-derived and that have highly viral susceptibility, this clone name is called Spex II, be deposited in China Committee for Culture Collection of Microorganisms common micro-organisms center on July 11st, 2005, preserving number is CGMCC No.1411.
Another object of the present invention is to provide the construction process of this clone.
Another purpose of the present invention be to provide this clone baculovirus scale operation and use purposes on the rhabdovirus expression vector express recombinant protein.
The present invention utilizes beet exigua larvae fatty body cell to be experiment material, adopts a kind of method that can set up insect cell line fast, has built up a strain to the high responsive beet armyworm fatty body clone of laphygma exigua nuclear polyhedrosis virus, called after Spex II.Preserving number is CGMCC No.1411.
It is as follows that the present invention sets up the concrete grammar of this clone:
(1) beet armyworm end instar larvae is immersed in the ethanolic soln 10-20 minute, carries out surface sterilization, clean insect with sterile distilled water then, blot the polypide surface again;
(2) dissect insect, take out complete beet armyworm fatty body tissue;
(3) clean organizing 2-3 time of obtaining in (2) 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 every 7-10 days sucking-off 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;
Whole process is all carried out under aseptic condition.
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 that adopts and the mixture of penicillin, Streptomycin sulphate and foetal calf serum, and the cell culture fluid pH that is made into is between 6.0-6.8.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 preferred 100U/mL; Animal serum content is 10% (volume ratio) of cell culture fluid.
The biological property of two strain beet armyworm 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.IgnoffoJ.Invertebr.Pathol.1989,54:97～102), measure the 10th generation cell growth curve and population doubling time.With 3 * 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 108.8 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.Because the chromosome number n=31 (Hara of beet armyworm, Tsuda, Funakoshi andKawarabata In Vitro Cell.Dev.Biol.1993,29A (12): 904～907), and the chromosome number of Spex II is between 116-131, therefore, newly-established clone Spex II forms (Fig. 2) by 4 times of somatocyte.
4. use the method (McIntosh, Grasela and Matteri (1996), InsectMol.Biol.5:187～195) of DAF-PCR to identify that clone Spex II derives from beet armyworm really, but not the pollution of other clone (Fig. 3).Banding pattern main after the DNA that is extracted by Spex II and the beet exigua larvae DNA cloning is identical, and to derive from the SL2 cell banding pattern of fruit bat obviously different with contrast.
5.Spex II, can observe typical cell pathology feature to the laphygma exigua nuclear polyhedrosis virus sensitivity, 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=6.01 * 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.
Description of drawings
Fig. 1 is the cultivation form of Spex II;
Fig. 2 is the caryogram of Spex II;
Fig. 3 is same as the dna fingerprint amplification collection of illustrative plates of beet armyworm S.exigua, and is different from the collection of illustrative plates of the clone SL2 of fruit bat for the dna fingerprint amplification collection of illustrative plates that DAF-PCR identifies Spex II;
Fig. 4 obtains a large amount of viral polyhedroies for Spex II after infecting laphygma exigua nuclear polyhedrosis virus.
Embodiment
The foundation of embodiment 1. beet exigua larvae fatty body clones
The beet exigua 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 Spex II.Spex II is deposited in China Committee for Culture Collection of Microorganisms common micro-organisms center on July 11st, 2005, and preserving number is CGMCC No.1411.
The biological characteristics of embodiment 2.Spex II 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 85.7% among the Spex II, mean diameter 14.7 μ m; Spindle cell accounts for 12.7%, is about 16.0-41.9 μ m (average 25.3 μ m), wide about 8.4-16.0 μ m (average 11.2 μ m); About 1.6% cell is that diameter range is between 18.2-31.4 μ m like scavenger cell.
(2) growth of cell: under 27 ℃, in 2 the 10th generations of clone,, population doubling time was respectively 108.8hr and 156.8hr 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 1.3 * 10 of cell
6Individual cell/mL.
(3) karyotyping: Spex II the 10th generation cell is 4 times of somatocyte, and chromosome number scope 116-131 (2n=62) sees Fig. 2.
(4) DAF-PCR identifies: clone Spex II derives from beet armyworm really, but not the pollution of other clone (Fig. 3).Banding pattern main after the DNA that is extracted by Spex II and the beet exigua larvae DNA cloning is identical, and derives from SL2 (Schneider ' s Drosophila Line 2, the ATCC preserving number: CRL-1963) the obvious difference of the banding pattern of cell of fruit bat with contrast.
(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
Spex II is to the laphygma exigua nuclear polyhedrosis virus sensitivity: with the concentration inoculation Spex II of SeNPV 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 SeNPV of Spex II amplification as a result can reach every milliliter of nutrient solution and produce 1.8 * 10
8The 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 beet exigua larvae in 2 latter stages in age.Its method is as follows: SeNPV is after viral 10 days in Spex II inoculation, collecting cell and nutrient solution thereof, 10% sodium lauryl sulphate (SDS) of adding 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 SeNPV.With distilled water the SeNPV 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 beet armyworm 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 beet exigua 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 beet armyworms of each concentration experiment.The mortality ratio of four days " Invest, Then Investigate " beet armyworms of experiment is calculated LC
50The 4th day LC as a result
50Be 1.99 * 10
5PIB/mL (95%CI=1.68 * 10
5PIB/mL to 8.65 * 10
5PIB/mL), use the polyhedron that increases by beet exigua larvae to carry out biological assay, its LC
50Be 4.32 * 10
5PIB/mL (95%CI=3.08 * 10
5PIB/mL to 6.07 * 10
5PIB/mL).With the same SeNPV of the SeNPV of Spex II cell amplification, its LC with the polypide amplification
50On statistical significance, do not have significant difference, illustrate that the virulence of the laphygma exigua nuclear polyhedrosis virus of producing with Spex II (SeNPV) does not change, can be used for the production of SeNPV.
Claims (3)
1. an insect cell line SpexII is deposited in China Committee for Culture Collection of Microorganisms common micro-organisms center on July 11st, 2005, and preserving number is CGMCC No.1411.
2. the described insect cell of claim 1 ties up to the purposes in the scale operation of baculovirus.
3. purposes according to claim 2, baculovirus wherein is a laphygma exigua nuclear polyhedrosis virus.
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| CN105176911A (en) * | 2015-01-07 | 2015-12-23 | 中国计量学院 | Chilo suppressalis larva midgut cell line with high yield of baculovirus |
| CA3003477A1 (en) * | 2015-11-01 | 2017-05-04 | Glycobac, Llc | Virus-free cell lines and methods for obtaining same |
| CN108588003B (en) * | 2018-04-27 | 2020-06-26 | 中国科学院动物研究所 | Method for establishing insect cell line |
| CN108531442B (en) * | 2018-04-28 | 2021-10-01 | 青岛农业大学 | Insect cell line cultured in serum-free suspension manner and application thereof |
| CN115851574B (en) * | 2022-12-27 | 2024-04-19 | 青岛农业大学 | Beet armyworm cell line |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN87106266A (en) * | 1986-09-08 | 1988-06-29 | 戴维·H·L·毕晓普 | The expression of hepatitis B virus antigen in the recombinant baculovirus carrier |
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Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN87106266A (en) * | 1986-09-08 | 1988-06-29 | 戴维·H·L·毕晓普 | The expression of hepatitis B virus antigen in the recombinant baculovirus carrier |
Non-Patent Citations (2)
| Title |
|---|
| 甜菜夜蛾核型多角体病毒(SeNPV)对家蚕细胞株(BmN)的感染. 吴福泉等.中国病毒学杀虫微生物专刊，第15卷. 2000 |
| 甜菜夜蛾核型多角体病毒(SeNPV)对家蚕细胞株(BmN)的感染. 吴福泉等.中国病毒学杀虫微生物专刊，第15卷. 2000 * |
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