CN109837236A - The Pomacea canaliculata spermary cell system of high yield baculoviral and its construction method and purposes - Google Patents
The Pomacea canaliculata spermary cell system of high yield baculoviral and its construction method and purposes Download PDFInfo
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- CN109837236A CN109837236A CN201910044518.8A CN201910044518A CN109837236A CN 109837236 A CN109837236 A CN 109837236A CN 201910044518 A CN201910044518 A CN 201910044518A CN 109837236 A CN109837236 A CN 109837236A
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Abstract
The invention discloses a kind of Pomacea canaliculata spermary cell system of high yield baculoviral and its construction method and purposes; Pomacea canaliculata spermary cell system is to organize from Pomacea canaliculata spermary; and to the high sensitive cell line of baculoviral; the invention also discloses the purposes of the method for building up of the cell line and the cell line in baculoviral scale growth.The cell line can be used to replicate this viroid, for the large-scale production of baculovirus insecticides, and building rod string design, express the protein with business or scientific value, thus have high business and scientific research value.
Description
Technical field
The invention belongs to a kind of Pomacea canaliculata of limnobios technical field of cell culture more particularly to high yield baculoviral essences
Nest cell line and its construction method and purposes.
Background technique
Since the Harrison and Carrel at the beginning of last century foundes the extracorporeal culturing method of animal tissue and cell, carefully
Born of the same parents' culture technique has obtained significant progress, and irreplaceable important work is all played in fundamental research and application study
With.However up to the present, vertebrate is focused primarily upon for the research of cell culture technology itself (especially lactation is dynamic
Object) and insect, it is relatively fewer for the concern of aquatic invertebrate cell culture.In the related cell that open publication is delivered
In the paper of culture problem, the overwhelming majority is the of vertebrate and insect
Since the 1960s, researcher begins to focus on the cell culture problem of aquatic invertebrate, aquatic nothing
The Various Tissues and cell of vertebrate are attempted carry out in vitro culture, including epithelial cell, haemocyte, glandula digestive, the young and
Embryonic tissue etc..The double navel spiral shell embryo (Biomphalaria of aquatic invertebrate are just established in the 1970s
Glabrataembryonie, BGE) cell line (Hansen, 1976).BGE cell line is Hansen to study blood fluke born of the same parents
What col infection cell mechanism was established in 1976 from the double navel spiral shell embryos of fresh water, after by the perfect cryopreservation methods such as Bayne and submit
Give ATCC (CRL-1494).
Cell line is always the weight of the scientific researches such as physiology, molecular biology and Developmental Biology as research material
Want tool, and important component of the insect cell line as rod string design, expressing a large amount of has weight
The exogenous proteins of big economic significance, while as bioreactor, it expands insect baculovirus and is used as biological insecticides, especially
It is to expand the recombinant baculovirus insecticides containing foreign gene.From the cell line of variety classes insect or from same
The cell line amplification virus or recombinant expression ability at kind insect different tissues position are had nothing in common with each other.Therefore establish and screening is new,
More baculoviral sensitive cell lines (strain), which are one, is highly desirable and has theoretical and practice significance work.Although
There are many reports using insect cell line amplification baculoviral, however up to the present, it yet there are no and utilize fresh water mollusk
The report of cell line production baculoviral
Pomacea canaliculata also known as Ampullaria gigas, apple spiral shell, originate in South America Amazon River basin.Before and after 1980, because of its albumen
Matter rich content, nutritional ingredient is high and fertility is strong, and is introduced into Philippine and Japan as a kind of aquatic economic animal,
And rapidly diffuse into East Asia and Southeast Asia remaining national (Halwart, 1994).Later due to marketization failure, Pomacea canaliculata is abandoned
It supports and spreads result population outbreak rapidly, seriously endanger crop production (Naylor, 1996).2000, world's conservation of nature connection
Alliance (World Conservation Union； International Union for Conservation of Nature
and Natural Resources；IUCN) it is pernicious outer to be classified as 100 kinds of the world by the Invasive Alien Species Committee of Experts for Pomacea canaliculata
Come one of invasive species (Lowe et al., 2000), and a kind of wherein unique freshwater snails.In China mainland, Pomacea canaliculata
It was introduced into Guangdong cultivation in 1981, starts to cultivate extensively on Guangdong, Guangxi, Fujian and other places after 1984, is then generalized to Zhejiang
The ground such as river, Jiangxi, Yunnan, Sichuan, have become on the south the Changjiang river at present most of provinces and regions serious agricultural pests (Yu Xiao equality,
2001).2003, Pomacea canaliculata was included in outside 16 kinds of first batch of invasion China by State Environmental Protection Administration and the Chinese Academy of Sciences (2003)
Come " blacklist " of species
During the prevention and treatment of Pomacea canaliculata, in addition to cultural control and physical control, the screening and identification of novel biopesticide
Have become research emphasis.The active bio measurement of biological pesticide needs largely to develop consistent Pomacea canaliculata, has to raising place
Strict requirements, heavy workload, and if have the Pomacea canaliculata cell of in vitro culture, biological pesticide can be studied in vitro
Effect to Pomacea canaliculata helps currently to kill to help to study insecticide to the mechanism of action of Pomacea canaliculata in cellular level
The improvement of worm agent and the invention of new pesticides.Therefore, through the invention, Pomacea canaliculata cell line is established, it is thin not only to can increase China
The quantity and type of born of the same parents system, moreover it is possible to offer reference experience for the foundation of such biological cell system in the future.Currently, thin about Pomacea canaliculata
The Vitro Culture Techniques and Pomacea canaliculata cell line of born of the same parents, relevant report is less, and only Pomacea canaliculata is organized and outer embrane group enough both at home and abroad
Knit culture (Nanjing Normal University, Jiang Dexun, fresh water fishery, 2008,38, the 2:54-59) report and Pomacea canaliculata flesh of cell
Meat, outer embrane, kidney and brain tissue originally culture (Hanshan Normal College, Li Xujie, guangdong agricultural science, 2017,44,8:
127-132) the Pomacea canaliculata heart tissue cell line (grant number: ZL2014108388759) established with laboratory where applicant.Mesh
Before, it there are no the report of Pomacea canaliculata spermary tissue lines foundation, also there are no raw using fresh water mollusk tissue lines
Produce the report of baculoviral.Spermary is the important sexual gland of Pomacea canaliculata, through the invention, constructs the Pomacea canaliculata sensitive to baculoviral
Spermary cell system will provide important research in relation to gene function, Sex Differentiation and disease control for research Pomacea canaliculata gender and put down
Platform.To identify that aquatic virus provides basis using the separation of fresh water mollusk cell line.
Summary of the invention
Cell tissue-derived the object of the present invention is to provide a kind of Pomacea canaliculata spermary and with height viral susceptibility
System, the entitled BIQ-Pc-II of the cell line.
Another object of the present invention is to provide the construction methods of the cell line.
Another object of the present invention is to provide the cell line in the large-scale production of baculoviral and use baculoviral
Expression vector expresses the purposes on recombinant protein.
The present invention establishes the Pomacea canaliculata spermary cell system of high yield baculoviral a kind of, and specific step is as follows:
(1) cell primary culture solution and cell secondary culture liquid are prepared；
(2) 20-30 grams of weight male Pomacea canaliculata is chosen to raise in sterile water 12 hours；
(3) spiral shell is immersed in mass percent concentration is 0.1%KMnO4In aqueous solution, surface sterilization 10- is carried out
15 minutes；
(4) it is cleaned Pomacea canaliculata 3~5 times with sterile distilled water, blots breechblock surface moisture with aseptic filter paper；
(5) Pomacea canaliculata is placed in the dissecting pan crossed with volume fraction 70% or 75% ethanol disinfection, dissects Pomacea canaliculata, takes
Pomacea canaliculata spermary is organized out, and spermary tissue is cleaned 3-5 times with physiological saline, then is cleaned 2-3 times with PBS, will with sterilizing scissors
Spermary tissue is cut into 0.5mm3-1mm3Small tissue blocks；
(6) small tissue blocks for handling (5) are placed in the T-25cm for filling 1ml-1.5ml cell primary culture solution in advance2Carefully
In born of the same parents' culture bottle, trypsin digestion is added, is then sufficiently suspended with cell primary culture solution, precipitating is collected by centrifugation；
(7) cell primary culture solution is added into precipitating, culture bottle is placed in incubator, 28 DEG C of constant temperature incubations；
(8) overnight after, wait organize it is adherent after, add 2ml-2.5ml cell secondary culture liquid, make tissue largely submerge
In the secondary culture liquid, do not make tissue suspension in cell secondary culture liquid when liquid feeding；
(9) every 5-7 days be sucked out 50%~70% amount cell secondary culture liquid, and change to simultaneously suction amount newly it is thin
Born of the same parents' secondary culture liquid, up to the cell for extending and being proliferated is full of culture bottle.
(10) it containing the individual cells being newly proliferated out, is put into new culture bottle together with whole cell culture fluid suctions,
And new cell secondary culture liquid is added, and same suction amount as much new thin is added in the former culture bottle containing tissue block
Born of the same parents' secondary culture liquid, two culture bottles are put into incubator and cultivate, and cell starts to pass on, Establishment of Cell Line success.
Whole process all aseptically carries out.
Cell primary culture solution of the present invention be Insect cellculture liquid and penicillin, streptomysin, amphotericin B,
The mixture of gentamicin and animal blood serum, pH value 7.0-7.8；The cell secondary culture liquid is Insect cellculture liquid
With the mixture of animal blood serum, pH value 7.0-7.8；The Insect cellculture liquid is selected from the DF12 culture medium of commercialization,
The animal blood serum is fetal calf serum；The Penicillin Content is 400U/ml, and content of streptomycin is 400 μ g/ml, both sexes
Mycin B content is 500 μ g/ml, and Study on Determination of Gentamycin is 100 μ g/ml；Animal blood serum content in the cell primary culture solution
For 10%~20% (volume ratio)；Animal blood serum content in the cell secondary culture liquid is 10%-20% (volume ratio)；Institute
The Pomacea canaliculata spermary cell for stating high yield baculoviral ties up to purposes in the large-scale production of baculoviral.The baculoviral
It is prawn baculovirus.
The beneficial effects of the present invention are: using the above scheme, having carried out originally culture and passage to Pomacea canaliculata spermary cell
Culture, achieves preferable culture effect.By being inoculated with baculoviral, the discovery present invention is sensitive to baculoviral, and the present invention is first
Secondary confirmation baculoviral has infection ability to fresh water mollusk cell line.The present invention is quickly, effective, repeatability is strong, is to light
The important supplement of water mollusk cell system.Pomacea canaliculata is important invasion pest, and the present invention helps to study in vitro
Pomacea canaliculata provides help for the research of its controlling way and the exploitation of novel biopesticide.
The biological property observation and measurement of Pomacea canaliculata spermary cell system in the present invention
1. through Germicidal efficacy, the cell line major part adherent growth, the shape of cell is most of round, small part shuttle shape,
It is less like macrophage-shaped (Fig. 1).
2. with 2x105Cell/mL concentration is inoculated into T-25cm2In culture bottle, 27 DEG C of no light cultures take one bottle daily
Cell concentration is measured, is drawn growth curve (Fig. 2), and according to formula T=tlg2/ [lg (N/N0)] population doubling time is calculated,
The cell population doublings time by calculating eighth generation is 86 hours.Wherein
T=time needed for one times of logarithmic phase balanced growth
T=is inoculated into the time of measurement cell number
N0Cell number when=inoculation
The total number of cells that N=moment t is measured
3. use interleukin-1 ' beta ' primer, using the method for DAF-PCR identify the cell line established of the present invention really come
Spermary derived from Pomacea canaliculata is organized, rather than the pollution of other cell lines (Fig. 3).The DNA band and Pomacea canaliculata provided by cell line
Banding pattern after DNA cloning is identical, and significantly different with control cell lines sf9 and s2 banding pattern.
3. the present invention is built, cell line is sensitive to prawn baculovirus, it can be observed that typical nucleus increases, includes
There is a large amount of polyhedrosis virus (Fig. 4).
Detailed description of the invention
The culture form of cell line after Fig. 1 culture 30 days；
The growth curve of Fig. 2 cell line；
The DAF-PCR of Fig. 3 cell line identifies map；
A large amount of viral polyhedron is obtained after Fig. 4 cell line infection nuclear polyhedrosis virus.
Specific embodiment
The foundation of 1. Pomacea canaliculata spermary cell system of embodiment
(1) cell primary culture solution and cell secondary culture liquid are prepared；
(2) 20-30 grams of weight male Pomacea canaliculata is chosen to raise in sterile water 12 hours；
(3) spiral shell is immersed in 0.1%KMnO4In solution, carry out surface sterilization 10 minutes；
(4) it is cleaned Pomacea canaliculata 3 times with sterile distilled water, blots breechblock surface moisture with aseptic filter paper；
(5) Pomacea canaliculata is placed in the dissecting pan sterilized with 70% alcohol, dissects Pomacea canaliculata, take out Pomacea canaliculata spermary group
It knits, spermary tissue is cleaned 3 times with physiological saline, then is cleaned 2 times with PBS, spermary tissue is cut into 0.5mm with sterilizing scissors3
Small tissue blocks；
(6) small tissue blocks for handling (5) are placed in the T-25cm for filling 1ml cell primary culture solution in advance2Cell culture
In bottle, trypsin digestion is added, is then sufficiently suspended with cell primary culture solution, precipitating is collected by centrifugation；
(7) cell primary culture solution is added into precipitating, culture bottle is placed in incubator, 28 DEG C of constant temperature incubations；
(8) overnight after, wait organize it is adherent after, add 2ml cell secondary culture liquid, tissue made to be mostly submerged in the biography
It is commissioned to train in nutrient solution, does not make tissue suspension in cell secondary culture liquid when liquid feeding；
(9) the cell secondary culture liquid of 50% amount was sucked out every 5-7 days, and changes to the new cell passage of suction amount simultaneously
Culture solution, up to the cell for extending and being proliferated is full of culture bottle.
(10) it containing the individual cells being newly proliferated out, is put into new culture bottle together with whole cell culture fluid suctions,
And new cell secondary culture liquid is added, and same suction amount as much new thin is added in the former culture bottle containing tissue block
Born of the same parents' secondary culture liquid, two culture bottles are put into incubator and cultivate, and cell starts to pass on, Establishment of Cell Line success.
Whole process all aseptically carries out.
The foundation of 2 Pomacea canaliculata spermary cell system of embodiment
(1) cell primary culture solution and cell secondary culture liquid are prepared；
(2) 20-30 grams of weight male Pomacea canaliculata is chosen to raise in sterile water 12 hours；
(3) spiral shell is immersed in 0.1%KMnO4In solution, carry out surface sterilization 15 minutes；
(4) it is cleaned Pomacea canaliculata 3~5 times with sterile distilled water, blots breechblock surface moisture with aseptic filter paper；
(5) Pomacea canaliculata is placed in the dissecting pan crossed with 75% ethanol disinfection, dissects Pomacea canaliculata, take out Pomacea canaliculata spermary group
It knits, spermary tissue is cleaned 5 times with physiological saline, then is cleaned 3 times with PBS, spermary tissue is cut into 1mm with sterilizing scissors3's
Small tissue blocks；
(6) small tissue blocks for handling (5) are placed in the T-25cm for filling 1.5ml cell primary culture solution in advance2Cell training
It supports in bottle, trypsin digestion is added, is then sufficiently suspended with cell primary culture solution, precipitating is collected by centrifugation；
(7) cell primary culture solution is added into precipitating, culture bottle is placed in incubator, 28 DEG C of constant temperature incubations；
(8) overnight after, wait organize it is adherent after, add 2.5ml cell secondary culture liquid, tissue made to be mostly submerged in this
In secondary culture liquid, do not make tissue suspension in cell secondary culture liquid when liquid feeding；
(9) the cell secondary culture liquid of 70% amount was sucked out every 5-7 days, and changes to the new cell passage of suction amount simultaneously
Culture solution, up to the cell for extending and being proliferated is full of culture bottle.
(10) it containing the individual cells being newly proliferated out, is put into new culture bottle together with whole cell culture fluid suctions,
And new cell secondary culture liquid is added, and same suction amount as much new thin is added in the former culture bottle containing tissue block
Born of the same parents' secondary culture liquid, two culture bottles are put into incubator and cultivate, and cell starts to pass on, Establishment of Cell Line success.
Whole process all aseptically carries out.
The Observation of biological characteristics of 3 cell line of embodiment and measurement
(1) morphological feature: through micro- sem observation, the cell line adherent growth, there are mainly three types of cell shapes: most of circle
Shape, small part shuttle shape are less like macrophage-shaped (Fig. 1).Round cell accounts for 90%, and 11.45 ± 1.10 μm of average diameter；Shuttle shape
Cell is about 22.0--38 μm, wide about 11 μm average；Between 20-21 μm of macrophage diameter.
(2) growth of cell: growth curve is in typical " S ", and population doubling time is 86 hours (Fig. 2).
(3) DAF-PCR is identified: being used interleukin-1 ' beta ' design primer, is identified the present invention using the method for DAF-PCR
The cell line of foundation derives from the spermary cell of Pomacea canaliculata really, rather than the pollution of other cell lines (Fig. 3).It is provided by cell line
The amplification of DNA band and Pomacea canaliculata spermary tissue DNA after banding pattern it is identical, and obviously not with control cell lines sf9 and s2 banding pattern
Together
The measurement of embodiment 4 viral susceptibility and yield
Cell line is to prawn baculovirus polyhedrosis virus sensitivity testing: logarithmic growth phase cell is deployed into
1x106/ mL concentration, is inoculated into culture bottle, every bottle of 10mL.After culture 3 hours, culture medium is discarded, attached cell is left, will be made
The poison disease vaccination got ready is in the cell, every bottle of 100uL.After culture 7 days, cell and viral polyhedron are harvested, blood count is used
Plate counts viral polyhedron number under the microscope, and as a result the built cell line of the present invention produces polygonal bulk concentration up to 16.5x106/
PIB/mL.Meanwhile it can be observed that typical cell pathology feature, i.e. nucleus increase are interior to contain a large amount of polyhedra particles
(Fig. 4).Illustrate that the built cell line of the present invention is sensitive to baculoviral.
Claims (10)
1. a kind of construction method of the Pomacea canaliculata spermary cell system of high yield baculoviral, which comprises the steps of:
(1) cell primary culture solution and cell secondary culture liquid are prepared；
(2) 20-30 grams of weight male Pomacea canaliculata is chosen to raise in sterile water 12 hours；
(3) spiral shell is immersed in mass percent concentration is 0.1%KMnO4In aqueous solution, carry out surface sterilization 10-15 minutes；
(4) it is cleaned Pomacea canaliculata 3~5 times with sterile distilled water, blots breechblock surface moisture with aseptic filter paper；
(5) Pomacea canaliculata is placed in the dissecting pan sterilized with volume fraction 70% or 75% ethanol solution, dissects Pomacea canaliculata, takes
Pomacea canaliculata spermary is organized out, and spermary tissue is cleaned 3-5 times with physiological saline, then is cleaned 2-3 times with PBS, will with sterilizing scissors
Spermary tissue is cut into 0.5mm3-1mm3Small tissue blocks；
(6) small tissue blocks for handling step (5) are placed in the T-25cm for filling 1ml-1.5ml cell primary culture solution in advance2Carefully
In born of the same parents' culture bottle, trypsin digestion is added, is then sufficiently suspended with cell primary culture solution, precipitating is collected by centrifugation；
(7) cell primary culture solution is added into precipitating, culture bottle is placed in incubator, 28 DEG C of constant temperature incubations；
(8) overnight after, wait organize it is adherent after, add 2ml-2.5ml cell secondary culture liquid,
(9) the cell secondary culture liquid of 50%~70% amount was sucked out every 5-7 days, and the new cell for changing to suction amount simultaneously passes
It is commissioned to train nutrient solution, until extending and the cell that is proliferated is full of culture bottle；
(10) it the individual cells being newly proliferated out, is put into new culture bottle, and is added new together with whole cell culture fluid suctions
Cell secondary culture liquid, and the new cell passage training of same suction amount as much is added in the former culture bottle containing tissue block
Nutrient solution, two culture bottles are put into incubator and cultivate, and cell starts to pass on, Establishment of Cell Line success.
2. the method according to claim 1, wherein the cell primary culture solution is Insect cellculture liquid
With the mixture of penicillin, streptomysin, amphotericin B, gentamicin and animal blood serum, pH value 7.0-7.8.
3. the method according to claim 1, wherein the cell secondary culture liquid is Insect cellculture liquid
With the mixture of animal blood serum, pH value 7.0-7.8.
4. the method according to claim 1, wherein the construction method whole process aseptically carries out.
5. according to the described in any item methods of Claims 2 or 3, which is characterized in that the Insect cellculture liquid is selected from quotient
The DF12 culture medium of product, the animal blood serum are fetal calf serum.
6. according to the method described in claim 2, it is characterized in that, the Penicillin Content is 400U/ml, content of streptomycin
For 400 μ g/ml, amphotericin B content is 500 μ g/ml, and Study on Determination of Gentamycin is 100 μ g/ml.
7. according to the method described in claim 2, it is characterized in that, the animal blood serum content is 10%~20% (volume
Than).
8. according to the method described in claim 3, it is characterized in that, the animal blood serum content is 10%~20% (volume
Than).
9. a kind of Pomacea canaliculata spermary cell of the high yield baculoviral of claim 1 the method building ties up to the big of baculoviral
Purposes in large-scale production.
10. purposes according to claim 8, baculoviral therein is prawn baculovirus.
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Cited By (1)
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| CN112852716A (en) * | 2021-03-12 | 2021-05-28 | 集美大学 | Cell line of testis tissue of large yellow croaker and application thereof |
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