CN110551689B

CN110551689B - Channa argus brain cell line and construction method and application thereof

Info

Publication number: CN110551689B
Application number: CN201910743467.8A
Authority: CN
Inventors: 王英英; 曾伟伟; 王庆; 李莹莹; 尹纪元; 石存斌; 常藕琴
Original assignee: Pearl River Fisheries Research Institute CAFS
Current assignee: Pearl River Fisheries Research Institute CAFS
Priority date: 2019-08-13
Filing date: 2019-08-13
Publication date: 2020-08-04
Anticipated expiration: 2039-08-13
Also published as: CN110551689A

Abstract

The invention discloses a channa argus brain cell line and a construction method and application thereof, aiming at the characteristics of channa argus brain tissue cells, the brain tissue cells are separated by adopting a collagenase I and pancreatin combined digestion method, primary culture is carried out, then the channa argus brain cell line is successfully constructed, the cells are continuously transferred to more than 100 generations, a large number of channa argus brain source cells can be provided, the cells can maintain a good growth state, and the channa argus brain cell line can be subjected to cryopreservation. The snakehead brain cell line has sensitivity to various fish viruses, and has cytopathy after virus inoculation, and can be used for adhesion characteristic research of snakehead source aeromonas schubertii, and can be directly applied to pathogenic characteristic research and vaccine development.

Description

Channa argus brain cell line and construction method and application thereof

Technical Field

The invention belongs to the technical field of biology, and particularly relates to a channa argus brain cell line and a construction method and application thereof.

Background

Both snakeheads and zebra fishes are relatively common economic fishes, and both belong to the order Perciformes, the suborder Channoidei and the snakehead family Channidae. Snakeheads are commonly named as snakeheads, are relatively violent in nature, good in appetite and strong in fertility and are mostly distributed in the Yangtze river basin and the water system in the north area, and the snakeheads are mainly distributed in the areas south of the Yangtze river basin, such as Guangdong, Hainan, Guangxi, Fujian, Yunnan and the like. The male parent of the channa argus is mostly from channa argus in Hunan province, Shandong province and other areas, the female parent of the channa argus is mostly local channa argus in Guangdong province, a first filial generation cultivated by hybridization of the two is called channa argus, and the hybridization technology belongs to interspecies hybridization of distant hybridization of fishes. The applicant successfully cultured the first channa maculata. The individual has obvious hybrid advantages, the individual has high growth speed and high muscle quality and nutritive value, and in addition, the adult individual has large size and also has strong disease resistance, can live in a low-oxygen environment, has relatively low feed coefficient and the like.

Under natural environment, the disease resistance of snakehead fishes is very strong, and the disease phenomenon hardly occurs. However, as the degree of intensification of snakehead fish culture increases and the scale of culture and culture density gradually expand, the number of infectious diseases of snakehead fish increases. With the frequent occurrence of various diseases and the outbreak of bacterial and viral diseases, the rapid development and popularization of the snakehead fish breeding industry are restricted. In order to further understand the mechanism of the channa maculata infected virus, a cell line with high sensitivity needs to be established, and a foundation is laid for the diagnosis of virus diseases and the development of virus vaccines in the future. The establishment of a primary cell line of fish is a mature technology at present, but obtaining a cell line sensitive to viruses has contingency and difficulty. Aiming at the existing problems, an effective way for establishing a cell line needing fish is to prepare a large batch of primary cell lines by quantity-to-quality, so as to screen out a sensitive cell line.

The cell line is an ideal material for gene function analysis, and has great significance for the discovery of fish immune gene functions. Fish is a vertebrate with a specific immune system and a non-specific system, and the non-specific immunity plays an important role in defense reactions of the fish to external stimuli and pathogenic organism invasion.

Aeromonas schubertii (Aeromonas schubertii), belonging to the family of Aeromonas, genus Aeromonas, also known as Aeromonas schubertii. It is a gram-negative bacillus, a monopolar flagellum, and is extremely active in locomotion. The bacteria are widely distributed in fresh water, brackish water, soil, fish and vertebrate intestinal tracts, and can cause wound infection of human and important pathogenic bacteria causing diarrhea. In aquaculture industry, especially snakehead fish culture, diseases caused by the bacteria frequently occur, and the bacteria can cause the snakehead fish to suffer from sarcoidosis. The bacterium is reported to have adhesion effect on fish cells.

At present, the primary culture technology of fish cells can be basically divided into two types, namely a tissue block culture method and a digestion culture method. But has the defects that mechanical damage is easy to generate and cells are not easy to migrate. At present, no report of a snakehead brain-derived cell line is found. Therefore, the construction method of the ophthallophora argus brain cell line needs to be researched at present, and a solid foundation is laid for establishing other histiocyte lines of the ophthallophora argus and carrying out virus research in the later period.

Disclosure of Invention

The invention mainly aims to provide a channa argus brain cell line (CAMB).

A secondary object of the invention is to provide a method for constructing a channa argus brain cell line.

Still another object of the present invention is to provide the use of the channa argus brain cell line.

In order to achieve the purpose, the technical scheme adopted by the invention is as follows:

a channa argus brain cell line CAMB is preserved in a China center for type culture collection in 2018, 6 months and 13 days, wherein the preservation number is CCTCC NO: C2018125.

further, the method comprises the following steps: and (2) placing the snakehead brain tissue into collagenase I for digestion, then adding pancreatin for digestion, adding a snakehead brain cell culture solution for culture, placing the snakehead brain cell culture solution into the pancreatin for digestion, collecting the digested cells, and performing primary culture and subculture to obtain the snakehead brain cell line.

Further, the method comprises the following steps:

1) placing the snakehead brain tissue into collagenase I for digestion, collecting cells, adding pancreatin for digestion, and collecting the digested cells;

2) adding the cells collected in the previous step into a snakehead brain cell culture solution to prepare a cell suspension, and performing primary culture at the temperature of 26-28 ℃;

3) when the primary culture cells fully grow on the bottom of a culture flask, removing the culture solution, digesting with pancreatin solution, removing the enzyme solution after digestion, adding a culture solution of the snakehead brain cells for resuspending the cells, and carrying out subculture on the resuspended cells.

Further, the snakehead brain tissue is firstly soaked in a tertiary-antibody solution, and the tertiary-antibody solution is a penicillin-streptomycin-amphotericin B tertiary-antibody solution.

Furthermore, the snakehead brain tissue is chopped sterile snakehead brain tissue.

Further, the collagenase I is 0.1% w/v, and is digested for 10-15 min; the pancreatin is 0.25% w/v. The digestion time is 10-15 min.

Further, the culture solution of the snakehead brain cells is an M199 culture medium; the M199 culture medium further comprises penicillin-streptomycin-amphotericin B three-antibody solution with the final concentration of 100u/ml and 20% fetal calf serum, and the pH value is 7.2-7.6.

Further, the pancreatin liquid in the step (3) contains 0.25% w/v pancreatin and 0.02% w/v EDTA.

The application of the snakehead brain cell line in culturing fish viruses; the virus is at least one of spring viremia of carp virus, channa argus rhabdovirus, frog iridovirus, genotype I grass carp reovirus and tilapia lake virus.

The application of the snakehead brain cell line in the adhesion of aeromonas is provided; the aeromonas is schuberite aeromonas of snakehead origin.

In recent years, researches on cloning of fish nonspecific related functional genomes and immune regulation mechanisms become hot spots, and the invention carries out nonspecific immune function research between pathogen and host through bacterial adhesion cell experiments.

The invention has the beneficial effects that:

the snakehead brain cell line is successfully cultured in vitro for the first time, the brain tissue cells are digested and separated by adopting pancreatin and collagenase, and the extended growth obviously occurs when the brain tissue cells are cultured for 7 days; after 15 days, the culture dish is fully paved, and after digestion, the culture dish has stronger reproductive capacity and presents a typical epithelial cell form; the cells are continuously subcultured for 16 months and are transferred to 50 generations, and the morphological characteristics and growth characteristics of epithelial cells are still maintained; even 100 generations of continuous culture still maintain strong growth characteristic and proliferation characteristic, and can provide a large amount of snakehead brain-derived cells.

The cell line of the invention has stable characteristics, which are up to 100 generations, and the cells can maintain good growth state, and can be frozen for storage. The channa argus brain cell line has sensitivity to various fish viruses, shows cytopathic effect after virus inoculation, and can be directly applied to pathogen research.

Drawings

FIG. 1 shows the morphology of a channa argus brain cell line under a phase contrast microscope; a is primary cells; b is 20-generation channa argus brain cells; c is 50-generation channa argus brain cells; d is 80-generation channa argus brain cells;

FIG. 2 shows the parameters of detection of suspended CAMB, i.e.cell count: 12215, mean volume: 1.28p L, mean diameter: 13.5. mu.m;

FIG. 3 is the optimum culture conditions of the Channa argus brain cell line; a is a culture medium: b is the serum concentration; c is temperature;

FIG. 4 shows chromosomes of brain cells of channa argus; wherein A is a chromosome of a brain cell of the channa argus, Number of cells is the Number of the cell, and Number of chromosome is the Number of the chromosome;

FIG. 5 shows the viral susceptibility of the kidney cells of channa maculata, wherein A. blank control, B.Ti L V, C.SVCV, D.HSHRV, E.FV3, F.GCRV;

FIG. 6 shows PCR electrophoretograms of virus infection of CAMB cells, wherein lane 1: CAMB cells infected with Ti L V-2017A, lane 3: CAMB cells infected with SVCV (nested PCR product), lane 5: CAMB cells infected with SVCV (general PCR product), lane 7: CAMB cells infected with HSHRV, lane 9: CAMB cells infected with FV3, lane 11: CAMB cells infected with genotype I GCRV, and

lanes

2, 4, 6, 8, 10 and 12 are blank controls (PBS added group) for each virus-infected group, respectively;

FIG. 7 shows the adhesion of aeromonas schubertii JW-I strain from snakehead to the brain cells of snakehead (1000 ×), wherein A is blank control, and B is aeromonas schubertii JW-I adhesion cells from snakehead.

Detailed Description

preferably, the method comprises the following steps: and (3) placing the snakehead brain tissue into collagenase I for digestion, adding pancreatin for digestion, adding the collagenase I into a snakehead brain cell culture solution for primary culture, and carrying out subculture on the cells to obtain the snakehead brain cell line.

Preferably, the method comprises the following steps:

1) placing the snakehead brain tissue into pancreatin for digestion, collecting cells, adding collagenase I for digestion, and collecting the digested cells;

2) adding the cells collected in the previous step into a snakehead brain cell culture solution to prepare a cell suspension, and adding 4.5-5.5% CO at the temperature of 26-28 DEG C₂Performing primary culture under the condition;

Preferably, the snakehead brain tissue is firstly soaked in a three-resistant solution, and the three-resistant solution is a penicillin-streptomycin-amphotericin B three-resistant solution.

Preferably, the snakehead brain tissue is chopped sterile snakehead brain tissue.

Preferably, the collagenase I is 0.1% w/v, and is digested for 10-15 min; the pancreatin is 0.25% w/v, and the digestion time is 10-15 min.

Preferably, the culture solution of the snakehead brain cells is an M199 culture medium; the M199 culture medium further comprises penicillin-streptomycin-amphotericin B three-antibody solution with the final concentration of 100u/ml and 20% fetal calf serum, and the pH value is 7.2-7.6.

Preferably, the pancreatin liquid in step (3) contains 0.25% w/v pancreatin and 0.02% w/v EDTA.

The technical solution of the present invention is clearly and completely illustrated below with reference to the following examples, but is not limited thereto.

example 1 isolation and Primary culture of brain cells of channa maculata

Test materials and reagents

Experimental animals: 10g-30g of channa argus (the channa argus is verified to be healthy channa argus which is not infected with aquatic animal viruses by a repeated molecular method).

Experimental apparatus: scalpels, ophthalmic scissors, ophthalmic forceps, gauze, and the like.

Culture media and related reagents

The concentration of the penicillin-streptomycin-amphotericin B three-resistant solution is 10000 u/ml;

culture medium: m199 medium (Gibco) containing penicillin-streptomycin-amphotericin B triantibody at a concentration of 100U/ml and 20% fetal bovine serum (Gibco);

pancreatin digestive juice: 2.3g of disodium hydrogen phosphate, 0.1g of monopotassium phosphate, 8.0g of sodium chloride, 0.2g of potassium chloride,

EDTA0.2g, pancreatin 0.6g and water 1000 ml; medical alcohol;

0.01M PBS buffer (NaCl 80g, Na)₂HPO₄·12H₂O 29g，KH₂PO₄2g, KCl 252 g, water 1000 ml).

And (3) detecting healthy channa maculata, temporarily culturing for 15 days in a laboratory, and starting an experiment if no other diseases exist.

The specific method comprises the following steps:

(1) soaking the channa argus in medical alcohol for 5min, shearing the brain of the channa argus under the aseptic condition, and soaking the sheared sterile brain tissue of the channa argus in a penicillin-streptomycin-amphotericin B three-resistant solution with the concentration of 200u/ml for 5 min;

(2) cut into small pieces (about 1 mm)³) Then 0.1% collagenase I is digested for 10-15min, the mixture is resuspended in a culture medium, centrifuged, the supernatant is discarded, 0.25% pancreatin digestive fluid is added, the mixture is digested for 10-15min, the supernatant is centrifuged, finally, the culture fluid of the goby snakehead brain cells is added, and the mixture is resuspended and mixed into a cell suspension;

(3) transferring the cell suspension into a culture bottle, and putting the culture bottle into an incubator at 27 ℃ for culture;

(4) observing the cell adherence condition after culturing for 2 days, and properly adding the culture solution of the ophthalmia argus brain cells (ensuring the pH of the culture solution to be 7.2-7.6). When the cells are plated to 1/3 (3-4 days), replacing the fresh culture solution once;

(5) after the cells are fully paved on the bottom of the culture dish (7-8 days), primarily digesting the cells by pancreatin digestive juice, and then placing the digested cells into the culture solution of the ophyta argus brain cells for subculture, wherein the subculture ratio is 1:2-1: 3.

Freezing and storing the snakehead brain cell line for later use; after recovery, the cell can continue to stably proliferate and grow. After that, the cells are passaged 1 time every 3-5 days according to the growth condition of the cells.

Culture solution of the snakehead brain cells: the kit comprises penicillin-streptomycin-amphotericin B three-antibody solution with the final concentration of 100u/ml, 20% of M199 culture medium of fetal calf serum and 7.2-7.6 of pH.

The experimental results are as follows: FIG. 1 shows the morphology of a channa argus brain cell line under a phase contrast microscope, showing a typical epithelial-like cell morphology. FIG. 2 shows the suspension characteristics of the Channa argus brain cell line, and the result shows that the Channa argus brain cell line has good uniformity and the diameter is mostly 12-18 microns.

Example 2 subculture and cryopreservation of the Channa argus brain cell line

Reagent

EDTA0.2g, pancreatin 0.6g and water 1000 ml.

Freezing and storing liquid: and (3) an M199 culture medium containing 10% of DMSO and 25% of special fetal bovine serum.

Experimental methods

When the cells grow to 90%, discarding the old culture medium, and adding 1ml of pancreatin digestive juice for digestion; when the cells are in a white fog shape, 2ml of the snakehead brain cell culture solution is added to stop the action of the pancreatin, the cells are mixed evenly by shaking lightly, and the cell count is carried out. Centrifuging at 1000rpm for 5min, removing culture medium, adding 1.5ml cell freezing medium, and resuspending cells.

Collecting the cells into a freezing tube, and annotating the name, the number and the freezing date of the cells on the freezing tube.

After one month, cell recovery is carried out according to a conventional cell recovery method, the cell proliferation capacity is still strong, and the state is good. A channa argus brain cell line CAMB is preserved in a China center for type culture collection in 2018, 6 months and 13 days, wherein the preservation number is CCTCC NO: C2018125.

example 3 growth characteristics assay of the brain cell line of channa maculata

Reagent

Pancreatin digestive juice: 2.3g of disodium hydrogen phosphate, 0.1g of potassium dihydrogen phosphate, 8.0g of sodium chloride, 0.2g of potassium chloride, 0.2g of EDTA0.2g, 0.6g of pancreatin and 1000ml of water.

Culture medium, M199 culture medium of special-grade fetal bovine serum with different concentrations, DMEM, MEM, L-15 culture medium

Experimental methods

Determination of optimal culture medium for 1 channa argus brain cells

Selecting four cell culture media of DMEM, M199, MEM and L-15, adding FBS with final concentration of 10% to prepare cell culture solution, adjusting cell density to 2 × 10⁵m L-1, four media were inoculated into 6-well plates at 2.5m L/well, and cultured in an incubator at 27 ℃ every 1 day, 3-well cells were collected from each experimental group by Trypsin-EDTA digestion and counted for 7 days, and the growth curve was plotted by counting 7 times continuously.

As a result, the optimal medium for the Channa argus brain cells was determined to be M199 or L-15 medium (FIG. 3A).

M199 Medium, according to the specification of Medium 199 Medium (containing Earle's balanced salt solution), adding 1L water into the container, placing the container on a magnetic stirrer, adding dry powder of Medium 199 Medium (containing Earle's balanced salt solution) while stirring, adding 10% fetal bovine serum after fully stirring and dissolving, and using powdered NaHCO₃Adjusting the pH of the culture solution to 7.2-7.6. Filtering to remove bacteria as soon as possible, subpackaging, and storing at-20 deg.C.

L-15 Medium, according to the requirements of the specification of L eibovitz's L-15 Medium (containing L-Glutamine), 1L water is added into a container, the container is placed on a magnetic stirrer, L-15 medium (containing L-Glutamine) dry powder is added while stirring, 10% fetal bovine serum is added after fully stirring and dissolving, and powdered NaHCO is used₃Adjusting the pH of the culture solution to 7.2-7.6. Filtering to remove bacteria as soon as possible, subpackaging, and storing at-20 deg.C.

Determination of 2-channa argus brain cell optimal serum concentration

The culture medium containing fetal bovine serum FBS at 5%, 10%, 15% and 20% concentration was prepared, and the cell density was adjusted to 2 × 10⁵mL^-1Four serum concentration media were inoculated at 2.5m L/well into 6-well plates and incubated at 27 ℃ in an incubator from each experiment every 1 dayThe cells in 3 wells were removed from the group, and the cells were collected by Trypsin-EDTA digestion and counted for 7 days in total, and the growth curve was plotted by counting 7 times continuously.

As a result: the FBS concentration of 10-20% is found to be suitable for the culture of tilapia brain cells (figure 3B).

Determination of optimal culture temperature of 3-channa argus brain cells

Selecting four different culture temperatures of 15 deg.C, 22 deg.C, 27 deg.C and 32 deg.C, using DMEM culture solution containing 10% FBS, and adjusting cell density to 2 × 10⁵mL^-1Taking 3-hole cells out of each experimental group every 1d, collecting and counting the cells by a Trypsin-EDTA digestion method, culturing for 7 days, continuously counting for 7 times, drawing a growth curve, finding that the cells are suitable for culturing tilapia brain cells at 22 ℃ and 27 ℃ (figure 3C), collecting and counting the cells, culturing for 7 days, continuously counting for 7 times, and drawing the growth curve.

The experimental results are as follows: the optimum culture conditions of the snakehead brain cells are as follows: the cells were cultured at 27 ℃ in M199 medium containing 10% fetal bovine serum.

Example 4 chromosome analysis of brain cells of channa argus

Adding colchicine with the final concentration of 8 mug/m L into 60 th generation of channa maculata kidney cells in a logarithmic growth phase, treating for 16h at 27 ℃, collecting the cells, performing hypotonic treatment for 20min by KCl with the concentration of 0.075 mol/L, adding 1m L precooled Carnot fixing liquid, centrifuging for 5min at 1000r/min, removing supernatant, fixing for 3 times by using the precooled Carnot fixing liquid, dripping for 15min each time, drying, staining for 25min by 5% Giemsa, performing microscopic examination, and respectively selecting 100 division phases for karyotyping and statistics.

The results showed (fig. 4A) that 43% of chromosomes of the northern snakehead kidney cells of the 60 th generation were 2 n-64, and 2 n-45 chromosomes of the northern snakehead somatic cells.

Example 5 infection experiment of the Channa argus brain cell line on different aquatic animal viruses

Two bottles of the goby snakehead brain cells with good growth condition are subjected to passage with the passage ratio of 1:3, fully and uniformly mixed and then are subpackaged to six 25cm²In the cell culture bottle, when the cells just overgrow the bottom of the culture bottle, namely when the cells are paved with 80-90%, the culture medium is sucked away, the HBSS buffer solution is washed twice, and 1m L fish viruses are respectively inoculated into the cells, wherein the fish viruses are Spring Viremia of Carp Virus (SVCV), goby spot fish rhabdovirus (HSHRV), Frog iridovirus (Frog virus3, FV3), gene I type grass Reovirus (GrassCarp Reovirus, GCRV), tilapia lake virus (Ti L V) and PBS as blank control groups, and blank controls are respectively set according to different culture temperatures.

After 1h of virus incubation, abandoning virus liquid, adding 5M L cell maintenance liquid (M199 culture medium containing 5% FBS), placing a cell culture bottle of SVCV into a 22 ℃ constant temperature incubator, placing another cell bottle of four viruses into a 28 ℃ incubator, placing a blank control into a 28 ℃ incubator, culturing at 28 ℃, observing cell growth and cytopathic effect CPE every day, collecting cell suspension before cells completely drop once CPE lesions appear, extracting RNA, detecting RT-PCR, collecting samples for RT-PCR experiment after 3 generations of virus inoculation groups without lesions are blindly transmitted, and analyzing whether viruses proliferate in CAMB cells by detecting virus transcripts.

When collecting samples, the culture bottle is taken out, placed in a refrigerator at minus 20 ℃ overnight, taken out and beaten with force after the culture medium is melted to ensure that the cells completely fall off, beaten, mixed and frozen, taken out 200 mu L cell suspension, put in a centrifuge tube for RNA extraction and PCR detection, and agarose gel electrophoresis is used for detecting PCR results.

As a result, after five aquatic animal viruses are respectively inoculated to the CAMB cells, as shown in figure 5, obvious cytopathic effect (CPE) appears in the cells, and RNA is extracted by collecting 200 mu L of each virus suspension on the 5 th day of inoculation, wherein, a 3 target band of FV is 531bp, a HSHRV band is 480bp, the detection of SVCV is a half-nested reaction, 714bp and 606bp fragments can be respectively detected by products of two PCR, a target band of gene I type GCRV is 661bp, and a band of Ti L V-2017A meshes is 700bp (figure 6).

According to the PCR and gel electrophoresis experiment results, target bands can be detected in cells inoculated with different viruses, which indicates that the channa argus brain cell line can proliferate the following viruses Ti L V, FV3, HSHRV, GCRV and SVCV, and provides a powerful material for research of aquatic animals.

Example 6 Acutionous maculatus brain cell line adhesion experiment to Aeromonas schubertii JW-I of snakehead origin

The snakehead source aeromonas schubertii contains flagella, flagellin participates in adsorption, invasion and colonization of bacteria, plays an important role in adhesion of adhesin to a host, and is one of important virulence factors of the bacteria.

Transferring cultured channa argus brain cells into a 6-hole cell culture plate (a cell slide is placed in advance), culturing for 24 hours at 28 ℃, washing for 3 times by using PBS after the cells grow into a single layer, respectively adding cell culture solution with the bacterial concentration of 1.0m L being 5x10 cfu/ml, simultaneously adding PBS into a blank group, enabling three groups to be parallel, incubating the cell line for 90 minutes at 28 ℃, washing for 3-4 times by using PBS, removing non-adhered bacteria, drying a cover glass, fixing for 5 minutes at room temperature (the volume ratio of fixing solution: methanol to glacial acetic acid is 3:1), naturally drying, dyeing by using Freund rapid cell staining solution, washing, sealing, randomly selecting 30 cells under an oil microscope, counting the bacteria adhered to each cell, and repeating the test for 3 times.

As shown in FIG. 7, the aeromonas schubertii JW-I from snakehead has strong adhesion to the brain cells of the snakehead, and the average number of adhered bacteria under the oil lens is 40 +/-2.

The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.

Claims

1. A channa argus brain cell line CAMB is preserved in a China center for type culture collection in 2018, 6 months and 13 days, wherein the preservation number is CCTCC NO: C2018125.

2. use of the channa argus brain cell line of claim 1 for culturing fish viruses; the virus is at least one of spring viremia of carp virus, channa argus rhabdovirus, frog iridovirus, genotype I grass carp reovirus and tilapia lake virus.