TWI417387B - S. exigua cell line, protein expression system containing the same, and method for virus incubation by using the same - Google Patents

Description

Spodoptera exigua cell line, protein expression system containing the same, and method for cultivating virus using the same

The invention relates to a beet armyworm cell line and an application thereof, in particular to a beet armyworm cell strain with high sensitivity to nuclear polyhedrosis virus and microsporidia and application thereof.

Spodoptera exigua belongs to the family Lepidoptera, and it feeds on leaves, flowers and tender stems, which seriously affects the growth of crops. In Taiwan, beet armyworm is a pest of vegetable crops, causing growing agricultural losses and has become a key epidemic prevention target.

Conventional control methods are mainly carried out by spraying chemicals to kill the larvae of beet armyworm. However, chemical agents are not only prone to residue, but also cause food safety problems, and are more likely to cause irreparable damage to the environmental soil. At the same time, the widespread use of toxic chemicals also has doubts about the ecological balance. According to this, there is indeed room for improvement in the conventional control methods.

A relatively new approach in the field is the biological control method, which controls the number of natural enemies of beet armyworm. Nuclear polyhedrosis virus (NPV) and microsporidia are common pathogenic microorganisms that infect beet armyworm, and are therefore a viable option for biological control.

The nuclear polyhedrosis virus is a baculovirus that, after infection of the host, carries out two different infection patterns. First, the budding virus (BV) is propagated by budding to cause infection between cells in the individual; secondly, multiple occlusion bodies (OB) are formed in the nucleus of the host cell, and the embedding body is formed. It contains viral particles and is highly tolerant to the environment. When the infected individual dies, the embedded body will be released from the dead individual, and the expansion will cause infection between the groups.

Microsporidiosis is a common epidemic of insects, and its main pathogen is Microsporidia of the genus Nosema . Microsporidia are infected and proliferated in two types of merionts and spores, which have a great influence on the growth and decline of insect populations.

The fundamental element of the biological control method for the epidemic prevention of beet armyworm is to obtain a large number of pathogenic microorganisms, such as the nuclear polyhedrosis virus or microsporidia mentioned above. Taking nuclear polyhedrosis virus as an example, since the life form of the virus must be propagated in living cells, it is customary to artificially capture the infected insects in the field and then separate the desired virus from the body. In order to be able to multiply nuclear polyhedrosis virus, it is possible to adopt a large number of breeding of Spodoptera exigua for the nuclear polyhedrosis virus required for mass reproduction. However, such a living reproduction method requires not only a large amount of manpower, but also many details are difficult to break through at the current technical level.

In summary, in order to use the biological control method to carry out epidemic prevention of beet armyworm, it is necessary to improve the way in which a large number of pathogenic microorganisms are obtained.

Accordingly, it is an object of the present invention to provide a Spodoptera exigua cell line which is highly susceptible to pathogenic microorganisms infected with Spodoptera exigua, and thus can be used for large-scale cultivation of the pathogenic microorganism in vitro.

Still another object of the present invention is to provide a method for culturing nuclear polyhedrosis virus in vitro, which achieves the purpose of mass culture of nuclear polyhedrosis virus in vitro by the high sensitivity of the cell strain of the present invention to nuclear polyhedrosis virus.

It is still another object of the present invention to provide a protein expression system which utilizes the specificity of the cell line of the present invention and nuclear polyhedrosis virus to improve the efficiency of transfection.

For the purpose of the present invention, the present invention provides a cell strain of Spodoptera exigua, which is deposited under the registration number of the Republic of China Food Industry Development Institute: BCRC 960429.

The present invention further provides a method of culturing a nuclear polyhedrosis virus, which is characterized in that the nuclear polyhedrosis virus is infected with the aforementioned cell strain.

The present invention further provides a protein expression system comprising: the aforementioned cell strain; and a viral vector.

Preferably, the aforementioned cell line is established from the carcass of Spodoptera exigua.

Preferably, the aforementioned cell strain is highly susceptible to nuclear polyhedrosis virus.

Preferably, the nuclear polyhedrosis virus is Spodoptera exigua NPV (SpeiNPV) or Autographa californica nucleopolyhedrovirus TWN4 (AcMNPV-TWN4).

Preferably, the cell strain has a sensitivity to nuclear polyhedrosis virus of 80 to 100%.

Preferably, after the aforementioned cell strain is infected with the aforementioned nuclear polyhedrosis virus, an average of 30 to 100 embedding bodies per cell are produced.

Preferably, the cell strain has a sensitivity to microsporidia of 80 to 100%.

Preferably, the aforementioned microsporidia are Bombyx mori.

Preferably, the inner transcribed spacer of the aforementioned cell line has the sequence of SEQ ID NO:01.

Preferably, the aforementioned viral vector carries the nucleic acid sequence of the protein to be expressed.

In summary, the present invention provides a Spodoptera exigua cell strain which is highly sensitive to nuclear polyhedrosis virus and microsporozoites, and thus is particularly suitable for the cultivation of such pathogenic microorganisms in vitro for use in pest control of agriculture. In addition, the cell strain of the present invention and the nuclear polyhedrosis virus are highly specific, and the above cell strain can be applied to protein expression to improve the efficiency of transfection.

A protein expression system constructed using the specificity of baculovirus and insect cell strains is considered to be an extremely efficient genetic engineering tool. Such protein expression systems typically use a polyhedrin promoter or a p10 promoter to express a foreign gene. Both promoters are highly efficient, so different types of foreign proteins can be produced quickly and in large quantities. Although the protein expression system of baculovirus-insect cell lines is known to have high potential, scientists are still working to develop more specific or susceptible cell lines to create more efficient expression systems.

The term "cell strain" as used in the present invention is as defined in the art; in short, a cell line refers to a single cell that is stably and continuously subcultured in a laboratory environment by manual isolation, transformation, and cultivation. Attribute cells. The present invention relates to a novel cell line isolated from the corpus callosum of Spodoptera exigua. Although other beet armyworm cell lines have existed in the field, they have never been isolated from carcass tissues. The cell strain of the present invention is highly sensitive to nuclear polyhedrosis virus, so that the cell strain of the present invention is particularly suitable for the propagation of nuclear polyhedrosis virus in vitro, and the cell strain of the present invention can be matched with a nuclear polyhedrosis virus vector as an effective protein. Performance system.

The following examples are intended to illustrate the details and spirit of the invention, but are not intended to limit the scope of the invention.

Example 1: Establishment of a cell strain of the present invention

[Experimental Materials]

The beet armyworm used in the present invention is provided by the Taiwan Provincial Drug Toxicology Laboratory or collected from the field, and is cultivated at room temperature by artificial feed according to the formula of the scientist Hill Light Man.

The culture solution used in the present invention is a TNM-FH culture solution, and the preparation method thereof is briefly described as follows: 228.5 g of Grace's insect cell culture medium (GIBCO ^TM ), 15 g of yeast extract (Bacto ^TM TC Yeastolate, Becton, Dickinson and Company), respectively. And 15 grams of milk protein hydrolysate (Lactalbumin Enzymatic Hydrolysate, ) 4740 ml of deionized water was added to form a mixed solution. After stirring uniformly, 1.75 g of sodium hydrogencarbonate (NaHCO ₃ ) was added, and the pH of the mixture was adjusted to 6.3 with 10% sodium hydroxide (NaOH), and then deionized water was added to 5000 ml. Then with a filter (Gelman Laboratory; 90Filter unit 0.2 μm) filtered. Finally, the culture solution was stored at 4 ° C for storage.

[primary cell culture]

The larvae of the beet armyworm, which were harvested, were incubated with artificial diet until they were phlegm, and worms were collected for 2 to 4 days. The carcass is first wiped with 10% sodium hypochlorite solution (Clorox solution) and 70% iodine. After air drying, the carcass is cut with ophthalmology. Next, the internal tissues of the corpus callosum were collected by a micropipette into a 25T culture flask pre-filled with 2 ml of TNM-FH culture solution, and subjected to primary culture at 28 °C. Then, the type change of the cells was observed every two days. TNM-FH medium as used herein is added 100 IU / ml penicillin ^{(penicillin, Gibco TM), 100} mg / ml Streptomycin ^{(Streptomycin, Gibco TM), 1.25} mg / ml antimycotic agents (amphotericin B, And 16% of fetal calf serum (FBS, Hyclone) with complement activity.

[subculture]

After one month of cultivation in the first generation, the first subculture was carried out. First, tap the bottom of the plate to detach the attached cells. Take 2 ml of cell suspension and transfer to a clean 25T flask. Subsequently, the addition of fresh culture dishes in TNM-FH medium, which is supplemented with 50 IU / ml penicillin (penicillin, Gibco ^TM), streptomycin 50 mg / ml of ^{(Streptomycin, Gibco TM), 1.25} mg / ml Antifungal agent (amphotericin B, And 16% of fetal calf serum (FBS, Hyclone) with complement activity, and the cells were cultured at 28 °C.

Subculture was carried out every 14 days, and after the cells were stably propagated, they were subcultured every 4 to 7 days. After subculture for 25 times, the cells were cultured in TNM-FH medium containing 8% fetal bovine serum, and the subculture interval was changed to once every 5 days. Most of the cells cannot be subcultured more than 50 times, and those who have been subcultured for 50 to 80 times are the cell lines of the present invention; some of them can be successively subcultured for one year. Here, the cell line of the present invention is named NTU-SE, and its registration number is BCRC 960429 in the Republic of China Food Industry Development Institute.

Example 2: Cell type observation and growth curve measurement of the cell strain of the present invention [Cell type observation]

The type of the cell line (NTU-SE) of the present invention was observed by a phase contrast microscope (Olympus IX-71). Please refer to the first figure. In the laboratory culture, NTU-SE cells may have different types such as round, polymorphic, spindle-shaped, comma, etc. state. As shown in Table 1 below, this example calculates the number of cells of each type and measures the average size of the cells. As can be seen from the results in the table, NTU-SE cells mostly showed the morphology of round cells or multi-spine cells, while the type of comma cells was the least.

[Growth curve]

The NTU-SE cells of the present invention were cultured at 28 ° C in a TNM-FH medium containing 8% fetal bovine serum, and the number of cells was recorded every hour to obtain a growth curve of NTU-SE cells, such as Shown in the second picture. Based on the rate of cell proliferation in the exponential growth phase in this growth curve, it is further estimated that the doubling time of the NTU-SE cells is about 35.5 hours.

Example 3: Isomerase map and DNA marker of the cell strain of the present invention [Iso Isomerase Map]

Isoisomerase refers to an enzyme that has different molecular weights in different species but exhibits the same enzyme activity. In other words, these enzymes have the same specificity substrate, but their molecular forms are not the same. The production of isomeric isomerases may be due to genetic mutations in the evolution process. These mutations, although not affecting the original activity of the enzyme, change their structural properties and are distinguished. The isotropy map can be used to know the similarities and differences between the species to be identified and the comparative species, and the relationship between them.

This experiment was carried out according to a conventional experimental method in the field. Briefly, a cell suspension of 1.5 × 10 ⁸ cells/ml was taken and repeatedly placed in a liquid nitrogen and a 37 ° C water bath several times to make The cells burst. Subsequently, it was centrifuged at 4 ° C for 10 minutes (90 g; KUBOTA 1300) to obtain a supernatant containing a soluble protein. The supernatant was mixed with 10 μl of 1% Bromophenol blue (BPB), placed in a 10% polypropylene guanamine gel, and electrophoresed at a voltage of 50 volts for 5 hours. After electrophoresis, the film was immersed in a substrate containing three enzymes: esterase, lactate dehydrogenase (LDH), and malate dehydrogenase (MDH) for staining.

Please refer to the third figure, which is a comparison of isoforms of NTU-SE cells (SE) of the present invention with other insect cell strains; wherein Sf9 represents a S. frugiperda cell line and MV represents a pod. The Maruca vitrata cell line, YB represents the yellow butterfly cell line, and LY represents the Lymantria xylina cell line. As can be seen from the figure, the isoform isomerase pattern of the NTU-SE cells of the present invention is not the same as that of the above four insect cell lines, and is clearly consistent with the prior knowledge in the field, and the isomerase map can be made. A basis for identifying NTU-SE cells of the invention.

[DNA Mark]

The internal transcribed spacers region (ITS region) is a highly variable DNA sequence in evolution, and is therefore often used for the identification of species identification and evolution. In this experiment, the ribosomal DNA internal transcribed spacer segment of the NTU-SE cell line of the present invention is propagated by polymerase chain reaction (PCR) according to a conventional step in the field, and nucleic acid sequencing is performed to obtain NTU- The ITS sequence of the SE cell line. The ITS sequence of the NTU-SE cell line of the present invention is shown in SEQ ID NO: 1, which can also be used as a basis for identifying NTU-SE cells of the present invention.

Example 4: Infection test of pathogenic microorganisms of the cell strain of the present invention

In the present example, the NTU-SE cell line, the autumn army insect cell line (Sf9), the eucalyptus urophyllin cell line (NTU-PN-HH), and the gypsy moth cell strain (IPLB-LD652Y) of the present invention will be tested. ), black horned butterfly cell line (NTU-LY), pea pod cell line (NTU-MV), silkworm cell line (BmN) and yellow butterfly cell line (NTU-YB) for nuclear polyhedrosis virus or microsporidia sensitivity. The aforementioned Sf9 cell line is a commercially available strain; the IPLB-LD652Y cell line is provided by Professor Dr. MJ Fraser of the University of Notre Dame, USA; the BmN cell line is provided by Dr. Zhao Yuzhan of the Institute of Biochemistry, Academia Sinica; the remaining cell lines are all in the laboratory. set up. All cell lines were maintained in TNM-FH medium containing 8% fetal calf serum, and cultured at 28 ° C, subcultured every 3 to 4 days.

The nuclear polyhedrosis virus used in the present embodiment includes: Spodoptera nuclear polyhedrosis virus (SpeiNPV-1), California genus Cryptocaryon nucleopolyhedrovirus (AcMNPV-TWN4), Eucalyptus urophylla nuclear polyhedrosis virus (PenuNPV), black The genus Lyxy MNPV, the silkworm nuclear polyhedrosis virus (BmNPV), the pea nucleus polyhedrosis virus (MaviNPV); the microsporidian strain Nosema Bombysis EB isolate. The aforementioned BmNPV system was provided by Dr. Zhao Yuzhan of the Institute of Biochemistry of the Academia Sinica. The other pathogenic microorganisms used in this example were isolated from the rickets of the laboratory in the wild (Taiwan Island).

The experimental steps are briefly described as follows: First, 1.2×10 ⁶ cells/flask of the cells to be infected are planted in a 25T culture flask, and after the cells are attached, an appropriate amount of virus solution (MOI=1) or 1 ml of microsporidia is added respectively. Inoculate the solution and shake it for one hour on a horizontal shaker. Thereafter, the virus solution or the microsporidia inoculum was removed, and the cells were transferred to TNM-HF medium (8% fetal bovine serum) and cultured at 28 °C. Then, observe the cytopathic condition every day. In the virus infection experiment, continuous observation for 14 days; in the microsporidia infection experiment, continuous observation for 30 days.

The results of the experiment are shown in Table 2 below. In the table, "+" indicates high sensitivity; "-" indicates low sensitivity or no obvious sensitivity.

It can be seen from the experimental results that the NTU-SE cell line of the present invention has high sensitivity to the beet armyworm nuclear polyhedrosis virus, the genus Helicoverpa armigera nuclear polyhedrosis virus and the silkworm microsporidia yellow butterfly isolate. In contrast, the NTU-SE cell line showed no significant sensitivity to the eucalyptus urophyllin nuclear polyhedrosis virus, the hornbeam moth nuclear polyhedrosis virus, the silkworm nuclear polyhedrosis virus, and the pod scorpion nuclear polyhedrosis virus.

Referring again to the fourth panel, it is shown that the NTU-SE cell line of the present invention is infected with Spodoptera exigua nuclear polyhedrosis virus (A) for 5 days and (B) for 10 days. After 5 days of infection, approximately 98% of the cells developed viral inclusions (as indicated by the arrows) in the nucleus due to viral infection, indicating the high sensitivity of the NTU-SE of the present invention to Spodoptera exigua nuclear polyhedrosis virus. After 10 days of infection, it was observed that most of the cells broke due to infection, and the embedded body was released into the culture solution. Here, the cells in the culture flask are further scraped off, poured into a centrifuge tube together with the culture solution, and uniformly shaken with an oscillator. Then, an average of 90 virus-embedded bodies per cell were calculated by a hemocytometer (the experimental data obtained after repeating the experiment to obtain the least amount of the embedded body is about 33 virus-embedded bodies per cell), meaning that the NTU- of the present invention SE cell strains have great potential as a tool for the commercial production of viral implants.

On the other hand, the fifth graph shows the case where the NTU-SE cell strain of the present invention was infected with the microsporidia spp. The mature spores in the photo are reflective and have a long oval shape. From the fifth figure, it can be observed that a large number of mature spores appear in the cytoplasm of almost all NTU-SE cells, and it is unexpected that the cell line of the present invention is also highly sensitive to the microspore isolate of the silkworm.

Example 5: Protein Expression System of the Invention

In the present embodiment, a Vibrio californica nuclear polyhedrosis virus vector (AcMNPV) carrying red fluorescent protein or green fluorescent protein is transfected into the NTU-SE cells of the present invention to observe the present invention. The efficiency of the protein expression system established by NTU-SE cells. The Sf9 cell strain is a cell strain which has been commercialized and is known to have high sensitivity to the Cryptosporidium parvum nuclear polyhedrosis virus vector, and thus was selected as a control group of the present example.

The experimental steps are briefly described below. First, the Brassica californica nuclear polyhedrosis virus vector carrying red fluorescent protein (DsRed) or green fluorescent protein (eGFP) was genetically engineered. Then, 1.2 × 10 ⁶ cells/flask of the cells to be infected were seeded in a 25T culture flask, and after the cells were attached, an appropriate amount of the suspension containing the aforementioned viral vector (MOI = 1) was separately added. After shaking for one hour on a horizontal shaker, the virus solution was removed, and the cells were transferred to TNM-HF medium (8% fetal bovine serum) and cultured at 28 °C. Five days later, protein performance was observed under a fluorescence microscope and a phase contrast microscope.

The experimental results are shown in Figure 6, where (c) and (f) are phase-contrast microscopic fields indicating the relative positions of the cells; (a), (b), (d) and (e) are the fluorescence microscope fields. , showing the status of fluorescent protein expression (DsRed and eGFP exposure time are 0.1 second, the scale is 100 μm in the figure). As can be seen from the results in the figure, the ability of the NTU-SE cells of the present invention as a protein expression system is inferior to that of the conventional commercial Sf9 cell line.

Example 6: Comparison of the NTU-SE cell strain of the present invention with the conventional Spodoptera exigua cell line

In the present example, the characteristics of the cell line of the present invention and the conventional Spodoptera exigua cell line in the field will be compared to understand the novel and progressive characteristics of the cell line of the present invention. The comparison is shown in Table 3 below. It should be noted that the values listed in the table for the cell line of the present invention such as the sensitivity and the yield of the embedded body are based on the experimental data obtained in the examples exemplified in the specification, and do not represent the present. The absolute value of the characteristic of the cell strain of the invention.

As can be seen from the table, only IOZCAS-Spex-II-A and Se301 have similar susceptibility to the cell strain of the present invention, and most of the beet armyworm cell lines in the field are less sensitive to nuclear polyhedrosis virus than the cells of the present invention. The strain is excellent. However, IOZCAS-Spex-II-A and Se301 are not derived from corpus callosum, and it is apparent that these two cell lines are not identical to the cell line of the present invention. On the other hand, the ability of the cell line of the present invention to produce an embedded body is also excellent. By comprehensively reviewing these characteristics, it can be understood that the cell line of the present invention is not identical to the conventional beet armyworm cell line, and has outstanding advantages in industrial utilization.

<110> National Taiwan University

<120> Spodoptera exigua cell line, protein expression system containing the same, and method for cultivating virus using the same

<130>

<160> 1

<170> PatentIn version 3.5

<210> 1

<211> 1408

<212> DNA

<213> Spodoptera exigua

<400> 1

The first figure shows the type of the cell strain of the present invention;

The second graph is the growth curve of the cell strain of the present invention;

The third panel is an isomerase map of the cell line of the present invention and other conventional insect cell strains; wherein (A) esterase; (B) lactate dehydrogenase; (C) malate dehydrogenase;

The fourth panel shows that the cell strain of the present invention receives the infection of Spodoptera exigua nuclear polyhedrosis virus (A) for 5 days and (B) for 10 days;

The fifth figure shows the condition of the cell line of the present invention after being infected with the microsporidia isolate of the silkworm, 21 days; and

Figure 6 shows the efficiency of the protein expression system of the present invention; (a) fluorescence microscopy field, NTU-SE cells, red fluorescent protein; (b) fluorescence microscopy field, NTU-SE cells, green fluorescent protein; c) phase contrast microscopy field, NTU-SE cells; (d) fluorescence microscopy field, Sf9 cells, red fluorescent protein; (e) fluorescence microscopy field, Sf9 cells, green fluorescent protein; (f) phase contrast microscope Field of view, Sf9 cells.

Claims (12)

A beet armyworm cell line, which is registered in the Republic of China Food Industry Development Institute: BCRC 960429. The beet armyworm cell strain described in claim 1 is established from the carcass of beet armyworm. For example, the beet armyworm cell strain described in claim 1 is 80-100% sensitive to nuclear polyhedrosis virus; the nuclear polyhedrosis virus is a Spodoptera exigua nuclear polyhedrosis virus or a California genus Body virus. The Spodoptera exigua cell line described in claim 3, which is infected by the aforementioned nuclear polyhedrosis virus, produces an average of 30 to 100 embedding bodies per cell. The Spodoptera exigua cell line according to claim 1, which is susceptible to microsporidia. The Spodoptera exigua cell line according to claim 5, wherein the microsporidian strain is a silkworm microsporidia. The Spodoptera exigua cell line according to claim 1, wherein the inner transcribed spacer has the sequence of SEQ ID NO: 01. A method for culturing a nuclear polyhedrosis virus, which causes a nuclear polyhedrosis virus to infect a cell strain according to claim 1; the nuclear polyhedrosis virus is a Spodoptera exigua nuclear polyhedrosis virus or a California genus Polyhedrosis virus. The method of claim 8, wherein the cell strain has a sensitivity to nuclear polyhedrosis virus of 80 to 100%. The method of claim 8, wherein the cell line is infected by the nuclear polyhedrosis virus, and an average of 30 to 100 cells per cell are produced. An embedded body. A protein expression system comprising: the cell strain according to claim 1; and a viral vector; wherein the virus is a nuclear polyhedrosis virus, and the nuclear polyhedrosis virus is a Spodoptera exigua nuclear polyhedrosis virus or California Helminthospora nuclear polyhedrosis virus. The protein expression system of claim 11, wherein the viral vector carries a nucleic acid sequence of a protein to be expressed.