Method for transfecting symbiotic bacteria Wolbachia into lepidoptera insect cell line for culture

Abstract

The invention relates to the technical field of microbial cells, and discloses a method for transfecting a symbiotic bacterium Wolbachia into a lepidoptera insect cell line for culture, which comprises the following steps: collecting insect eggs infected with a Wolbachia host; culturing a lepidoptera insect cell line; adding eggs of the insect infected with the Wolbachia host into a centrifugal tube containing PBS (Phosphate Buffer Solution); washing the eggs; adding a cell culture medium into the centrifugal tube after the eggs are washed, and fully grinding the eggs by using a sterilization grinding rod to obtain an egg homogenate; according to the method, bacteria and insect cells are tightly combined together by utilizing weak centripetal force generated by low-speed centrifugation, and the bacteria are absorbed into the cells by combining the endocytosis and exocytosis characteristics of the cells, so that the cross-host transfection of the symbiotic bacteria is realized. The technology is of great significance to establishment of new insect strains infected with specific symbiotic bacteria.

Description

Technical Field

[0001] The invention relates to the technical field of microbial cells, in particular to a method for transfecting symbiotic bacteria Wolbachia into a lepidopteran insect cell line culture. Background Art

[0002] The symbiotic bacterium Wolbachia is widely distributed in insects and lives inside insect cells. It is an intracellular symbiotic bacterium.

[0003] Studies have shown that Wolbachia can regulate host reproduction by killing male insects infected with the bacteria (male killing), feminizing the host (feminization), inducing host parthenogenesis (parthenogenesis) and inducing host cytoplasmic incompatibility (CI). In particular, the cytoplasmic incompatibility induced by Wolbachia causes the fertilized eggs of cytoplasmic incompatibility offspring to die during embryonic development. Using the principle of cytoplasmic incompatibility to release male insects carrying specific Wolbachia strains to mate with female insects that are not infected with the bacteria in nature, causing the death of host offspring, can effectively reduce the target pest population. This technology has been successfully applied to the prevention and control of dipteran pests and mosquitoes.

[0004] The use of the above technology requires obtaining an insect population infected with Wolbachia. Currently, artificial transfection technology is usually used to inject Wolbachia into new insect species. However, due to the different compatibility of different insect species with Wolbachia, Wolbachia needs to be cultured in the cell line of the same type of host for a period of time, which helps the symbiotic bacteria adapt to the physiological environment of the new host and improves the success rate of Wolbachia cross-host transfection.

[0005] However, current technology cannot cultivate symbiotic bacteria in vitro, making it difficult for researchers to effectively understand the mechanism of interaction between Wolbachia and insects. Summary of the invention

[0006] The purpose of the present invention is to solve the problems existing in the prior art and to propose a method for transfecting symbiotic bacteria Wolbachia into a lepidopteran insect cell line culture.

[0007] In order to achieve the above object, the present invention adopts the following technical solutions:

[0008] A method for transfecting symbiotic bacteria Wolbachia into a lepidopteran insect cell line culture, comprising the following steps:

[0009] Collect eggs of Wolbachia-infected host insects;

[0010] Cultivation of lepidopteran insect cell lines;

[0011] Add eggs of Wolbachia -infected host insects to a centrifuge tube containing PBS;

[0012] Washing insect eggs;

[0013] Adding cell culture medium to the centrifuge tube after washing the eggs, and fully grinding the eggs with a sterilized grinding rod to obtain an egg homogenate;

[0014] The egg homogenate is added into a culture container of lepidopteran insect cells and then centrifuged. After centrifugation, the homogenate is transferred to a cell culture incubator for culture.

[0015] Wolbachia is an intracellular symbiotic bacterium that lives inside the egg. The present invention utilizes this characteristic to disinfect the surface of insect eggs, remove other microorganisms that may stick to the egg surface, and reduce contamination by miscellaneous bacteria. The weak centripetal force generated by low-speed centrifugation is utilized to tightly combine the bacteria and cells. Combined with the endocytosis and exocytosis characteristics of the cells, the cells can easily absorb the bacteria, thereby realizing cross-host transfection of the symbiotic bacteria.

[0016] Preferably, the specific process of washing insect eggs is:

[0017] Wash the eggs three times with 1× PBS for 30 seconds each time;

[0018] Wash the eggs three times with 0.5% sodium hypochlorite and 75% alcohol for 30 seconds each;

[0019] Rinse three times with 1× PBS for 30 seconds each.

[0020] Preferably, after washing the insect eggs, the insect eggs are stored in a -4°C temperature environment.

[0021] Preferably, the lepidopteran insect cell line is cultured in a T25 culture flask.

[0022] Preferably, the specific process of adding the egg homogenate into a culture container of lepidopteran insect cells and then centrifuging is:

[0023] Aspirate the old culture medium in the culture bottle and add new culture medium;

[0024] The lepidopteran insect cells were resuspended in the new culture medium by pipetting lightly and evenly distributed into six-well plates;

[0025] The ground egg homogenate was evenly distributed into six-well plates, numbered, and sealed with sealing film;

[0026] The six-well plate was centrifuged at 4°C and 350 g for 3 hours, and then transferred to a cell culture incubator for culture.

[0027] Preferably, the cell culture incubator is a constant temperature cell culture incubator, and the culture conditions in the constant temperature cell culture incubator are: 24-26°C.

[0028] Preferably, the lepidopteran insect is fall armyworm.

[0029] Preferably, the centrifuge tube is sterilized at high temperature when in use.

[0030] Preferably, the number of eggs infected with Wolbachia host insects added to the centrifuge tube containing PBS is not less than 500.

[0031] Preferably, the sealing film is a plastic sealing film.

[0032] Compared with the prior art, the present invention has the following beneficial effects:

[0033] The method of the present invention utilizes the weak centripetal force generated by low-speed centrifugation to tightly bind bacteria and insect cells together, and combines the endocytosis and exocytosis characteristics of cells to absorb bacteria into cells, thereby achieving cross-host transfection of symbiotic bacteria. This technology is of great significance for establishing new insect strains infected with specific symbiotic bacteria.

[0034] Compared with endosymbiotic bacteria cultured in vivo, the method of the invention is more convenient for observing the morphology and physiological response of bacteria.

[0035] The present invention establishes an in vitro culture system, which is convenient for enriching endosymbiotic bacteria and provides the possibility for the symbiotic bacteria to transfect other pests for biological control.

[0036] The present invention provides a technical means for efficiently and conveniently transfecting the symbiotic bacteria Wolbachia into lepidopteran insect cells, more effectively understands the mechanism of action between Wolbachia and insects, and lays a theoretical foundation for future biotechnology applications. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 This is the PCR detection result of Wolbachia in the F1 transfected cells in Example 1.

[0038] Figure 2 This is the PCR detection result of Wolbachia in F5 transfected cells in Example 1.

[0039] Figure 3 This is the PCR detection result of Wolbachia in F11 transfected cells in Example 1. DETAILED DESCRIPTION

[0040] The technical solutions in the embodiments of the present invention will be described clearly and completely below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all the embodiments.

[0041] Example 1

[0042] This example uses Wolbachia transfection technology to introduce the Wolbachia strain that infects the tomato leafminer into the fall armyworm cell line. The specific process is as follows:

[0043] Equipment and component preparation:

[0044] 1. Low-speed centrifuge: produces centripetal force to combine Wolbachia bacteria with insect cells.

[0045] 2. Biological safety cabinet: provides a sterile environment to avoid external contamination.

[0046] 3. Incubator: Provides suitable temperature and gas environment to maintain cell growth.

[0047] 4. Microscope: used to observe cell status and transfection effect.

[0048] 5. Syringes and microsyringes: Injecting Wolbachia bacteria into cells.

[0049] The specific cultivation steps are as follows:

[0050] (1) Collection of Wolbachia host insect eggs:

[0051] Add 1xPBS solution into a 1.5 ml centrifuge tube and pick 800 fresh eggs that are 100% infected with Wolbachia host insect eggs into the centrifuge tube using a brush or a picking needle.

[0052] (2) Preliminary washing and storage of Wolbachia host insect eggs:

[0053] Add 1xPBS solution to the centrifuge tube for washing;

[0054] The eggs were washed three times with 1× PBS for 30 seconds each;

[0055] Wash the eggs three times with 0.5% sodium hypochlorite and 75% alcohol for 30 seconds each;

[0056] Rinse with PBS three times, 30 seconds each time;

[0057] Store at -4℃;

[0058] Add 200 µl of Sf-900 III SFM cell culture medium (brand: Gibco; catalog number: 12658027) to the centrifuge tube containing the clean eggs and grind the eggs thoroughly with a sterilized grinding rod for 5 minutes.

[0059] (3) Adherent culture of Lepidoptera insect cell lines:

[0060] The Spodoptera frugiperda SF9 cell line was cultured in a suitable incubator or T25 cell, and the temperature (25°C) and CO2 concentration (5%) were controlled for pre-culture;

[0061] When using the Spodoptera frugiperda SF9 cell line, aspirate the old culture medium and add 6 ml of new culture medium;

[0062] Resuspend the SF9 cells in the new culture medium by gently pipetting and evenly distribute them into six-well plates;

[0063] The ground egg homogenate was evenly distributed into six-well plates, numbered, and sealed with sealing film;

[0064] The six-well plate was centrifuged at 4°C and 350 g for 3 h, and then transferred to a cell culture incubator for culture;

[0065] After culturing for 2 to 3 days, transfer the cells in the six-well plate to a new T25 culture flask or incubator to promote the adaptation and reproduction of bacteria in the cells.

[0066] Compared with the prior art, the present invention utilizes the cytoplasmic incompatibility phenomenon caused by Wolbachia and releases male insects carrying specific Wolbachia strains to mate with female insects that are not infected with the bacteria in nature, which can significantly reduce the population density of target pests, thereby providing an efficient biological control method for agricultural pest management.

[0067] By growing Wolbachia in cell lines of the same type of host, the symbionts can adapt to the physiological environment of the new host. This process can increase the success rate of transfecting Wolbachia into new insect populations and ensure the feasibility and practicality of the technology.

[0068] In the technical implementation, by disinfecting the surface of insect eggs, potential microbial contamination can be effectively removed, the purity of Wolbachia during the transfection process can be ensured, and the reliability of the experiment can be improved.

[0069] This technical method combines low-speed centrifugation with the endocytosis and exocytosis characteristics of cells, making the combination between bacteria and cells tighter, promoting the cross-host transfection of Wolbachia, and thus expanding the application potential of this technology in different insect species.

[0070] Then use a microscope to observe the state of the cells to confirm the success rate of transfection, and evaluate the impact of transfection on the cells by observing the growth and division of the cells.

[0071] And 100 μl of culture medium can be taken to extract DNA, and Wolbachia-specific wsp (Wolbachia surface protein) primers can be used for PCR detection to detect the transfection effect of the symbiotic bacterium Wolbachia.

[0072] The extraction process of transfected cell DNA is as follows:

[0073] 100ul of the cell fluid after 3 days of culture was taken out, and the supernatant was removed after simple centrifugation. 30ul STE buffer + 2ul proteinase K were added, and the mixture was incubated at 56℃ for 2h in a PCR instrument, and then inactivated at 95℃ for 7min to obtain a crude extract of transfected cell DNA.

[0074] PCR detection of transfected cells:

[0075] The transfected cells were tested for Wolbachia infection by PCR. The transfected cell DNA was used as a template and WSP-FP and WSP-RP were used as primers to amplify the Wolbachia sp gene. The sequence of WSP-FP was 5'-TGGTCCAATAAGTGATGAAGAAAC-3', and the sequence of WSP-RP was 5'-AAAAATTAAACGCTACTCCA-3'. The PCR program was set to activate at 94°C for 5 min, followed by 35 cycles of 98°C for 30 s, 55°C for 30 s, and 72°C for 1 min, and then extended at 72°C for 7 min. The PCR products were subjected to gel electrophoresis at 150 V for 20 min, and the bands were observed in a UV gel imager.

[0076] The results are as follows Figure 1 , Figure 2 , Figure 3 As shown, Wolbachia was still stably detected in F1, F5, and F11 cells. Wolbachia could be artificially transfected into lepidopteran insect cells with an infection rate of 100%.

[0077] The above description is only a preferred specific implementation manner of the present invention, but the protection scope of the present invention is not limited thereto. Any technician familiar with the technical field can make equivalent replacements or changes according to the technical scheme and inventive concept of the present invention within the technical scope disclosed by the present invention, which should be covered by the protection scope of the present invention.

Claims

1. A method for transfecting symbiotic bacteria Wolbachia into a lepidopteran insect cell line culture, characterized in that: The following steps are involved: Collect eggs of Wolbachia-infected host insects; Cultivation of lepidopteran insect cell lines; Add eggs of Wolbachia -infected host insects to a centrifuge tube containing PBS; Washing insect eggs; Adding cell culture medium to the centrifuge tube after washing the eggs, and fully grinding the eggs with a sterilized grinding rod to obtain an egg homogenate; The egg homogenate is added into a culture container of lepidopteran insect cells and then centrifuged. After centrifugation, the homogenate is transferred to a cell culture incubator for culture.

2. A method for transfecting the symbiotic bacterium Wolbachia into a lepidopteran insect cell line culture according to claim 1, characterized in that: The specific process of washing worm eggs is as follows: Wash the eggs three times with 1× PBS for 30 seconds each time; Wash the eggs three times with 0.5% sodium hypochlorite and 75% alcohol for 30 seconds each; Rinse three times with 1× PBS for 30 seconds each.

3. A method for transfecting the symbiotic bacterium Wolbachia into a Lepidoptera insect cell line culture according to claim 2, characterized in that: After the eggs are washed, they are stored in a -4°C environment.

4. A method for transfecting symbiotic bacteria Wolbachia into lepidopteran insect cell line culture according to claim 2, characterized in that: The lepidopteran insect cell line was cultured in a T25 culture flask.

5. A method for transfecting symbiotic bacteria Wolbachia into Lepidoptera insect cell line culture according to claim 1, characterized in that: The specific process of adding the egg homogenate into the culture container of the lepidopteran insect cells and then centrifuging is as follows: Aspirate the old culture medium in the culture bottle and add new culture medium; The lepidopteran insect cells were resuspended in the new culture medium by pipetting lightly and evenly distributed into six-well plates; The ground egg homogenate was evenly distributed into six-well plates, numbered, and sealed with sealing film; The six-well plate was centrifuged at 4°C and 350 g for 3 hours, and then transferred to a cell culture incubator for culture.

6. A method for transfecting symbiotic bacteria Wolbachia into Lepidoptera insect cell line culture according to claim 5, characterized in that: The cell culture box adopts a constant temperature cell culture box, and the culture condition in the constant temperature cell culture box is: 24-26°C.

7. A method for transfecting symbiotic bacteria Wolbachia into Lepidoptera insect cell line culture according to claim 1, characterized in that: The lepidopteran insect is fall armyworm.

8. A method for transfecting symbiotic bacteria Wolbachia into a lepidopteran insect cell line culture according to claim 1, characterized in that: When the centrifuge tube is in use, it is also subjected to high temperature sterilization.

9. A method for transfecting symbiotic bacteria Wolbachia into Lepidoptera insect cell line culture according to claim 1, characterized in that: The number of eggs of the Wolbachia host insect infected with the virus added to the centrifuge tube containing PBS is not less than 500.

10. A method for transfecting symbiotic bacteria Wolbachia into Lepidoptera insect cell line culture according to claim 5, characterized in that: The sealing film is a plastic sealing film.

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