A high-yield baculovirus of the yponomeuta evonymellus pupa ovary cell line and its construction method and use
Patent Information
- Application Number
- CN202610746946.5
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2026-05-28
- Publication Date
- 2026-08-21
AI Technical Summary
到目前为止，Cellosaurus数据库（https://web.expasy.org/cellosaurus）没有记录原始建立的来自柚木驼蛾Hyblaea puera的细胞系，目前还没有对柚木驼蛾核型多角体病毒（Hyblaea puera Nucleopolyhedrovirus，HypuNPV）高度敏感的柚木驼蛾卵巢来源细胞系的报道
本发明成功构建了一种高产杆状病毒的柚木驼蛾蛹卵巢细胞系（CGMCCNo.46769），该细胞系以卵巢组织为原料构建，形态均一、贴壁性能优良，传代30代以上仍保持遗传稳定性，群体倍增时间为31.65小时，可长期冻存与高效复苏，满足工业化连续培养需求。经DAF‑PCR鉴定，确证该细胞系来源于柚木驼蛾，无外源细胞污染，种属特异性明确。该细胞系对杆状病毒，尤其是柚木驼蛾核型多角体病毒（HypuNPV），高度敏感。
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Abstract
Description
Technical Field
[0001] This invention relates to the field of biotechnology, and in particular to a high-yield baculovirus-producing ovarian cell line of the teak camel moth pupa, its construction method, and its uses. Background Technology
[0002] It has been reported that since the successful establishment of the first insect cell line in 1965, more than 1,000 insect cell lines have been established in the past 60 years. These cell lines originate from insects of various orders, including Lepidoptera, Diptera, Homoptera, Hymenoptera, Orthoptera, and Coleoptera, with the majority originating from Lepidoptera and Diptera. Insect cell lines have long been important tools in scientific research, including physiology, developmental biology, cell biology, molecular biology, and biochemistry. Furthermore, insect cells serve as a crucial component of baculovirus expression vector systems, expressing a large number of exogenous proteins of significant economic and scientific importance. Simultaneously, insect cells play a vital role as bioreactors in amplifying insect baculoviruses for use as biological insecticides, particularly in amplifying recombinant baculovirus insecticides containing exogenous genes.
[0003] Lepidoptera cell lines are derived from many tissues, including ovaries, embryos, hemocytes, and fat bodies. Cell lines derived from different insect species, or from different tissue sites of the same insect, exhibit varying expression capabilities for amplified viruses or recombinant proteins. Insect ovaries are usually paired and serve as the site of oogenesis and development. Since Grace established the first insect cell line using ovarian tissue, the ovary has remained a crucial tissue source for cell line establishment. Therefore, establishing and screening new and more baculovirus-sensitive cell lines (strains) is a necessary and theoretically and practically significant endeavor.
[0004] Teak camel moth Hyblaea puera (Cramer, 1777) is a lepidopteran pest that has caused serious damage to mangroves in recent years. To date, the Cellosaurus database (https: / / web.expasy.org / cellosaurus) does not contain any original records of the original strain from the teak camel moth. Hyblaea puera Currently, there are no reports of teak moth ovary-derived cell lines that are highly susceptible to teak moth nucleopolyhedrovirus (HypuNPV). Summary of the Invention
[0005] The purpose of this invention is to provide a high-yield baculovirus-producing ovarian cell line of the teak moth pupa, its construction method, and its applications, thereby addressing the problems existing in the prior art. The high-yield baculovirus-producing ovarian cell line of the teak moth pupa provided by this invention can serve as a highly efficient host system for the large-scale preparation of baculovirus insecticides, research on viral pathogenic mechanisms, and recombinant protein expression, providing crucial material support for the biological control of the teak moth and the application of insect biotechnology.
[0006] To achieve the above objectives, the present invention provides the following solution: This invention provides a high-yield baculovirus-producing ovarian cell line of the teak camel moth pupa, which was deposited on December 18, 2025, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 46769.
[0007] This invention also provides a method for constructing a high-yield baculovirus-producing ovarian cell line from the pupa of the teak camel moth, comprising the following steps: After surface disinfection of the teak camel moth pupa, the ovarian tissue was removed, cleaned, and cultured in cell culture medium. After passage, the teak camel moth pupa ovarian cell line was obtained.
[0008] Furthermore, the surface of the teak camel moth pupa was disinfected using a 75% (v / v) ethanol aqueous solution.
[0009] Furthermore, the cleaning process includes: first washing with physiological saline or PBS buffer, and then washing with cell culture medium.
[0010] Furthermore, the cell culture medium is Insect-XPRESS containing antibiotics and fetal bovine serum. TM Culture medium.
[0011] Furthermore, the antibiotics are penicillin and streptomycin.
[0012] Furthermore, the content of penicillin in the cell culture medium is 25-500 U / mL; the content of streptomycin in the cell culture medium is 25-500 μg / mL; the volume content of fetal bovine serum in the cell culture medium is 5-20%; and the pH of the cell culture medium is 5.9-6.9.
[0013] Preferably, the penicillin content in the cell culture medium is 100 U / mL; the streptomycin content in the cell culture medium is 100 μg / mL; and the fetal bovine serum content in the cell culture medium is 15% by volume.
[0014] The present invention also provides the application of the above-mentioned teak camel moth pupa ovary cell line in the production of baculovirus.
[0015] Furthermore, the baculovirus is a nucleopolyhedrovirus of the teak camel moth.
[0016] The present invention discloses the following technical effects: This invention successfully constructed a high-yield baculovirus-producing ovarian cell line from the pupa of the teak moth (CGMCC No. 46769). Constructed from ovarian tissue, this cell line exhibits uniform morphology, excellent adhesion, and maintains genetic stability even after more than 30 passages. Its population doubling time is 31.65 hours, allowing for long-term cryopreservation and efficient thawing, meeting the requirements for industrial continuous culture. DAF-PCR identification confirmed that the cell line originates from the teak moth, is free from exogenous cell contamination, and demonstrates clear species specificity. This cell line is highly sensitive to baculoviruses, especially teak moth nucleopolyhedrovirus (HypuNPV).
[0017] The high-yield baculovirus-producing ovarian cell line of the teak camel moth pupae provided by this invention can serve as an efficient host system for the large-scale preparation of baculovirus insecticides, research on viral pathogenic mechanisms, and recombinant protein expression, providing key material support for the biological control of the teak camel moth and the application of insect biotechnology. Attached Figure Description
[0018] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.
[0019] Figure 1 This is a microscopic observation of the adherent ovarian tissue of the teak camel moth pupa during primary culture; the scale bar is 100 μm. Figure 2 Cell morphology of Hp-1, the ovarian cell line of the teak camel moth pupa; scale bar is 50 μm. Figure 3 Growth curve of Hp-1, the ovarian cell line of the teak camel moth pupa; Figure 4 The image shows the DAF-PCR comparison of the ovary cell line Hp-1 of the teak camel moth pupa, the ovary of the teak camel moth pupa, the commercial sf9 cell line, and the rice leaf folder; where M is the marker; 1-4 are the PCR amplification products of the DNA of the ovary cell line Hp-1 of the teak camel moth pupa, the ovary of the teak camel moth pupa, the commercial sf9 cell line, and the rice leaf folder, respectively. Figure 5Microscopic observation of the Hp-1 cell line of the pupa of the teak camel moth after infection with nucleopolyhedrovirus (HypuNPV). Detailed Implementation
[0020] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.
[0021] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.
[0022] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.
[0023] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.
[0024] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.
[0025] A high-yield baculovirus-producing ovarian cell line from the pupa of the teak camel moth can be constructed using insect cell culture medium containing antibiotics and fetal bovine serum, at a pH of 5.9-6.9; the insect cell culture medium is Insect-XPRESS. TM(BioWhittaker, Lonza, Walkersville, MD, USA, 12-730Q); the antibiotics selected are penicillin and streptomycin; the content of penicillin in the insect cell culture medium is 25-500 U / mL, preferably 100 U / mL; the content of streptomycin in the insect cell culture medium is 25-500 μg / mL, preferably 100 μg / mL; the content of fetal bovine serum is 5-20% (v / v) of the insect cell culture medium, preferably 15% (v / v).
[0026] Example 1: Establishment of the ovarian cell line of the teak moth pupa Collect female pupae of the teak moth during the pre-emergence stage, immerse them in 75% (v / v) ethanol aqueous solution for 15 minutes for surface disinfection, and remove the ovarian tissue, keeping it as intact as possible during the operation. Wash the tissue three times with physiological saline (or PBS buffer), and then twice with insect cell culture medium. Place the tissue into a cell culture tube containing 0.5 mL of insect cell culture medium and incubate at 28°C in a dark incubator. Replace the insect cell culture medium with fresh insect cell culture medium every 3 days, and continue culturing until the cells proliferate to fill the bottom of the cell culture tube. Figure 1 After being passaged for 10 generations, the ovarian cell line of the teak camel moth pupa was successfully established and named Hp-1. Hp-1 maintained genetic stability even after more than 30 generations of continuous passage.
[0027] The insect cell culture medium used in this embodiment is: Insect-XPRESS TM (BioWhittaker, Lonza, Walkersville, MD, USA, 12-730Q), containing 100 U / mL penicillin, 100 μg / mL streptomycin and 15% (v / v) fetal bovine serum, pH=6.5.
[0028] The ovarian cell line Hp-1 from the pupa of the teak camel moth was deposited on December 18, 2025, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 46769.
[0029] Example 2: Observation and determination of the biological characteristics of Hp-1 (1) Morphological characteristics Under an inverted microscope, Hp-1 cells consisted of adherent cells and a small number of suspension cells, with cell shapes including round, spindle-shaped, and macrophage-like. Figure 2 ).
[0030] (2) Cell growth With 2×10 5The 20th generation Hp-1 cells were seeded into 24-well plates containing insect cell culture medium at a concentration of cells / mL and cultured at 28°C. The cell growth curve of the 20th generation Hp-1 cells was then measured. Figure 3 And calculate the cell population doubling time using the formula T=lg2 / [lg(N / N0)] (where T=time required for the average growth phase to double; N0=0 number of cells; N=number of cells measured at time t; t=time from inoculation to measurement of cell number).
[0031] The insect cell culture medium used in this embodiment is: Insect-XPRESS TM (BioWhittaker, Lonza, Walkersville, MD, USA, 12-730Q), containing 100 U / mL penicillin, 100 μg / mL streptomycin and 15% (v / v) fetal bovine serum, pH=6.5.
[0032] The population doubling time of Hp-1 in insect cell culture medium at generation 20 was 31.65 hours. This cell line remained phenotypically stable after more than 30 passages.
[0033] (3) Identification using DAF-PCR method DNA extracted from Hp-1, DNA extracted from the ovaries of the teak moth pupa, DNA extracted from the sf9 cell line, and DNA extracted from the rice leaf folder were subjected to PCR amplification and electrophoresis using 1% agarose gel. The results are shown in the figure below. Figure 4 .
[0034] The primers used for PCR amplification are: aldolaseF: CCGGAGCAGAAGAAGGAGCT (SEQ ID NO.1); aldolaseR: CACATACTGGCAGCGCTTCA (SEQ ID NO. 2).
[0035] The results showed that the Hp-1 cell line did indeed originate from the teak moth, rather than from other contaminants.
[0036] (4) Cryopreservation and thawing Traditional cell cryopreservation methods are used to freeze and preserve cells, which can then be successfully thawed.
[0037] Example 3: Sensitivity determination of Hp-1 to baculovirus Budding virus particles (BV) of the teak camel moth nucleopolyhedrovirus (HypuNPV) (the supernatant obtained by removing blood cells from the hemolymph of teak camel moth larvae after feeding on HypuNPV for 3 days) were inoculated with Hp-1 and cultured for 7 days. Viral polyhedromes could be harvested, with an infection rate of approximately 60.2%. Under an inverted microscope, typical cytopathological features could be observed, namely enlarged cell nuclei containing numerous polyhedral granules. Figure 5 ).
[0038] The test results above show that Hp-1 is sensitive to the nucleopolyhedrovirus of the teak moth and has the characteristic of producing high levels of baculovirus.
[0039] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.
Claims
1. A high-yield baculovirus-producing ovarian cell line from the pupa of the teak camel moth, characterized in that, The ovarian cell line of the teak camel moth pupa was deposited on December 18, 2025, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 46769.
2. A method for constructing a high-yield baculovirus-producing ovarian cell line from the pupa of the teak camel moth, characterized in that, Includes the following steps: After surface disinfection of the teak camel moth pupa, the ovarian tissue was removed, cleaned, and cultured in cell culture medium. After passage, the teak camel moth pupa ovarian cell line was obtained.
3. The construction method according to claim 2, characterized in that, The surface of the teak camel moth pupa was disinfected using a 75% (v / v) ethanol aqueous solution.
4. The construction method according to claim 2, characterized in that, The cleaning process includes: first washing with physiological saline or PBS buffer, and then washing with cell culture medium.
5. The construction method according to claim 2, characterized in that, The cell culture medium was Insect-XPRESS containing antibiotics and fetal bovine serum. TM Culture medium.
6. The construction method according to claim 5, characterized in that, The antibiotics mentioned are penicillin and streptomycin.
7. The construction method according to claim 6, characterized in that, The content of penicillin in the cell culture medium is 25-500 U / mL; the content of streptomycin in the cell culture medium is 25-500 μg / mL; the volume content of fetal bovine serum in the cell culture medium is 5-20%; and the pH of the cell culture medium is 5.9-6.
9.
8. The construction method according to claim 7, characterized in that, The penicillin content in the cell culture medium is 100 U / mL; the streptomycin content in the cell culture medium is 100 μg / mL; and the fetal bovine serum content in the cell culture medium is 15% by volume.
9. The application of the teak moth pupa ovary cell line as described in claim 1 in the production of baculovirus.
10. The application according to claim 9, characterized in that, The baculovirus is a nucleopolyhedrovirus of the teak camel moth.