A culture medium and culture method for promoting the development of Haemonchus contortus larvae
By adding compound vitamins and bovine liver extract to the culture medium of Haemonchus contortus, the problem of in vitro culture of Haemonchus contortus to adult worms has been solved, achieving efficient adult development and culture, which has important research and control significance.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- HUAZHONG AGRI UNIV
- Filing Date
- 2026-03-03
- Publication Date
- 2026-05-26
AI Technical Summary
Existing in vitro culture systems for Haemonchus contortus cannot culture to the adult stage, and there is a lack of effective culture media and methods.
Using compound vitamins and/or bovine liver extract as auxiliary components, combined with specific concentrations and ratios of culture medium components, including basal culture medium and antibiotics, promotes the development of Haemonchus contortus larvae from the early stage of stage IV to the late stage of stage IV and into adult worms.
The in vitro culture of adult Haemonchus contortus has been successfully achieved, with high development rate, short culture cycle, simple operation, low cost, and readily available raw materials.
Smart Images
Figure CN121753760B_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of biotechnology, specifically relating to a culture medium and culture method for promoting the development of Haemonchus contortus larvae. Background Technology
[0002] *Haemonchus contortus* is a blood-sucking parasitic nematode that infests the abomasum of more than 40 species of animals, including cattle and sheep. Because its red digestive tract and white reproductive tract intertwine after feeding on blood, it is also known as the "stomach worm." *Haemonchus contortus* is distributed worldwide, including tropical, subtropical, temperate, and subarctic zones, with the widest distribution in tropical and subtropical regions. This parasite is extremely harmful to its host; when it feeds heavily on the host's blood, it causes anemia and even death, becoming a significant factor affecting the development of ruminant animal husbandry. Control of *Haemonchus contortus* mainly relies on a limited number of chemical drugs, including benzimidazoles, thiabendazoles, and macrolides. These three classes of drugs have been widely used to treat gastrointestinal nematode infections since their introduction; however, the overuse of these drugs by many herders and livestock farmers has led to a global trend of drug resistance in *Haemonchus contortus*, and in many areas, there are no effective treatments available for *Haemonchus contortus* disease.
[0003] Currently, the development of drugs and vaccines related to Haemonchus contortus infection heavily relies on large animal models, which suffer from significant individual variability and high costs. Therefore, establishing an in vitro culture system for Haemonchus contortus has significant research and application value. Because parasitic nematodes are extremely dependent on the host's nutritional supply, they have developed unique developmental patterns through long-term evolution. The in vitro environment cannot completely simulate the host's internal environment, resulting in a general lack of stable in vitro culture systems for most animal parasitic nematodes during their parasitic period. Therefore, establishing an in vitro culture system for Haemonchus contortus is not only important for the research and control of Haemonchus contortus infection, but can also provide a reference for the research and control of other animal parasitic nematode diseases.
[0004] However, research on the in vitro culture system of Haemonchus contortus has progressed slowly. To date, its parasitic stage can only be cultured up to the end of the fourth stage (CN118542292A), and there is a lack of applicable adult culture media and culture methods. Summary of the Invention
[0005] The purpose of this invention is to solve the problem that existing in vitro culture systems for Haemonchus contortus cannot be cultured to the adult stage, and to provide the application of auxiliary components such as compound vitamins and / or bovine liver extract powder in promoting the development of Haemonchus contortus larvae, as well as to provide a culture medium and culture method for promoting the development of Haemonchus contortus larvae.
[0006] The above objective is achieved through the following technical solution:
[0007] In a first aspect, the present invention provides the application of compound vitamins and / or bovine liver extract powder in promoting the development of Haemonchus contortus larvae or in the preparation of a culture medium for promoting the development of Haemonchus contortus larvae, wherein promoting the development of Haemonchus contortus larvae includes: promoting the development of early stage IV larvae to late stage IV larvae, and promoting the development of early stage IV larvae to adults.
[0008] Preferably, the concentration of the compound vitamin is 1-3% (v / v); the concentration of the bovine liver extract powder is 50-500 μg / mL. The compound vitamin contains D-biotin, choline chloride, folic acid, inositol, nicotinamide, D-calcium pantothenate, pyridoxal hydrochloride, riboflavin, thiamine hydrochloride, etc., and is preferably a compound vitamin from Sigma.
[0009] More preferably, the concentration of the compound vitamins is 2% (v / v); and the concentration of the bovine liver extract powder is 250 μg / mL.
[0010] In a second aspect, this invention provides a culture medium for promoting the development of Haemonchus contortus larvae. The promotion of Haemonchus contortus larvae development includes: promoting the development of early stage IV larvae to late stage IV larvae, and promoting the development of early stage IV larvae to adults; the culture medium includes a basal culture medium and auxiliary components. The auxiliary components include a complex vitamin and / or bovine liver extract powder. Preferably, the concentration of the complex vitamin is 1%-2% (v / v); the concentration of the bovine liver extract powder is 50-250 μg / mL. More preferably, the concentration of the complex vitamin is 2% (v / v); the concentration of the bovine liver extract powder is 250 μg / mL.
[0011] In some embodiments, the auxiliary components further include: pyruvate, non-essential amino acids, L-glutamine, fetal bovine serum, defibrinated sheep blood, and L-glutathione. The defibrinated sheep blood is preferably processed as follows: fresh defibrinated sheep blood is repeatedly freeze-thawed, then centrifuged at high speed, and the supernatant is collected to obtain processed defibrinated sheep blood. The non-essential amino acids include L-alanine, L-asparagine, L-aspartic acid, L-glutamic acid, glycine, L-proline, and L-serine, preferably Sigma-Aldrich non-essential amino acids.
[0012] Preferably, the concentration of pyruvate is 0.5-2 mM, the concentration of non-essential amino acids is 0.05-0.3 mM, the concentration of L-glutamine is 1-5 mM, the concentration of fetal bovine serum is 10-30% (v / v), the concentration of defibrinated sheep blood is 1-5% (v / v), and the concentration of L-glutathione is 0.1-0.5 mg / mL.
[0013] More preferably, the concentration of pyruvate is 1 mM, the concentration of non-essential amino acids is 0.1 mM, the concentration of L-glutamine is 2 mM, the concentration of fetal bovine serum is 20% (v / v), the concentration of defibrinated sheep blood is 2.5% (v / v), and the concentration of L-glutathione is 0.3 mg / mL.
[0014] In some embodiments, the culture medium further includes antibiotics for preventing contamination, including penicillin, streptomycin, and amphotericin B.
[0015] Preferably, the concentration of penicillin is 50-150 U / mL, the concentration of streptomycin is 0.05-0.2 mg / mL, and the concentration of amphotericin B is 0.1-0.5 μg / mL.
[0016] More preferably, the concentration of penicillin is 100 U / mL, the concentration of streptomycin is 0.1 mg / mL, and the concentration of amphotericin B is 0.25 μg / mL.
[0017] Preferably, the culture medium comprises a basal culture medium and the following components at the following concentrations: 1-3% (v / v) of compound vitamins, 50-250 μg / mL of bovine liver extract, 0.5-2 mM of pyruvate, 0.05-0.3 mM of non-essential amino acids, 1-5 mM of L-glutamine, 10-30% (v / v) of fetal bovine serum, 1-5% (v / v) of defibrinated sheep blood, 0.1-0.5 mg / mL of L-glutathione, 50-150 U / mL of penicillin, 0.05-0.2 mg / mL of streptomycin, and 0.1-0.5 μg / mL of amphotericin B.
[0018] More preferably, the culture medium comprises a basal culture medium and the following components at the following concentrations: 2% (v / v) compound vitamins, 250 μg / mL bovine liver extract, 1 mM pyruvate, 0.1 mM non-essential amino acids, 2 mM L-glutamine, 20% (v / v) fetal bovine serum, 2.5% (v / v) defibrinated sheep blood, 0.3 mg / mL L-glutathione, 100 U / mL penicillin, 0.1 mg / mL streptomycin, and 0.25 μg / mL amphotericin B.
[0019] When the promotion of *Haemaphysalis contortus* larval development is described as promoting the development of early-stage IV larvae to late-stage IV larvae, the basal culture medium comprises: a culture medium consisting of NCTC-135 / NCTC-109 and LB in a volume ratio of 0.9-1.1:1.8-2.2 (preferably 1:2), and insect cell culture medium. The insect cell culture medium includes SF medium and Grace's supplemented insect cell culture medium, preferably SF medium.
[0020] When the promotion of Haemonchus contortus larval development refers to promoting the development of early fourth-stage larvae into adults, the basal culture medium is preferably insect cell culture medium. The insect cell culture medium includes SF medium and Grace's supplemented insect cell culture medium, preferably SF medium.
[0021] In a third aspect, this invention provides a method for culturing early fourth-stage larvae of *Haemaphysalis contortus* into late fourth-stage larvae or adults, comprising the following steps: adding early fourth-stage larvae of *Haemaphysalis contortus* to the aforementioned culture medium for cultivation to obtain late fourth-stage larvae or adults of *Haemaphysalis contortus*. The early fourth-stage larvae are obtained from molted third-stage larvae.
[0022] Preferably, the culture conditions are: temperature 38.5-40℃, CO2 content 18-22%.
[0023] More preferably, the cultivation conditions are: temperature 39°C and CO2 content 20%.
[0024] The present invention has the following advantages and beneficial effects:
[0025] This invention discovers that compound vitamins and / or bovine liver extract powder, as excipients, can promote the development of early-stage fourth-stage larvae of *Haemaphysalis contortus* into late-stage fourth-stage larvae and adults. The culture medium provided by this invention enables the in vitro culture of *Haemaphysalis contortus* adults for the first time, which is beneficial for promoting the development of related drugs and vaccines for this parasite and has significant implications for parasite control. The adult development rate cultured by this invention is high (up to 40% or more), and it also has advantages such as short culture cycle, simple operation, readily available culture medium raw materials, and low production cost. Attached Figure Description
[0026] Figure 1 This study compares the effects of different concentrations of lipoprotein-cholesterol on the culture of fourth-stage larvae of Haemaphysalis contortus.
[0027] Figure 2 This study compares the effects of different concentrations of compound vitamins on the culture of fourth-stage larvae of Haemaphysalis contortus.
[0028] Figure 3 This study compares the effects of different concentrations of bovine liver extract powder on the culture of fourth-stage larvae of Haemaphysema contortus, based on the addition of 2% (v / v) multivitamins.
[0029] Figure 4 This study compares the effects of different basal culture media on the development of adult Haemonchus contortus.
[0030] Figure 5 This involves morphological observation of adult female Haemonchus contortus worms cultured in vitro.
[0031] Figure 6 This involves morphological observation of adult male Haemonchus contortus worms cultured in vitro. Detailed Implementation
[0032] The technical solution of the present invention will be described in detail below with reference to specific embodiments and examples. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention. Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods. Unless otherwise specified, the materials and reagents used in the following embodiments are commercially available.
[0033] Material:
[0034] (1) Laboratory animals and insect strains
[0035] Healthy goats aged 3-6 months were purchased from the Hubei Academy of Agricultural Sciences, and the Haemaphyseudomonas contortus was preserved in our laboratory.
[0036] (2) Main materials and reagents
[0037] NCTC-135 medium (Thermo Fisher, USA), M-199 medium (Sigma, USA), Grace's supplemented insect cell medium (Gibco, USA), SF medium (Gibco, USA), multivitamins (Sigma, USA, B6891, BME vitamin solution [100×]), non-essential amino acids (Sigma, USA, M7145, MEM non-essential amino acid solution [100×]), bovine liver extract (Yuanye Biotechnology), lipoprotein-cholesterol (Lee Biosolutions, USA), defibrinated sheep blood (Dening Biotechnology), fetal bovine serum (Gibco, USA), penicillin-streptomycin-amphoteric acid B solution (Sigma, USA), L-glutathione (Sigma, USA).
[0038] Multivitamin: Each liter contains 0.1 g of D-Biotin, 0.1 g of choline chloride, 0.1 g of folic acid, 0.2 g of inositol, 0.1 g of nicotinamide, 0.1 g of D-calcium pantothenate, 0.1 g of pyridoxal hydrochloride, 0.01 g of riboflavin, and 0.1 g of thiamine hydrochloride.
[0039] Non-essential amino acids: Each liter contains 0.89 g of L-alanine, 1.5 g of L-asparagine, 1.33 g of L-aspartic acid, 1.47 g of L-glutamic acid, 0.75 g of glycine, 1.15 g of L-proline, and 1.05 g of L-serine.
[0040] (3) Obtaining infective third-stage larvae (iL3) of Haemonchus contortus
[0041] Healthy goats aged 3-6 months were artificially dewormed with ivermectin. Fourteen days after deworming, feces were examined for eggs for three consecutive days. After confirming the absence of eggs, the goats were artificially infected with 7000 *Haemaphysema contortus* iL3 worms. Feces were examined for eggs 28 days later. Once egg production stabilized, feces were collected and cultured in a 26°C, humidified incubator for 7 days until the eggs developed into iL3 worms. After 7 days of culture, iL3 worms were collected from the feces using the Bellman method, the specific steps of which are as follows:
[0042] ① Place two layers of filter paper on the gauze and wrap the feces inside.
[0043] ② Place the wrapped feces into a Bellman funnel and add dechlorinated water at a temperature below 40°C until the feces are submerged.
[0044] ③ After standing for 4 hours, collect the lower liquid in a 50 mL centrifuge tube, let it stand for another 4 hours, discard the upper 45 mL liquid, and collect the bottom 5 mL liquid.
[0045] ④ Add 2 mL of BU solution (each liter of BU solution contains 4.04 g NaCl, 2.99 g KH2PO4, and 17.91 g Na2HPO4·12H2O) to a glass test tube, add filter paper to the top of the liquid, add the liquid retained in the previous step to the glass test tube, let it stand for 4 hours, collect the bottom iL3, and store it in 10 times the amount of BU solution in a 16℃ saturated humidity incubator.
[0046] Other materials whose sources are not specified are all conventional materials in this field.
[0047] Example 1: Screening for key auxiliary components that promote adult development
[0048] 1. Obtaining the third-stage molted larvae of *Haemaphysalis contortus*:
[0049] (1) Place 1.2 mL of BU solution containing iL3 into a 1.5 mL centrifuge tube, add 0.15% NaClO, and desheath at 37℃ for 10 min;
[0050] (2) After desheathing is completed, centrifuge at 1300 g for 3 min at room temperature;
[0051] (3) After centrifugation, discard 1 mL of supernatant, add 1 mL of 0.85% NaCl solution containing three antibodies (100 U / mL penicillin, 0.1 mg / mL streptomycin, and 0.25 μg / mL amphotericin B) and mix well. Centrifuge at 1300 g for 3 min. Repeat this step five times.
[0052] (4) Discard 1 mL of supernatant, add 1 mL of M-199 medium containing three antibiotics and mix well, centrifuge at 1300 g for 3 min; repeat this step three times to obtain pure molting third-stage larvae.
[0053] 2. Culture of three stages of molting larvae
[0054] (1) Preparation of culture medium for molting stage III larvae: M-199 medium was supplemented with 100 U / mL penicillin, 0.1 mg / mL streptomycin, 0.25 μg / mL amphotericin B, 1 mM pyruvate, 0.1 mM non-essential amino acids, 2 mM L-glutamine, and 20% (v / v) fetal bovine serum (FBS).
[0055] (2) The treated molting third-stage larvae were placed in a 24-well plate at a density of 100 larvae / well. 500 μL of culture medium containing third-stage larvae was added to each well. The plate was then incubated in a 39℃ 20% CO2 incubator for 7 days until the larvae developed into early fourth-stage larvae.
[0056] 3. Screening for key auxiliary components that promote the development of four larvae.
[0057] Early larvae of the fourth stage were collected by centrifugation at 1300 g for 3 min. They were then added to NCTC-135:LB=1:2 (v / v) medium containing different auxiliary components and placed in 24-well plates at a density of 100 larvae / well, with 500 μL of medium per well. The plates were incubated at 39℃ in a 20% CO2 incubator. The top 200 μL of medium was aspirated every two days for medium replacement.
[0058] This experiment used NCTC-135:LB=1:2 (v / v) basal medium supplemented with 100 U / mL penicillin, 0.1 mg / mL streptomycin, 0.25 μg / mL amphotericin B, 1 mM pyruvate, 0.1 mM non-essential amino acids, 2 mM L-glutamine, 20% (v / v) FBS, 2.5% (v / v) centrifuged blood, and 0.3 mg / mL L-glutathione. The effects of adding different concentrations of lipoprotein cholesterol, multivitamins, and bovine liver extract on the development rate of late-stage fourth-instar larvae were compared. Lipoprotein cholesterol, multivitamins, and bovine liver extract were tested sequentially, and each subsequent test was supplemented with a previously tested substance effective in improving the development rate of late-stage fourth-instar larvae to maximize the development rate of late-stage fourth-instar larvae in this culture system.
[0059] Preparation of the above-mentioned centrifuged blood: Fresh defibrinated sheep blood was frozen at -20℃ and thawed at room temperature twice. Then it was dispensed into 1.5 mL centrifuge tubes and centrifuged at 12000 g and 4℃ for 10 min. The supernatant of the centrifuged defibrinated sheep blood was collected and stored in a 4℃ freezer to obtain the centrifuged product of freeze-thawed defibrinated sheep blood (hereinafter referred to as: centrifuged blood).
[0060] The criteria for distinguishing between the early, middle, and late stages of the four larval stages are as follows: In the early stage, the reproductive primordia cells are 12, the tail is slender and curved towards the dorsal side, and the reproductive primordia of the female are farther from the excretory opening than those of the male; In the middle stage, the reproductive system of the larva is long and spindle-shaped, the tail of the female is slender and curved towards the dorsal side into a small hook shape, and the tail of the male is shorter and thicker; In the late stage, the reproductive system of the larva has developed into a tubular shape, and the various organs of the female reproductive system are clearly visible, especially the vulva under a microscope, and the mucus sac and copulatory umbilicus of the male tail are clearly visible.
[0061] Figure 1 This study compares the effects of different concentrations of lipoprotein-cholesterol on the culture of fourth-stage larvae. Cholesterol is an important nutrient that nematodes cannot synthesize de novo. Therefore, the study attempted to supplement lipoprotein-cholesterol, a natural animal-derived cholesterol, to observe its effect on larval development. The results showed that different concentrations of lipoprotein-cholesterol had no significant effect on the development of late-stage fourth-stage larvae after 15 days of in vitro culture, possibly because the cholesterol provided by serum was sufficient to meet the developmental needs of the larvae.
[0062] Figure 2 This study compares the effects of different concentrations of compound vitamins on the culture of fourth-stage larvae. It shows that when the compound vitamin supplementation amount is 2% (v / v) for 15 days in vitro culture of fourth-stage larvae, it can significantly improve the development rate of late-stage fourth-stage larvae.
[0063] Figure 3 The study compared the effects of different concentrations of bovine liver extract powder on the culture of fourth-stage larvae with the addition of 2% (v / v) compound vitamins. It can be seen that 50-250 μg / mL of bovine liver extract powder can significantly improve the development rate of late-stage fourth-stage larvae after 20 days of in vitro culture of fourth-stage larvae.
[0064] Example 2: In vitro culture methods and conditions for adult Haemonchus contortus
[0065] Early larvae of the fourth stage were collected by centrifugation at 1300 g for 3 min. The larvae were then added to the culture medium with different formulations as shown below and placed in 24-well plates at a density of 100 larvae / well, with 500 μL of culture medium per well. The plates were incubated at 39℃ in a 20% CO2 incubator. The top 200 μL of culture medium was aspirated every two days for medium replacement.
[0066] The effects of different basal media on adult development were compared by adding 2% (v / v) multivitamins, 250 μg / mL bovine liver extract, 100 U / mL penicillin, 0.1 mg / mL streptomycin, 0.25 μg / mL amphotericin B, 1 mM pyruvate, 0.1 mM non-essential amino acids, 2 mM L-glutamine, 20% (v / v) FBS, 2.5% (v / v) centrifuged blood, and 0.3 mg / mL L-glutathione to different basal media (NCTC-135:LB=1:2 medium, SF medium, and Grace's supplemented insect cell medium).
[0067] The criteria for distinguishing between the early, middle, and late stages of larvae and adults are as follows: In the early stage, the reproductive primordia cells are 12, the tail is slender and curved towards the dorsal side, and the reproductive primordia of the female are farther from the excretory opening than those of the male; In the middle stage, the reproductive system of the larva is long and spindle-shaped, the tail of the female is slender and curved towards the dorsal side into a small hook shape, and the tail of the male is shorter and thicker; In the late stage, the reproductive system of the larva has developed into a tubular shape, and the various organs of the female reproductive system are clearly visible, especially the vulva under a microscope. The mucus sac and the rudimentary copulatory umbrella of the male tail are clearly visible; In the adult male, the copulatory umbrella of the tail is unfolded, and the adult female has a characteristic ovary, in which the eggs are clearly visible.
[0068] Figure 4 This study compares the effects of different basal culture media on adult development. After 25 days of in vitro culture of fourth-stage larvae, no adults were obtained using NCTC-135:LB=1:2 (v / v) medium as the basal medium. Adults were successfully obtained using SF medium and Grace's supplemented insect cell medium as the basal medium, and the adult development rate of SF medium was significantly higher than that of Grace's supplemented insect cell medium.
[0069] Figure 5 , 6 Morphological observation of adult *Haemaphysalis contortus* worms cultured in vitro revealed that the adults are significantly larger than the larvae, with females being even longer and exhibiting a mature reproductive system. Females possess a clearly defined vulvar operculum, characteristic of adult females, while males possess a characteristic copulatory umbrella structure. This invention enables the in vitro culture of adult *Haemaphysalis contortus* worms, and the significant development of these characteristic adult structures represents a substantial advancement in *Haemaphysalis contortus* in vitro culture technology.
[0070] The above embodiments are only used to help illustrate the present invention and are not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.
Claims
1. The application of compound vitamins and bovine liver extract powder in promoting the development of Haemonchus contortus larvae or in the preparation of a culture medium for promoting the development of Haemonchus contortus larvae, characterized in that: The aforementioned promotion of Haemaphysalis contortus larval development includes: promoting the development of early stage IV larvae into late stage IV larvae, and promoting the development of early stage IV larvae into adults.
2. The application according to claim 1, characterized in that: The concentration of the compound vitamins is 1-3% (v / v); The concentration of the bovine liver extract powder is 50-500 μg / mL.
3. A method for culturing early fourth-stage larvae of *Haemaphysalis contortus* into late fourth-stage larvae or adults, characterized in that: Includes the following steps: Early stage IV larvae of *Haemaphysalis contortus* were added to a culture medium that promotes the development of *Haemaphysalis contortus* larvae and cultured to obtain late stage IV larvae or adults of *Haemaphysalis contortus*. The aforementioned promotion of Haemaphysalis contortus larval development includes: promoting the development of early stage IV larvae into late stage IV larvae, and promoting the development of early stage IV larvae into adults. The culture medium includes a basal culture medium and auxiliary components; The auxiliary ingredients include multivitamins and beef liver extract powder.
4. The method according to claim 3, characterized in that: The auxiliary ingredients also include: pyruvate, non-essential amino acids, L-glutamine, fetal bovine serum, defibrinated sheep blood, and L-glutathione.
5. The method according to claim 3, characterized in that: The culture medium also includes antibiotics to prevent contamination.
6. The method according to claim 3, characterized in that: The culture medium includes a basal culture medium and the following components at the following concentrations: compound vitamins 1-3% (v / v), bovine liver extract powder 50-250 μg / mL, pyruvate 0.5-2 mM, non-essential amino acids 0.05-0.3 mM, L-glutamine 1-5 mM, fetal bovine serum 10-30% (v / v), defibrinated sheep blood 1-5% (v / v), L-glutathione 0.1-0.5 mg / mL, penicillin 50-150 U / mL, streptomycin 0.05-0.2 mg / mL, and amphotericin B 0.1-0.5 μg / mL.
7. The method according to claim 3, characterized in that: The aforementioned method for promoting the development of Haemaphysalis contortus larvae is to promote the development of early stage IV larvae to late stage IV larvae. The basal culture medium includes: an insect cell culture medium composed of NCTC-135 / NCTC-109 and LB in a volume ratio of 0.9-1.1:1.8-2.
2. When promoting the development of Haemaphysema contortus larvae, specifically when promoting the development of early fourth-stage larvae into adults, the basal culture medium is an insect cell culture medium.
8. The method according to claim 7, characterized in that: The insect cell culture media include SF medium and Grace's supplemented insect cell culture medium.
9. The method according to claim 3, characterized in that: The cultivation conditions are: temperature 38.5-40℃, CO2 content 18-22%.