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Antheraea pernyi ovary cell culture medium and application thereof

A technology of ovarian cells and medium, applied in animal cells, vertebrate cells, artificial cell constructs, etc., can solve the problems affecting the enthusiasm of tussah silkworms, the reduction of production and the failure of silkworm farmers, etc., and achieve the effect of comprehensive nutrients in cell growth and convenient operation

Active Publication Date: 2013-04-17
辽宁省农业科学院大连生物技术研究所
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AI Technical Summary

Problems solved by technology

However, because tussah silkworms are kept in the wild, they are subjected to the invasion of various pathogenic microorganisms such as viruses, bacteria, and microsporidia, and diseases occur from time to time, resulting in reduced or even no harvest for some silkworm farmers. The development of tussah silkworm production, and tussah silkworm cells are the necessary tools and platforms to study the pathogenesis of these diseases and the law of pathogenic infection. Therefore, it is of great significance to carry out research on tussah silkworm cell culture methods and establish tussah silkworm ovary cell culture methods for the development of tussah silkworm production and related research

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  • Antheraea pernyi ovary cell culture medium and application thereof
  • Antheraea pernyi ovary cell culture medium and application thereof
  • Antheraea pernyi ovary cell culture medium and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] Example 1 Tussah silkworm ovary cell culture medium MLM-45 formula composition and preparation method

[0041] The composition of the medium formula and the preparation of the medium. The culture medium is composed of amino acids, sugars, inorganic salts, vitamins and cell growth-promoting additives.

[0042] Table 1. Prepare 5 times 1000mL amino acid and sugar mixed storage solution (×5, MLM-AAS), its composition and dosage:

[0043] 5 times MLM-AAS ingredients Dosage (g) L-Cystine (L-cystine) 0.28 L-Tyrosine (L-Tyrosine) 0.425 α-Alanine (α-Alanine) 1.55 β-Alanine (β-Alanine) 1.05 L-Arginine-HCl (L-Arginine) 3.45 L-Aspartic acid (L-Aspartic acid) 3.75 L-Asparagine (L asparagine)- 3.75 L-Glutamic acid (L-glutamic acid) 4.5

[0044] L-Glutamine (L-glutamine) 4.5 L-Glycine (L-Glycine) 2.3 L-Histidine (L-histidine) 4.25 L-Isoleucine (L-Isoleucine) 2.6 L-Leucine (L-Leucine) 0.405 L-Lysine-HCl (L-Lysine) 3.25 L-Methionine (L-methionine) 2.25 L-...

Embodiment 2

[0056] Example 2 Method for in vitro culture of tussah silkworm ovary cells

[0057] The material of Qing 6th Tussah silkworm variety (5th instar larvae and pupae) was provided by the Liaoning Provincial Sericulture Research Institute.

[0058] 1. Obtaining of Tussah Silkworm Ovary Cells:

[0059] (a) Obtaining ovarian cells from tussah silkworm pupa: Take healthy tussah silkworm pupa, disinfect the body surface with 75% ethanol for 20 minutes, wash 3 times with sterile distilled water in an ultra-clean workbench, dry the body surface water with sterile gauze, and use a pin Fix in a dissecting wax tray sterilized by UV for 2 hours. Cut the abdomen aseptically, use ophthalmic scissors and tweezers to dissect and remove the ovarian tissue in sterile Corlson buffer (1000ml Corlson solution formula: NaCl 7g, KCl 0.2g, CaCL 2 0.2g, MgCl 2 .6H2O 0.1g, NaH 2 PO4 0.2g, NaHCO 3 In a small petri dish containing 0.05 g and 8 g glucose, remove the adherent fatty tissue and capsule on the ovar...

Embodiment 3

[0064] Example 3 In Vitro Culture Conditions of Tussah Silkworm Ovary Cells

[0065] Use currently available commercial insect culture media (MM, TC-100, MGM-443, GRACE, IPL-41, MGM-448, TNM-FH, SF-900) and MLM-45 prepared in Example 1 Based on cell growth test.

[0066] After selecting the above different insect media and culturing tussah silkworm pupae and larval ovarian cells for 2 months under the same culture conditions, observation under a phase-contrast inverted microscope showed that the growth of MLM-45 media cells was better than that of the others, and counted by Trypan Blue staining Cell survival (see Table 5). Use MLM-45 medium to carry out cell growth experiments at different temperatures (4℃, 15℃, 20℃, 26℃, 27℃, 28℃, 30℃, 37℃) to determine the optimal cell growth temperature (Table 6 ). Using MLM-45 medium, different pH (pH 5.5, 6.0, 6.2, 6.3, 6.4, 6.5, 7.0) effects on cell growth were tested to determine the optimal pH for cell growth (Table 7). Use MLM-45 mediu...

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Abstract

The present invention discloses composing components of an antheraea pernyi ovary cell culture medium, a preparation method of the antheraea pernyi ovary cell culture medium, and an application of the antheraea pernyi ovary cell culture medium in antheraea pernyi ovary cell culture. According to the present invention, a prepared amino acid and sugar mixing storage solution (MLM-AAS), an inorganicsalt storage solution (MLM-Salt), a vitamin storage solution (MLM-Vit), bovine serum albumin, fetuin and the like are prepared according to a certain ratio, and the pH value is adjusted to 6.2-6.4 toobtain a MLM-45 culture medium, wherein the prepared MLM-45 culture medium is added with fetal bovine serum and a penicillin and streptomycin mixing solution according to a certain volume ratio before the prepared MLM-45 culture medium is used, the volume ratio of the fetal bovine serum is 20%, and the volume ratio of the penicillin and streptomycin mixing solution is 1%. With a plurality of experiments, the following in vitro cell growth conditions are determined that: the culture temperature is 26-28 DEG C, the pH value is 6.2-6.4, and the osmotic pressure is 315-350 mOsm / kg. With the present invention, technical services are provided for researches in antheraea pernyi biology, antheraea pernyi pathogenic microorganism and related fields.

Description

technical field [0001] The invention relates to the fields of insect anatomy, insect tissue culture and the like, in particular to a tussah ovary cell culture medium and its application in the in vitro culture technology of tussah ovary cells. Background technique [0002] Insect cell culture is a technology that removes cells from insects, simulates the physiological environment of insects, and enables cells to survive, grow, and maintain structure and function under sterile, suitable, and rich nutrient conditions. Because primary cells have just been separated from the tissue, the biological characteristics have not changed much, and they still retain the original genetic characteristics, and are the closest to and reflect the growth characteristics in vivo. They are suitable for experimental research such as drug sensitivity tests and cell differentiation. In the early 20th century, many entomologists tried to use insect cells cultured in vitro as tools for scientific res...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/071
Inventor 王林美李树英岳冬梅范琦
Owner 辽宁省农业科学院大连生物技术研究所
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