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Preparation and applications of giant panda follicle-stimulating hormone beta subunit monoclonal antibody

A monoclonal antibody and β subunit technology, applied in the field of monoclonal antibody preparation, can solve the problem of no specific anti-giant panda FSH specific antibody

Active Publication Date: 2016-10-26
CHENGDU RES BASE OF GIANT PANDA BREEDING
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  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] The purpose of the present invention is to provide an anti-giant panda FSH β subunit monoclonal antibody hybridoma cell line, anti-giant panda FSH β subunit monoclonal antibody and its preparation method, and solve the problem that there is no specific anti-giant panda FSH specific antibody in the prior art

Method used

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  • Preparation and applications of giant panda follicle-stimulating hormone beta subunit monoclonal antibody
  • Preparation and applications of giant panda follicle-stimulating hormone beta subunit monoclonal antibody
  • Preparation and applications of giant panda follicle-stimulating hormone beta subunit monoclonal antibody

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preparation example Construction

[0019] The preparation method of specific anti-giant panda FSHβ monoclonal antibody has the following steps:

[0020] 1) Design and synthesis of specific polypeptide fragments: According to the protein sequence of the giant panda FSH β subunit (NP_001291794), referring to the sequence analysis of other species, a specific sequence of the giant panda FSH β subunit was designed, as shown in SEQ ID NO: 1: LVYKDPARPNIQKICTFKELAYETVKVPGC, artificially synthesized This specific polypeptide fragment is coupled with KLH to enhance the immunogenicity as an immunogen (FSHβ-KLH) for the production of specific antibodies. This part is entrusted to Shanghai Sangon Biotechnology Co., Ltd. to complete.

[0021] 2) Immunization of mice: Adjuvant: antigen = 1:1 (coupling polypeptide FSHβ-KLH) mixed solution, after complete emulsification, intraperitoneal injection. The adjuvant for the first immunization is complete adjuvant, and the adjuvant for the subsequent immunization is incomplete adjuv...

Embodiment 1

[0030] 1. Preparation of anti-Panda FSHβ monoclonal antibody

[0031] 1.1 Design and synthesis of specific polypeptide fragments: According to the NCBI database giant panda FSHβ subunit amino acid sequence, accession number: NP_001291794, the sequence is:

[0032] MKSVQLCFLFCCWRAICCKSCELTNITITVEKEECRFCISINTTWCAGYCYTRDLVYKDPARPNIQKICTFKELAYETVKVPGCAHQADSLYTYPVATECHCGKCDSDSTDCTVRGLGPSYCSFNEMKE, and referring to the sequence analysis of other species, designed a giant panda FSHβ-specific sequence

[0033] LVYKDPARPNIQKICTFKELAYETVKVPGC (FSHβ-1) is artificially synthesized and coupled with KLH (spoon space hemocyanin) to produce specific antibody immunogen (FSHβ-KLH). For the synthesis of polypeptide fragments, this process is relatively mature. Through the sequence disclosed in this application, Sangon Bioengineering (Shanghai) Co., Ltd. can produce polypeptide fragment FSHβ-1 and immunogen FSHβ-KLH

[0034] 1.2 Immunization of mice with antigen: inoculate with a mixture of adju...

Embodiment 2

[0089] Application of anti-giant panda FSHβ subunit monoclonal antibody in western blot technique.

[0090] 1. Extraction of total protein from the target tissue

[0091] Application of protein extraction kit: C510004-0020, provided by Sangon Bioengineering (Shanghai) Co., Ltd., to extract total protein from adult giant panda testis and ovary tissue, and determine the total protein concentration by BCA method.

[0092] 2 Gel preparation: prepare polyacrylamide gel, stacking gel 5%, separating gel 12%.

[0093] 4. Sample preparation: protein sample volume is 80ug

[0094] 5. Electrophoresis: stacking gel 80V, 30min; separating gel 120V, 90min.

[0095] 6. Film transfer: 250mA, 40min.

[0096] 7. Sealing: 5% skimmed milk, shake slowly at 37°C for 2 hours.

[0097] 8. Incubate the primary antibody: Dilute hybridoma cell line 09199(2) and C12(3) antibodies at 1:500, shake slowly at 4°C overnight, and rewarm at 37°C for 1 hour the next day.

[0098] 9. Incubate the secondary a...

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Abstract

The present invention discloses preparation and applications of a giant panda follicle-stimulating hormone beta subunit monoclonal antibody, wherein an anti-giant panda FSH[beta] subunit monoclonal antibody hybridoma cell line 09199 is provided, and the preservation number is CCTCC NO: C2016144. According to the present invention, a cell fusion technology is used, the hybridoma cell line secreting the anti-FSH[beat] subunit monoclonal antibody is established, the cell line culture supernatant is collected and purified, the highly-specific FSH[beta] subunit monoclonal antibody is obtained, application of the highly-specific FSH[beta] subunit monoclonal antibody in Western Blot protein detection is established to provide information for FSH protein location and tissue expression, and the deep researches on the ligand, the antagonist and the anti-antagonist of the FSH receptor are performed with the highly-specific FSH[beta] subunit monoclonal antibody so as to create the conditions for the founding of the specific ligand, the antagonist and the anti-antagonist; and the new data and the new idea are provided for the further exploration of the FSH secretion, the further regulation of the genes, and the revealing of the reproductive physiological laws.

Description

technical field [0001] The invention relates to the preparation of a specific monoclonal antibody, especially for the detection of giant panda follicle-stimulating hormone protein. Background technique [0002] Follicle-stimulating hormone (FSH) is a glycoprotein hormone secreted by basophils of the anterior pituitary gland. In the ovary, follicle-stimulating hormone stimulates the growth of immature follicles, which release inhibin to block further follicle-stimulating hormone synthesis. This mechanism ensures the selectivity of ovulation. At the end of the luteinizing phase, there is also a small increase in follicle-stimulating hormone levels, possibly related to the beginning of the next ovulatory cycle. In the testis, follicle-stimulating hormone increases the level of Sertoli cells to synthesize male hormone-binding protein, induces Sertoli cells to bind tightly, and secretes inhibin, which plays a vital role in the process of sperm formation. The active form of fol...

Claims

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Application Information

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IPC IPC(8): C12N5/20C07K16/26G01N33/76G01N33/577C12R1/91
CPCC07K16/26
Inventor 蔡开来侯蓉叶尚勉张志和王涓刘玉良罗娌蔡志刚
Owner CHENGDU RES BASE OF GIANT PANDA BREEDING
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