Giant panda lutropin beta subunit monoclonal antibody preparation method and application thereof

A monoclonal antibody, β subunit technology, applied in the biological field, can solve the problem of no specific anti-giant panda LH specific antibody and so on

Pending Publication Date: 2018-11-16
CHENGDU RES BASE OF GIANT PANDA BREEDING
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  • Abstract
  • Description
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  • Application Information

AI Technical Summary

Problems solved by technology

[0006] The purpose of the present invention is to provide an anti-giant panda LHβ subunit monoclonal antibody hybridoma cell line, an anti-giant panda LHβ subunit monoclonal antibody and a preparation method, so as to solve the problem that there is no specific anti-giant panda LH specific antibody in the prior art

Method used

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  • Giant panda lutropin beta subunit monoclonal antibody preparation method and application thereof
  • Giant panda lutropin beta subunit monoclonal antibody preparation method and application thereof
  • Giant panda lutropin beta subunit monoclonal antibody preparation method and application thereof

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preparation example Construction

[0024] The preparation method of specific anti-giant panda LHβ monoclonal antibody has the following steps:

[0025] 1) Design and synthesis of specific polypeptide fragments: According to the giant panda LHβ subunit protein sequence (NP_001291788.1), referring to the sequence analysis of other species, the giant panda LHβ subunit-specific sequence SEQ ID NO: 1 was designed: CGGPRAQPLACDRPPLPGLL, This specific polypeptide fragment is artificially synthesized and coupled with KLH to enhance the immunogenicity as the immunogen (LHβ-KLH) for the production of specific antibodies. This part is entrusted to Shanghai Sangon Biotechnology Co., Ltd. to complete.

[0026] 2) Mice immunization: Adjuvant: antigen = 1:1 (coupling polypeptide LHβ-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 adjuvant. Before the first immunization of ...

Embodiment 1

[0035] 1. Preparation of anti-giant panda LHβ monoclonal antibody

[0036] 1.1 Design and synthesis of specific polypeptide fragments: According to the amino acid sequence of the giant panda LHβ subunit in the NCBI database, accession number: NP_001291788.1, the sequence is: MEMFQGLLLWLLLNTGGAWASRGPLRPLCRPINATLAAENEACPVCITFTTTICAGYCPSMVRVLPAALPPVPQPVCTYHELRFASIRLPGCPPGVDPMVSFPVALSCRCGPCRLFNSDCGGPRAQPLACDRPPLPPG

[0037] L, and referring to the sequence analysis of other species, the giant panda LHβ-specific sequence CGGPRAQPLACDRPPLPGLL (LHβ-1) was designed, artificially synthesized, and coupled with KLH (key empty hemocyanin), as an immunogen for the production of specific antibodies ( LHβ-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 LHβ-1 and immunogen LHβ-KLH

[0038] 1.2 Immunization of mice with antigen: i...

Embodiment 2

[0093] Application of anti-giant panda LHβ subunit monoclonal antibody in western blot technique.

[0094] Extraction of total protein from target tissue

[0095] Application of protein extraction kit: C510004-0020, provided by Sangon Bioengineering (Shanghai) Co., Ltd., to extract total protein from adult giant panda pituitary tissue, and determine the total protein concentration by BCA method.

[0096] 2 Gel preparation: prepare polyacrylamide gel, stacking gel 5%, separating gel 12%.

[0097] 4. Sample preparation: protein sample volume is 80ug

[0098] 5. Electrophoresis: stacking gel 80V, 30min; separating gel 120V, 90min.

[0099] 6. Film transfer: 250mA, 40min.

[0100] 7. Sealing: 5% skimmed milk, shake slowly at 37°C for 2 hours.

[0101] 8. Incubate the primary antibody: the antibodies of hybridoma cell line 14081-1-9(2) and C12(3) were diluted 1:500, shake slowly at 4°C overnight, and rewarm at 37°C for 1 hour the next day.

[0102] 9. Incubate the secondary an...

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Abstract

The invention discloses a giant panda lutropin beta subunit monoclonal antibody preparation method and an application thereof, which belong to the technical field of biology. A preservation number ofan anti-giant panda lutropin beta subunit monoclonal antibody hybridomas cell strain 14081-9 is CCTCC NO:C2018128. A cell fusion technology is used for establishing the hybridomas cell strain for secreting the anti-giant panda lutropin beta subunit monoclonal antibody, the cell strains are collected and purified to culture a supernatant, and the high-specificity lutropin beta subunit monoclonal antibody is obtained, the established high-specificity lutropin beta subunit monoclonal antibody is used for Western Blot protein detection, LH protein positioning and tissue expression information, anddeep researches on LH acceptor ligand, an antagonist, and an anti-antagonist by the LH beta submit high-specificity monoclonal antibody can be carried out, and conditions can be created for discoveryof the specific ligand, the antagonist, and the anti-antagonist. The method provides novel data for discussing LH secretion and gene further regulation and control, reveals a reproduction physiological law, reveals reproduction physiological law in giant panda, and grasps giant panda oestrus ovulation and pregnancy rules.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to the preparation and application of giant panda luteinizing hormone beta subunit monoclonal antibody. Background technique [0002] Luteinizing hormone (LH) is a glycoprotein hormone secreted by basophils in the anterior pituitary gland, which is controlled by gonadotropin-releasing hormone (GnRH) in the hypothalamus and regulated by positive and negative feedback from the ovary. At present, a large number of studies have shown that LH plays an important role in the completion of follicle development and maturation and the acquisition of oocyte development potential. The follicle consists of a central egg cell surrounded by granulosa cells and theca cells. Follicle formation is a highly coordinated physiological process regulated by many factors. FSH and LH are the main factors regulating the growth and development of follicles. The production of estrogen depends on the synergistic...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/20C07K16/26G01N33/76
CPCC07K16/26G01N33/76
Inventor 蔡开来侯蓉张志和张梦诗王涓刘玉良
Owner CHENGDU RES BASE OF GIANT PANDA BREEDING
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