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Method for producing pregnant mare serum gonactotropin by monoclonal antibody technology

A gonadotropin and monoclonal antibody technology, applied in hormone peptides, animal/human proteins, specific peptides, etc., can solve the problems of increasing gonadotropin protein loss, gonadotropin protein inactivation, long process flow, etc. The production process is simple, the purity is high, and the effect of reducing environmental pollution

Inactive Publication Date: 2013-02-13
INNER MONGOLIA BOGE FARMING ANIMAL HUSBANDRY DEV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The process uses acid treatment, multiple use of ethanol, resulting in the inactivation of part of the gonadotropin protein, and the whole process is longer, which increases the loss of the gonadotropin protein, so the recovery rate is low (the average active recovery rate is 60%)

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0022] Example 1 Production of Serum Gonadotropin in Pregnant Horses Using Monoclonal Antibody Technology

[0023] A. Preparation of anti-pregnant horse serum gonadotropin monoclonal antibody

[0024] (1) Animal immunity

[0025] Immune 18-24 g of BALB / c mice by conventional methods, inject 0.3 ml of 1000 IU / ml PMSG subcutaneously at multiple points, take eyeball blood for antibody titer determination after multiple immunizations, and select the antibody that produces high titer (> 1:9000) ) antibody mice were used for hybridoma preparation.

[0026] (2) cell fusion

[0027] Spleen cells were isolated from the spleen of mice 30 days after immunization and compared with logarithmic phase myeloma cells SP 2 / 0 mixed at 4:1, added 50% PEG6000 for 45 seconds to promote fusion to form hybridoma cells, the treated cells were diluted and placed in HAT culture medium, spread to 96-well plates and 24-well plates, in the containing 5%CO 2 , cultured at 37°C, 7-8 days after fusion, ...

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PUM

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Abstract

The invention discloses a method for producing pregnant mare serum gonactotropin by a monoclonal antibody technology. The method comprises the following steps of: taking pregnant mare serum as a raw material, separating and purifying gonadotropin once by a gonadotropin monoclonal affinity column, wherein the affinity column adopts a normal mouse immunity and monoclonal antibody screening technology to obtain the affinity column which is formed by that the monoclonal antibody is connected with the activated Sepharose2B, adsorbing the gonadotropin onto the affinity column after the pregnant mare serum passes through the affinity column, eluting by a high-salt solution, dialyzing, and purifying the gonactotropin once. According to the method, the immune affinity column is specifically combined by antigen and antibody, so that the impure protein can be effectively removed, the pregnant mare serum gonactotropin can be purified once, and the obtained pregnant mare serum gonactotropin is high in purity, good in quality, and high in yield.

Description

technical field [0001] The invention relates to the technical field of extraction and purification of small biological molecules, in particular to a method for producing serum gonadotropin in pregnant horses by using monoclonal antibody technology. Background technique [0002] Pregnant Mare Serum Gonadotropin (PMSG) molecule is composed of α and β subunits, α subunit is composed of 96 amino acids, β subunit is composed of 149 amino acids, and its molecular weight is roughly 39500 (determined by the content of Sugar content), the sugar content is about 45%, of which sialic acid is 10.8% to 12.1%, isoelectric point PI 2.6-2.65, easily soluble in water, and stable when dry. PMSG has the dual properties of follicle-stimulating hormone and luteinizing hormone used in livestock reproduction. For female animals, it can promote the development of follicles in the ovary and corpus luteum production, and for males, it can promote the production of sperm in the seminiferous tube of th...

Claims

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Application Information

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IPC IPC(8): C07K14/575C07K1/22
Inventor 王勇王秀丽高飞
Owner INNER MONGOLIA BOGE FARMING ANIMAL HUSBANDRY DEV