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Detection method for protein activity of fish growth hormone

A growth hormone and protein activity technology, applied in the field of bioengineering, can solve the problems of cumbersome injection, implantation and irrigation, long detection cycle, and difficult to standardize the experimental conditions

Inactive Publication Date: 2009-04-01
上海中科伍佰豪生物工程有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although these methods are the most direct, there are many unfavorable factors in actual detection
Injection, implantation and irrigation operations are cumbersome and time-consuming
Soaking and feeding have the disadvantage that the amount of fish growth hormone is too large, and the growth-promoting effect is interfered by many factors. This is because the fish growth hormone protein will be decomposed by protease in the digestive tract of fish and lose part of its biological activity.
The common disadvantage of these activity detection methods at the fish body level is that the detection cycle is long and the test results are not repeatable, because the growth of fish body is a complex process affected by many factors such as fish body itself, nutrition, environment, etc., so the detection It is difficult to unify and standardize the experimental conditions

Method used

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  • Detection method for protein activity of fish growth hormone
  • Detection method for protein activity of fish growth hormone
  • Detection method for protein activity of fish growth hormone

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0084] Construction of CHO-K1 cells stably expressing grass carp growth hormone receptor gene (CHO-gcGHR)

[0085] 1. Materials

[0086] A. Strains and vectors

[0087] Eukaryotic cell CHO-K1 (CCTCC number is GDC018) was purchased from Wuhan China Type Culture Collection Center; Escherichia coli XL1-Blue competent cells were purchased from Stratagene; the expression vector was purchased from pcDNA from Invitrogen TM 3.1(+).

[0088] B. Molecular Biology Reagents

[0089] Restriction enzymes Hind III, ApaI, and T4 DNA ligase were purchased from TaKaRa Company; KOD plus DNA polymerase was purchased from ToYoBo Company.

[0090] DNA Marker was purchased from GeneRuler of Fermentas Company TM , 100bp Ladder Plus (Product No.: SM0321) and λ-HindIII Digest from TaKaRa Company (Product No.: D3403A).

[0091] Protein Marker was purchased from Fermentas Company's pre-stained Marker (product number: SM0671).

[0092] RNA extraction kit was purchased from Qiagen.

[0093] RT-PCR k...

Embodiment 2

[0125] Embodiment 2 Preparation of silver carp growth hormone protein

[0126] 1. Materials

[0127] Vector: Escherichia coli HMS174(DE3), Origami TM (DE3) Competent cells were purchased from MERCK, Escherichia coli XL1-Blue competent cells were purchased from Stratagene; expression vector pET-21(+) was purchased from MERCK.

[0128] Molecular biology reagents: restriction enzymes EcoRI, HindIII, and T4 DNA ligase were purchased from TaKaRa; KOD plus DNA polymerase was purchased from ToYoBo; RNA extraction kit was purchased from Qiagen; RT-PCR kit was purchased from Invitrogen Company; Plasmid Extraction Kit and Gel Recovery Kit were purchased from Tiangen Company; nickel affinity column packing was purchased from Qiagen Company; primer synthesis was completed by Shanghai Saibaisheng Gene Technology Co., Ltd.; Complete; other reagents were of analytical grade.

[0129] 2. Method

[0130] (1) RT-PCR clone silver carp growth hormone gene (scGH)

[0131] The total RNA extrac...

Embodiment 3

[0141] Detection of the biological activity of fish growth hormone protein in promoting the proliferation of CHO-gcGHR cells

[0142] 1. Materials

[0143] A. Strains and vectors

[0144] The eukaryotic cell CHO-K1 (CCTCC number is GDC018) was purchased from Wuhan China Type Culture Collection; the expression vector was purchased from Invitrogen’s pcDNA TM 3.1(+).

[0145] B. Molecular Biology Reagents

[0146] F12 medium and fetal bovine serum were purchased from Gibco.

[0147] MTT was purchased from Amresco.

[0148] 2. Method

[0149] (1) Insert 4,000 CHO-gcGHR cells prepared above into each well of a 96-well cell culture plate, and culture the cells with F12 medium containing 10% fetal bovine serum for 48 hours. The cell culture temperature is 37°C, CO 2 The content is 5%;

[0150] (2) Remove the serum-containing medium, replace with serum-free medium to culture cells for 6 hours, and then replace with F12 medium containing fish growth hormone protein to incubate w...

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Abstract

The invention discloses a novel fish growth hormone receptor suitable for constructing a cell model used in the detection of fish growth hormone protein activity and a coding gene thereof. After a fish growth hormone receptor gene is switched to a host cell CH0, a reformed CH0 cell which can express the fish growth hormone receptor is obtained. The fish growth hormone receptor expressed by the cell reserves favorable biological construction, which can be favorably applied to detecting the activity of the fish growth hormone protein.

Description

technical field [0001] The invention belongs to the technical field of bioengineering, and more specifically, the invention relates to a protein, a polynucleotide, a recombinant cell and a detection method for detecting the activity of a fish growth hormone protein. Background technique [0002] Fish growth hormone is a single-chain protein secreted by fish pituitary anterior lobe cells. The most basic physiological function of fish growth hormone is to promote fish growth and participate in various anabolic effects. Fish growth hormone can promote fish protein Synthesis of nitrogen, resulting in a positive nitrogen balance, improving the absorption rate of liver or muscle cells to amino acids and the synthesis rate of RNA, so as to improve the conversion rate of food, thereby promoting the growth of fish. Moreover, the exogenous fish growth hormone obtained by the recombinant method can also increase the synthesis of protein in the fish body, enhance the absorption of some ...

Claims

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Application Information

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IPC IPC(8): C07K14/46C07K14/615C12Q1/02G01N33/74C12N15/12
Inventor 胡炜龚毅
Owner 上海中科伍佰豪生物工程有限公司
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