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Preparation method of polyclonal antibody capable of marking fish germline stem cells

A polyclonal antibody and reproductive stem cell technology, applied in the field of molecular biology, can solve problems that have not yet been used in antibody detection

Active Publication Date: 2021-07-27
INST OF AQUATIC LIFE ACAD SINICA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, the identification methods of fish reproductive stem cells are limited to morphological identification and gene detection, and there are no relevant reports on the use of antibodies for detection

Method used

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  • Preparation method of polyclonal antibody capable of marking fish germline stem cells
  • Preparation method of polyclonal antibody capable of marking fish germline stem cells
  • Preparation method of polyclonal antibody capable of marking fish germline stem cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0023] Embodiment 1 Preparation of Nanos2 full-length protein sequence

[0024] After analysis and research, the inventors found that the full-length protein sequence of zebrafish Nanos2 has high homology with at least 73 full-length protein sequences of Nanos2 in other common fish species. It is distributed from the N-terminus to the C-terminus, therefore, the inventors used the full-length protein sequence as the immunogen. The detailed preparation steps of the above-mentioned immunogen are as follows:

[0025] (1) Construction of the expression vector: After optimizing the cDNA sequence of the protein coding region of Nanos2 through Escherichia coli codons, the artificial synthesis method was used to synthesize the DNA sequence (see SEQ ID NO: 1), and at the same time carry The BamH1 / Xhol1 restriction site was cloned into the pET-28a expression vector by restriction restriction method.

[0026] (2) Plasmid transformation: The constructed pET-28a-Nanos2 plasmid was transfo...

Embodiment 2

[0032] Preparation and purification of embodiment 2 polyclonal antibody

[0033] Immunization of New Zealand big-eared rabbits

[0034] Animal selection: The rabbits are New Zealand white rabbits, young and middle-aged with a weight of about 2.5Kg. Animals were selected as healthy animals with shiny coat color and free movement. Choose good animals and pre-raise them for about 2 weeks. The purpose is to eliminate some unqualified animals so that the later experiments can be carried out smoothly.

[0035] Preparation before the experiment: Rabbits are marked.

[0036] Antigen preparation: (1) Take the antigen out of the -20 degree refrigerator, dissolve it at room temperature, avoid repeated freezing and thawing, and mark the syringe with the item number and animal number. (2) Extract the antigen (the antigen is completely mixed), the antigen concentration is 1mg / ml for the first immunization, 0.5ml / rabbit, and the antigen concentration is halved for the second immunization-...

Embodiment 3

[0057] Example 3 Functional Verification and Application of Polyclonal Antibody

[0058] The polyclonal antibody prepared in Example 2 was used to label the reproductive stem cells in the testis and ovary of zebrafish adult fish, the testis of Yellow River carp and the testis of crucian carp by immunofluorescence, and the results were as follows Figure 4-7 As shown, the positive signal of Nanos2 (green) is specifically highly expressed in the adult zebrafish testis ( Figure 4 ), zebrafish juvenile ovary ( Figure 5 ) and Yellow River carp testis ( Image 6 ), crucian carp testis ( Figure 7 ) in a small number of cells. Observation under the laser confocal microscope revealed that the nucleus of Nanos2-positive cells was larger, the chromatin color in the nucleus was lighter, and there were 2-3 cavities in the nucleus (this is the nucleolus structure), which was in line with the morphological characteristics of reproductive stem cells, so it was inferred that Nanos2 Able...

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Abstract

The invention relates to the field of molecular biology, in particular to a preparation method of a polyclonal antibody capable of marking fish germline stem cells. The antibody is prepared from zebra fish Nanos2 protein as an immunogen immune animal, the prepared antibody can specifically recognize the specific molecular marker protein Nanos2 of germline stem cells, can be used for immunohistochemistry and immunofluorescence detection, specifically marks and detects germline stem cells in zebra fish testis and ovaries, and fills the blank of the application of detecting the fish germline stem cells by using the antibody specificity.

Description

technical field [0001] The invention relates to the field of molecular biology, in particular to a preparation method of a polyclonal antibody capable of marking fish reproductive stem cells. Background technique [0002] The identification and evaluation of fish germline stem cells is fundamental to fish breeding efforts based on germ cell manipulation. In order to conveniently identify germline stem cells in fish adult gonads and effectively assess the self-renewal and maintenance status of germline stem cells, it is necessary to identify and evaluate germline stem cells in fish gonads. At present, the identification methods of fish reproductive stem cells are limited to morphological identification and gene detection, and there is no report on the use of antibodies for detection. Contents of the invention [0003] Based on this, the purpose of the present invention is to provide a method for preparing a polyclonal antibody that can label fish reproductive stem cells. T...

Claims

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Application Information

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IPC IPC(8): C07K16/06C07K16/18G01N33/569G01N33/68
CPCC07K16/065C07K16/18G01N33/56966G01N33/6851G01N2333/4603
Inventor 叶鼎孙永华张泉清何牡丹王厚鹏
Owner INST OF AQUATIC LIFE ACAD SINICA
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