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Milk caw PAG1 (Pregnancy-associated glycoprotein 1) polypeptide, and polyclonal antibody and application thereof

A polyclonal antibody, cow technology, applied in the direction of peptide, peptide source, specific peptide, etc., can solve the problem of not realizing localization, and achieve the effect of convenient research and development and debugging, strong application prospect and good specificity

Active Publication Date: 2017-06-13
SHANXI AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, at present, the PAG detection kit and related antibody products have not been localized, and there are no bovine PAG antigen and antibody products on the market.

Method used

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  • Milk caw PAG1 (Pregnancy-associated glycoprotein 1) polypeptide, and polyclonal antibody and application thereof
  • Milk caw PAG1 (Pregnancy-associated glycoprotein 1) polypeptide, and polyclonal antibody and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] Example 1: Design and synthesis of PAG1 polypeptide.

[0029]According to the amino acid sequence of PAG1 published by NCBI, the PAG1 recombinant protein shown in Seq ID No.1 was cloned through E. coli codon optimization. The secondary structure and spatial distribution of the PAG1 recombinant protein antigen were analyzed, and the 25 amino acids at the N-terminal 284-308 of the PAG1 protein antigen epitope were determined to be the target, and the amino acid sequence was Asn Asn Ile His Arg LeuIle Gly Ala Ile Pro Arg Gly Ser Glu His Tyr Val Pro Cys Ser Glu Val Asn Thr, as shown in Seq ID No.4. Finally, Beijing Huada Gene Co., Ltd. was entrusted to synthesize the above peptides, which were denoted as anti PAG1-3.

Embodiment 2

[0030] Example 2: Cross-linking of PAG1 polypeptides with carrier proteins.

[0031] After rewarming the cross-linking agent SPDP reagent at room temperature for 5 minutes, take 4.6 mg SPDP and dissolve it in 740 μl DMSO to obtain an SPDP solution with a final concentration of 20 mM.

[0032] Take 10mg of KLH, dissolve it in 2ml of PBS-EDTA solution, add 50µl of the above SPDP solution, let it stand at room temperature for 1h, then apply HiTrap TM Deaslting column desalting column to elute excess SPDP to obtain coupled BSA-SPDP.

[0033] Take 4 mg of the above synthesized polypeptide and add it into the coupled BSA-SPDP system overnight at room temperature to obtain the immunogen PAG1-3-KLH coupled with the polypeptide and KLH.

Embodiment 3

[0034] Example 3: PAG1 polypeptide immunization and preparation of multiple antisera.

[0035] Take 200 µg of the immunogen prepared in Example 2, dilute it to 200-500 µl with normal saline, add an equal volume of Freund's adjuvant (Freund's complete adjuvant for initial immunization, and Freund's incomplete adjuvant for booster immunization) and mix evenly to form Water in oil. New Zealand white rabbits of about 2.0 kg were used as animals for immunization, and the mixed immunogen was subcutaneously injected into the back for immunization, and 8 to 10 spots were injected, and the amount of immunization was 400 μg of protein. Two weeks later, the first booster immunization was given with a dose of 200 µg protein, the second booster immunization was given one month later with a dose of 200 µg protein, and the third booster immunization was given one month later with a dose of 200 µg protein.

[0036] Negative serum was collected from the ear vein of rabbits before immunization...

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PUM

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Abstract

The invention relates to a milk caw PAG1 (Pregnancy-associated glycoprotein 1) polypeptide and a polyclonal antibody of the polypeptide. The amino acid sequence of the milk caw PAG1 polypeptide is Asn Asn Ile His Arg Leu Ile Gly Ala Ile Pro Arg Gly Ser Glu His Tyr Val Pro Cys Ser Glu Val Asn Thr. The polypeptide and a carrier protein are crosslinked and are then used for immunizing animals; the immunized animal blood is collected for preparing antiserum; separation and purification are performed to obtain the polyclonal antibody. The antibody is applied to milk caw pregnancy detection, and has the advantages of high specificity, high valence and great application prospects.

Description

technical field [0001] The present invention relates to an antigen and its polyclonal antibody, in particular to a polypeptide for dairy cow pregnancy-associated glycoprotein, a polyclonal antibody obtained by immunizing animals with the polypeptide as an antigen, and a preparation method and application of the polyclonal antibody. Background technique [0002] With the development of my country's breeding industry towards intensification and modernization, the reproductive efficiency of female animals has been paid more and more attention. Mismatch, mismatch, early embryo death and repeated infertility during mating will prolong the empty pregnancy period of the female animal, thereby reducing the reproductive rate of the female animal, which is not conducive to the development of the breeding industry. Therefore, it is necessary to carry out early pregnancy diagnosis after mating, so as to strengthen the feeding and management of pregnant female animals, ensure the smooth ...

Claims

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Application Information

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IPC IPC(8): C07K14/47C07K16/18C07K16/06G01N33/68
CPCC07K14/4715C07K16/065C07K16/18G01N33/689G01N2333/471G01N2800/36
Inventor 赫晓燕张昱王海东牛姝曹校瑞
Owner SHANXI AGRI UNIV
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