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Preparation method of rabbit polyclonal antibody of pregnancy-specific glycoprotein 3

A polyclonal antibody, pregnancy-specific technology, applied in immunoglobulin, chemical instruments and methods, anti-animal/human immunoglobulin, etc., can solve the problem of lack of titer

Inactive Publication Date: 2019-04-05
BIOHIT BIOTECH HEFEI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] At present, there is no PSG3-related in vitro diagnostic kit on the market, let alone a titer of 1:10 6 PSG3 Related In Vitro Diagnostic Kit

Method used

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  • Preparation method of rabbit polyclonal antibody of pregnancy-specific glycoprotein 3
  • Preparation method of rabbit polyclonal antibody of pregnancy-specific glycoprotein 3
  • Preparation method of rabbit polyclonal antibody of pregnancy-specific glycoprotein 3

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0020] Example 1 Preparation of PSG3 recombinant protein

[0021] The preparation method of PSG3 recombinant protein refers to the steps in the patent application with application number 201610063556.4:

[0022] (1) Fermenting and inducing the recombinant bacteria of the recombinant PSG3 protein, collecting the precipitate and extracting the total protein after the bacterial cell is broken, the recombinant bacteria of the recombinant PSG3 protein is JM109(DE3) / pET-30α-rPSG3 engineering bacteria; wherein, The nucleotide sequence encoding PSG3 is shown in SEQ ID NO: 1, and the amino acid sequence of PSG3 is shown in SEQ ID NO: 2;

[0023] (2) The total protein can be purified by refolding and anion exchange chromatography to obtain a protein solution after purification of the recombinant PSG3 protein;

[0024] (3) Adding a lyoprotectant to the purified protein solution of the recombinant PSG3 protein, and vacuum freeze-drying to obtain the PSG3 recombinant protein.

Embodiment 2

[0025] Example 2 Preparation of PSG3 Antiserum

[0026] (1) Rabbit immunity

[0027] 1) The rabbits used for immunization are New Zealand white rabbits (purchased from the Animal Experiment Center of Anhui Medical University), female, with a body weight of 1.5-2.0kg. Before immunization, the rabbit's ear veins were wiped with 75% alcohol, and the hairs at the veins were plucked Gently tap the ear vein to expand the vein, use a syringe to draw 2 mL of blood from the ear vein, let the whole blood stand at 37°C for 1 hour, centrifuge at 4000rpm for 30 minutes, and collect negative serum;

[0028] 2) For the first immunization, mix and emulsify 1 mL of PSG3 antigen (protein content: 2 mg / mL) and Freund's complete adjuvant at a volume ratio of 1:1. After the emulsified antigen is dropped on cold water for a long time, it is judged that the emulsification is complete. Inject the emulsified antigen subcutaneously at multiple points on the back of the rabbit, the injection point is n...

Embodiment 3

[0036] Example 3 Purification and purity identification of PSG3 polyclonal antibody

[0037] 1) Affinity chromatography purification, purification with rProteinG column, used with a matching purification instrument (instrument model: AKTA primeplus, instrument manufacturer: GE);

[0038] 2) After purification, the antibody was dialyzed in PBS. After the dialysis was completed, the antibody was sterile filtered, subpackaged, and stored at 2-8°C;

[0039] 3) Identification of polyclonal antibody, 10 μg of purified polyclonal antibody was used for SDS-PAGE identification, and the identification result showed that the purity was greater than 95%.

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Abstract

The invention relates to the technical field of biology and in particular relates to a preparation method of a rabbit polyclonal antibody of pregnancy-specific glycoprotein 3 (PSG3). A prokaryotic expression recombinant bacterium of recombinant expression PSG3 is fermented and subjected to induction expression; after purification, high-purity PSG3 recombinant protein is obtained; the protein is used as an immunogen to immunize New Zealand white rabbits to obtain immune serum; affinity chromatography purification is carried out on antiserum to obtain the high-purity PSG3 rabbit polyclonal antibody. After the purified antibody is subjected to SDS-PAGE (Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis), the purity is greater than 95 percent; the antiserum polyclonal valence and the purified polyclonal valence are greater than 1 to 106; after being sterilely filtered, the antibody can be stored for a long period at 2 to 8 DEG C and can be stored for 2 years at room temperature.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a method for preparing a rabbit polyclonal antibody of pregnancy-specific glycoprotein 3. Background technique [0002] Pregnancy Specific Glycoprotein (PSG) is a placental polypeptide, which was isolated and purified from human placenta by German scientist Bohn in 1971, and scholars from various countries have conducted extensive research on it. It was found that PSG is closely related to early pregnancy complications, intrauterine fetal growth status and digestive tract tumor diseases, and it has an indicative role in the diagnosis of clinically related diseases. Japanese scholars reported that pregnancy-specific glycoprotein 1 (Pregnancy Specific Glycoprotein 1, PSG1) can be detected in the blood of patients with cervical cancer, uterine body cancer, ovarian cancer, breast cancer, lymphoma and gastrointestinal malignant tumors. Patients with PSG1 in the cytoplasm had a shorter su...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/18
CPCC07K16/18C07K2317/20
Inventor 鲁江徽刘峰谢龙生
Owner BIOHIT BIOTECH HEFEI
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