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Method for preparing pET-28a-SUMO-coagulation factor II protein antigen and polyclonal antibody thereof

A pet-28a-sumo-, polyclonal antibody technology, applied in the field of molecular biology, can solve the problem that the role and expression rules are not fully studied and the like

Active Publication Date: 2019-10-18
HUNAN AGRICULTURAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the role and expression of coagulation factor II, an important regulatory factor of coagulation and innate immunity, in grass carp immune response has not been fully studied.

Method used

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  • Method for preparing pET-28a-SUMO-coagulation factor II protein antigen and polyclonal antibody thereof
  • Method for preparing pET-28a-SUMO-coagulation factor II protein antigen and polyclonal antibody thereof
  • Method for preparing pET-28a-SUMO-coagulation factor II protein antigen and polyclonal antibody thereof

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0024] Synthesis of embodiment 1 grass carp coagulation factor II gene fragment

[0025] Based on the grass carp blood coagulation factor II gene sequence (retrieval number KF937688) in the GenBank database of the NCBI website, after using Beacon Desiner software to obtain potential primers, Oligo 6.0 was used to evaluate and finally determine the target gene sequence amplification primers, and then according to the vector homology arm sequence, enzyme The order of the cleavage site and the target gene sequence amplification primer constitutes the primer sequence (primers are synthesized by Wuhan Aibatek Biotechnology Co., Ltd.) that is finally used to amplify the grass carp coagulation factor II gene, wherein the enzyme cleavage sites are NdeI-NotI and Kan+, the primer sequence is as follows:

[0026] pET-28a-SUMO-FII-F: 5'-GAACAGATTGGTGGATCCAATAATAAGGAAGCCCTCT-3', SEQ ID NO.1;

[0027] pET-28a-SUMO-FII-R: 5'-TGCGGCCGCAAGCTTTCCTCCCACAATTCTGC-3', SEQ ID NO.2.

[0028] Using ...

Embodiment 2

[0029] Example 2 Obtaining of recombinant protein pET-28a-SUMO-coagulation factor II antigen

[0030] The amplified grass carp coagulation factor II gene fragment that meets the expectations was purified using the AxyPrepDNA gel recovery kit from Axygen Company, and was purified according to the instructions of the pET-28a-SUMO expression vector (purchased from Huayueyang Biological (Beijing) Technology Co., Ltd.) The purified grass carp blood coagulation factor II gene fragment was connected to the pET-28a-SUMO expression vector. After sequencing to confirm that the connection was correct, it was introduced into Escherichia coli competent cell strain E.coli Rosetta and cultivated to OD 600 0.5-0.6. Then add 0.8mM IPTG, induce for 4 hours at 37°C to achieve bacteriostasis, and use SDS-PAGE gel electrophoresis to purify the target protein to obtain pET-28a-SUMO-coagulation factor II protein (pET-28a-SUMO-coagulation factor II protein (pET -28a-SUMO-blood coagulation factor II ...

Embodiment 3

[0031] The acquisition of embodiment 3 polyclonal antibody

[0032] Select healthy 6-week-old experimental-grade Japanese white rabbits (provided by Wuhan Aibotec Biotechnology Co., Ltd.), and collect 10ml of blood from the ear vein for the first time after one week of stability as a negative control, and then use the prepared pET-28a-SUMO- Coagulation factor II protein was used as an antigen to immunize two experimental-grade Japanese white rabbits for multiple times: the immunization site was 4 parts on the back, each site was injected with 250 μl, the immune dose used for the first immunization was 0.3 mg, and complete Freund’s adjuvant was used. 12 days after the second immunization, the dose of antigen used was 0.15 mg, using incomplete Freund's adjuvant; the third immunization was performed 26 days after the first immunization, the dose of antigen used was 0.15 mg, and incomplete Freund's adjuvant was used. Freund's adjuvant; the fourth immunization was carried out 40 da...

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Abstract

The invention provides a method for preparing a pET-28a-SUMO-coagulation factor II protein antigen and a polyclonal antibody thereof. According to the method, a grass carp coagulation factor II gene sequence is synthesized by using a grass carp coagulation factor II specific primer through a PCR amplification technology, then cloned into a pET-28a-SUMO expression vector, then delivered into E. coli Rosetta for expression and then purified to obtain the pET-28a-SUMO-coagulation factor II protein antigen, the antigen is used for repeated immunization of laboratory-scale Japanese white rabbits, blood sampling is carried out, antiserum is collected, pET-28a-SUMO-coagulation factor II protein is used as the antigen for affinity purification, and the concentrated grass crap coagulation factor IIpolyclonal antibody is obtained. The method can obtain the concentrated grass carp coagulation factor II polyclonal antibody meeting the requirements of subsequent experiments, thereby providing an important experimental reagent for the subsequent detection of the expression of the grass carp coagulation factor II.

Description

technical field [0001] The invention relates to the field of molecular biology, in particular to a preparation method of pET-28a-SUMO-coagulation factor II protein antigen and polyclonal antibody thereof. Background technique [0002] Coagulation factor II, also known as prothrombin, is a vitamin K-dependent coagulation protein mainly produced in the liver. It can not only be converted into thrombin under the action of coagulation factor VII and calcium ions, but also can avoid the occurrence of thrombosis caused by excessive thrombin formation through negative feedback regulation, and is an important factor for balancing procoagulant and anticoagulant. In addition, studies have shown that human coagulation factor II gene is closely related to the recognition and interaction of different bacteria and extracellular proteins (such as phospholipids and coagulation enzymes), and both pathogen recognition and coagulation play important roles in the innate immune system. Therefor...

Claims

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Application Information

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IPC IPC(8): C12N15/70C07K14/745C07K16/36
CPCC07K14/745C07K16/36C12N15/70
Inventor 刘巧林许宝红肖调义陈蕾吕丽刚刘益
Owner HUNAN AGRICULTURAL UNIV
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