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Preparing method of grass carp complement 5 polyclonal antibody

A polyclonal antibody and complement technology, applied in the field of molecular biology, can solve the problems that the role and expression rules have not been fully studied

Active Publication Date: 2019-09-10
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 complement component 5, which is an important regulator of inflammatory response and immune response, in grass carp immune response has not been fully studied.

Method used

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  • Preparing method of grass carp complement 5 polyclonal antibody
  • Preparing method of grass carp complement 5 polyclonal antibody
  • Preparing method of grass carp complement 5 polyclonal antibody

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0022] Example 1 Synthesis of Grass Carp Complement Component 5 Gene Fragment

[0023] Based on the grass carp complement C5 gene sequence (retrieval number AUW36852.1) in the GenBank database of the NCBI website, primers were designed using Primer Premier 5 software to amplify the target gene sequence, and then amplified according to the vector homology arm sequence, restriction site and target gene sequence The order of the primers constitutes the primer sequence finally used to amplify the grass carp complement component 5 gene (primers were synthesized by Wuhan Aibotec Biotechnology Co., Ltd.), wherein the enzyme cutting sites are NdeI-NotI and Kan+, and the primer sequences are as follows:

[0024] pET-28a-SUMO-C5-F: 5'-GAACAGATTGGTGGATCCGAAAAAGTATACTTAATC-3', SEQ ID NO.1;

[0025] pET-28a-SUMO-C5-R: 5'-TGCGGCCGCAAGCTTAGCAGAAGAAATCGCCTGAAGGAAG-3', SEQ ID NO.2.

[0026] Using the Merck GenElute Animal Genomic DNA Extraction Kit, extract grass carp DNA according to the kit...

Embodiment 2

[0027] Example 2 Obtaining of recombinant protein pET-28a-SUMO-complement component 5 antigen

[0028] The expected amplified grass carp complement component 5 gene fragment was purified using a kit (AxyPrep DNA gel recovery kit from Axygen Company) and purified according to the pET-28a-SUMO expression vector (purchased from Huayueyang Biology (Beijing) Science and Technology Co., Ltd.) instructions to connect the purified grass carp complement component 5 gene fragment to the pET-28a-SUMO expression vector, after sequencing to confirm that the connection is correct, introduce it into the Escherichia coli competent cell strain E.coli Rosetta, and cultivate it 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 the grass carp complement component 5 antigen, pET-28a-SUMO-, whose protein concentration and purity meet the immune requirements. Complement component ...

Embodiment 3

[0029] Example 3 Obtaining of polyclonal antibodies

[0030] 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 obtained antigen to immunize two rabbits multiple times. Experimental grade Japanese big-eared white rabbits, the immunization site is 4 parts on the back, each site is injected with 250 μl, the immunization dose used for the first immunization is 0.6 mg, and complete Freund's adjuvant is used; the second immunization is performed 12 days later, using The dose of grass carp complement component 5 antigen was 0.3 mg, using incomplete Freund's adjuvant; the third immunization was performed 26 days after the first immunization, and the dose of grass carp complement component 5 antigen was 0.3 mg, using incomplete Freund's adjuvant ; The fourth immunization was carried out ...

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Abstract

The invention discloses a preparing method of a grass carp complement 5 polyclonal antibody. According to the preparing method, grass carp complement 5 primers are utilized for synthesizing a grass carp complement 5 gene sequence through a PCR amplification technology, then the gene sequence is cloned to a pET-28a-SUMO expression carrier, then the expression carrier is guided into E.coli Rosetta for expression, and then purification is conducted to obtain a pET-28a-SUMO-complement 5 protein antigen; the antigen immunizes experiment-grade Japanese white rabbits multiple times, then blood is sampled, antiserum is collected, the pET-28a-SUMO-complement 5 protein antigen is adopted for affinity purification, and then the concentrated complement 5 polyclonal antibody is obtained. By means of the method, the concentrated grass carp complement 5 polyclonal antibody meeting the subsequent experiment demand can be obtained, and important experiment materials are provided for subsequent detection of the expression of the grass carp complement 5.

Description

technical field [0001] The invention relates to the field of molecular biology, in particular to a preparation method of grass carp complement component 5 polyclonal antibody. Background technique [0002] Complement component 5 is the fifth effector molecule involved in the activation of the complement system, which plays an important role in regulating inflammation and apoptosis. The function of complement component 5 in inducing the expression of interleukin and tumor necrosis factor in mammals and enhancing the immune response of the body has been verified by a large number of experiments. [0003] Grass carp, as a large amount of freshwater fish cultured in my country, is an important source of aquatic protein for Chinese residents. However, grass carp disease has always threatened the healthy cultivation of grass carp, becoming one of the key bottlenecks restricting large-scale cultivation of grass carp, and an important factor affecting the quality of grass carp. Th...

Claims

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Application Information

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IPC IPC(8): C07K16/18C07K16/06
CPCC07K16/06C07K16/18
Inventor 许宝红苏建明兰时乐刘巧林肖调义
Owner HUNAN AGRICULTURAL UNIV
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