Preparation method of apostichopus japonicus AjCYTB polyclonal antibody

A polyclonal antibody, a technique for imitating Apostichopus japonicus, which is used in the preparation methods of peptides, botanical equipment and methods, biochemical equipment and methods, etc.

Pending Publication Date: 2021-05-11
DALIAN POLYTECHNIC UNIVERSITY
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  • Summary
  • Abstract
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  • Claims
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AI Technical Summary

Problems solved by technology

[0003] At present, there are few related studies on the AjCYTB protein level of imitation sea cucumber, especially the research on the preparation of AjCYTB antibody. The research on the preparation of polyclonal antibody against AjCYTB will be a great contribution to the research of the internal reference gene of sea cucumber Provide a powerful tool to provide experimental support for the biological research of Apostichopus japonicus

Method used

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preparation example Construction

[0020] The invention provides a method for preparing a polyclonal antibody of A. japonicus japonicus AjCYTB, comprising the following steps:

[0021] (1) Delete the sequence corresponding to the transmembrane region in the A. japonicus japonicus AjCYTB gene, and splicing the remaining non-transmembrane region sequences to obtain the spliced ​​AjCYTB gene sequence;

[0022] (2) Codon optimization is performed on the spliced ​​AjCYTB gene sequence, and the obtained amino acid sequence is shown in SEQ ID NO: 1;

[0023] (3) Construct the AjCYTB expression vector with the codon-optimized AjCYTB gene sequence;

[0024] (4) Transforming the AjCYTB expression vector into competent cells, followed by expanded culture, induced expression, bacterial cell collection, bacterial cell disruption, and protein purification to obtain the AjCYTB protein;

[0025] (5) Animals were immunized twice with AjCYTB protein to obtain AjCYTB polyclonal antibody.

[0026] In the present invention, the e...

Embodiment 1

[0045] (1) Delete the sequence corresponding to the transmembrane region in the A. japonicus japonicus AjCYTB gene, and splicing the remaining non-transmembrane region sequences to obtain the spliced ​​AjCYTB gene sequence;

[0046] (2) Codon optimization is performed on the spliced ​​AjCYTB gene sequence, and the obtained amino acid sequence is shown in SEQ ID NO: 1;

[0047] (3) construct AjCYTB expression vector with codon-optimized AjCYTB gene sequence and pCold II;

[0048] (4) Transform the AjCYTB expression vector into Escherichia coli BL21 competent thallus, expand the culture to OD600=0.4 with LB medium containing 90 μg / ml ampicillin under the condition of 36°C, and then incubate at 14°C with the IPTG concentration of Induced under the condition of 0.1mM for 20h, after the cells were collected, the cells were broken, and the protein was purified using the His tag protein purification kit to obtain the AjCYTB protein;

[0049] (5) The 6-week-old Kunming mice were immu...

Embodiment 2

[0051] (1) Delete the sequence corresponding to the transmembrane region in the A. japonicus japonicus AjCYTB gene, and splicing the remaining non-transmembrane region sequences to obtain the spliced ​​AjCYTB gene sequence;

[0052] (2) Codon optimization is performed on the spliced ​​AjCYTB gene sequence, and the obtained amino acid sequence is shown in SEQ ID NO: 1;

[0053] (3) construct AjCYTB expression vector with codon-optimized AjCYTB gene sequence and pCold II;

[0054](4) Transform the AjCYTB expression vector into Escherichia coli BL21 competent cells, expand the culture to OD600=0.45 with LB medium containing 100 μg / ml ampicillin under the condition of 37 °C, and then at 15 °C, the concentration of IPTG is Induced under the condition of 0.25mM for 22h, after the cells were collected and crushed, the protein was purified using the His-tag protein purification kit to obtain the AjCYTB protein;

[0055] (5) The 6-week-old Kunming mice were immunized twice with AjCYTB...

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Abstract

The invention relates to the technical field of preparation of polyclonal antibodies and provides a preparation method of an apostichopus japonicus AjCYTB polyclonal antibody. The preparation method comprises the following steps of: splicing non-transmembrane region sequences in an apostichopus japonicus AjCYTB gene, then carrying out codon optimization and constructing an AjCYTB expression vector; and transforming the expression vector into competent cells, carrying out enlarged culture, induced expression, thallus collection, thallus breaking and protein purification to obtain AjCYTB protein, and immunizing animals twice by using the AjCYTB protein to obtain the apostichopus japonicus AjCYTB polyclonal antibody. The prepared AjCYTB polyclonal antibody has high sensitivity and relatively high titer, provides a high-quality antibody for detection of the apostichopus japonicus AjCYTB protein, provides a new method and a research direction for preparation of the apostichopus japonicus antibody and plays an important role in promoting development of apostichopus japonicus breeding industry.

Description

technical field [0001] The invention relates to the technical field of polyclonal antibody preparation, in particular to a method for preparing a japonicus japonicus AjCYTB polyclonal antibody. Background technique [0002] Cytochrome b (cytochromeb, CYTB) exists in mitochondria, and participates in the oxidative phosphorylation electron transfer of mitochondria together with other cytochromes. In recent years, researchers have found that AjCYTB is stably expressed in 11 developmental stages of A. japonicus and in the body wall, coelomocytes, intestinal tract and respiratory tree tissues of A. japonicus through transcriptional expression analysis; and in bacterial lipopolysaccharide (LPS) Before and after stimulation, there was no significant difference in the expression of AjCYTB transcript levels in multiple developmental stages of A. japonicus and four tissues of juvenile ginseng, which provided a basis for its feasibility as an internal reference gene. [0003] At prese...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/18C07K16/06C07K14/435C07K1/22C12N15/12C12N15/70
CPCC07K16/18C07K16/065C07K14/43504C12N15/70C12N2800/22
Inventor 王晗王丽君郭荷椰王际辉赵清雅
Owner DALIAN POLYTECHNIC UNIVERSITY
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