CN110669131A - Preparation method of apostichopus japonicus AjAIF-1 polyclonal antibody - Google Patents
Preparation method of apostichopus japonicus AjAIF-1 polyclonal antibody Download PDFInfo
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Abstract
The invention discloses a preparation method of an apostichopus japonicus AjAIF-1 polyclonal antibody, which comprises the following steps: s1, carrying out codon optimization on the AjAIF-1 gene according to the apostichopus japonicus genomic database information; s2, constructing an expression vector, realizing the recombinant expression of the AjAIF-1 prokaryotic cell, and transforming the expression vector into an escherichia coli competent cell; s3, carrying out amplification culture, induction expression, thallus collection, thallus crushing and protein purification on the transformed escherichia coli in sequence to obtain the target protein expressed by the AjAIF-1 gene. The AjAIF-1 polyclonal antibody prepared by the invention has high specificity, high sensitivity, higher titer and certain specificity, provides a high-quality antibody for the detection of the AjAIF-1 protein of the apostichopus japonicus, provides a new method and a new research direction for the preparation of the apostichopus japonicus antibody, and plays an important role in promoting the development of the apostichopus japonicus breeding industry.
Description
Technical Field
The invention relates to the technical field of biological correlation, in particular to a preparation method of an apostichopus japonicus AjAIF-1 polyclonal antibody.
Background
The apostichopus japonicus is also called stichopus japonicus. In China, it is mainly distributed in coastal areas of the North, such as Liaoning, Shandong and Hebei. The apostichopus japonicus has high nutritive value and medicinal value, so the apostichopus japonicus becomes one of the important mariculture species in China. At present, the sea cucumber aquaculture industry in the yellow sea and the Bohai sea is rapidly developing. With the continuous expansion of the culture scale of the apostichopus japonicus in China, the probability of tissue injury caused by the sea cucumber in the culture and transportation process is increased, so that the sea cucumber is easy to be infected by bacteria to cause diseases, the health condition of the sea cucumber is influenced, and great economic loss is brought to farmers. Under the background, the research on the expression mode and immune defense mechanism of immune related genes of the apostichopus japonicus is very necessary for improving the disease resistance of the apostichopus japonicus and reducing the economic loss; allograft Inflammatory Factor (AIF-1), one of the genes that plays a major role in Inflammatory responses, was originally cloned by Vtans in 1995 from rat allocardiac transplants and was later discovered in human allocardiac transplants. Initial studies have shown that AIF-1 is involved in immune transplant rejection and inflammatory responses. Subsequent studies found that AIF-1 is highly expressed in tissue damage. At present, the AIF-1 has been widely and deeply researched in vertebrate animals such as human beings and mice, and also plays an important role in the immune reaction process of species such as spinaless animal sponges, sea urchins, oysters, apostichopus japonicus and the like.
At present, related researches aiming at the apostichopus japonicus allograft inflammatory factor AjAIF-1 are few, particularly, no related research report exists at present on the researches such as antibody preparation of the AjAIF-1, and the research on the preparation of the polyclonal antibody of the apostichopus japonicus immune related gene AjAIF-1 provides a powerful tool for the research on the apostichopus japonicus immune mechanism, so that the method plays an important role in reducing the economic loss caused by apostichopus japonicus diseases and promoting the development of the apostichopus japonicus breeding industry.
Disclosure of Invention
The invention aims to provide a preparation method of an apostichopus japonicus AjAIF-1 polyclonal antibody, which can prepare the polyclonal antibody of an apostichopus japonicus immunity related gene AjAIF-1 through research, provide a powerful tool for the research of an apostichopus japonicus immunity mechanism, and further play an important role in reducing economic loss caused by apostichopus japonicus diseases and promoting the development of the apostichopus japonicus breeding industry.
In order to achieve the purpose, the invention provides the following technical scheme: a preparation method of an apostichopus japonicus AjAIF-1 polyclonal antibody comprises the following steps:
s1, carrying out codon optimization on the AjAIF-1 gene according to the apostichopus japonicus genomic database information;
s2, constructing an expression vector, realizing the recombinant expression of the AjAIF-1 prokaryotic cell, and transforming the expression vector into an escherichia coli competent cell;
s3, carrying out amplification culture, induction expression, thallus collection, thallus crushing and protein purification on the transformed escherichia coli in sequence to obtain target protein expressed by AjAIF-1 gene;
s4, diluting the purified AjAIF-1 protein serving as an antigen with physiological saline, performing secondary immunization on an animal serving as an immunogen, and collecting serum of the immunized animal;
s5, ELISA titer detection is carried out on the serum containing the AjAIF-1 antibody, and an antibody with high titer is selected.
Preferably, in S2, the Escherichia coli susceptible strain is Escherichia coli BL21 susceptible strain.
Preferably, in S3, protein purification is performed using a His-tag protein purification kit in the protein purification process.
Preferably, in S4, the animal is selected from 6-8 weeks old Kunming mice.
Preferably, in S4, the AjAIF-1 protein as an antigen requires immunization of an animal twice, wherein the first immunization: the immune amount of each mouse is 20 mug of AjAIF-1 antibody protein, and an adjuvant is prepared by using a five-week standard mouse monoclonal antibody/polyclonal antibody; and (3) second immunization: on day 21, the immunization amount of each mouse was 20 μ g of AjAIF-1 antibody protein, and the adjuvant was prepared using five weeks of standard mouse monoclonal/polyclonal antibody.
Preferably, in S4, serum samples are collected after immunization of mice on day 35.
Preferably, in S5, the antibody is used as an immunogen and is induced at 20 ℃ with IPTG with the final concentration of 0.25mmol/L for 16-20 h.
The invention provides a preparation method of an apostichopus japonicus AjAIF-1 polyclonal antibody, which has the following beneficial effects:
the AjAIF-1 polyclonal antibody prepared by the method can be successfully applied to the detection of the AjAIF-1 protein of the apostichopus japonicus sample by a protein immunoblotting method, has high specificity and high sensitivity, has certain specificity and higher titer, provides a high-quality antibody for the detection of the AjAIF-1 protein of the apostichopus japonicus, provides a new method and a new research direction for the preparation of the apostichopus japonicus antibody, and further plays an important role in reducing the economic loss caused by the disease of the apostichopus japonicus and promoting the development of the apostichopus japonicus breeding industry.
Detailed Description
Example 1:
a preparation method of an apostichopus japonicus AjAIF-1 polyclonal antibody comprises the following steps:
s1, carrying out codon optimization on the AjAIF-1 gene according to the apostichopus japonicus genomic database information;
s2, constructing an expression vector, realizing the recombinant expression of the AjAIF-1 prokaryotic cell, and transforming the expression vector into an escherichia coli competent cell;
s3, carrying out amplification culture, induction expression, thallus collection, thallus crushing and protein purification on the transformed escherichia coli in sequence to obtain target protein expressed by AjAIF-1 gene;
s4, diluting the purified AjAIF-1 protein serving as an antigen with physiological saline, performing secondary immunization on an animal serving as an immunogen, and collecting serum of the immunized animal;
s5, ELISA titer detection is carried out on the serum containing the AjAIF-1 antibody, and an antibody with high titer is selected.
Preferably, in S2, the Escherichia coli susceptible strain is Escherichia coli BL21 susceptible strain.
Preferably, in S3, protein purification is performed using a His-tag protein purification kit in the protein purification process.
Preferably, in S4, the animal is selected from 6 weeks of Kunming mice.
Preferably, in S4, the AjAIF-1 protein as an antigen requires immunization of an animal twice, wherein the first immunization: the immune amount of each mouse is 20 mug of AjAIF-1 antibody protein, and an adjuvant is prepared by using a five-week standard mouse monoclonal antibody/polyclonal antibody; and (3) second immunization: on day 21, the immunization amount of each mouse was 20 μ g of AjAIF-1 antibody protein, and the adjuvant was prepared using five weeks of standard mouse monoclonal/polyclonal antibody.
Preferably, in S4, serum samples are collected after immunization of mice on day 35.
Preferably, in S5, the antibody is used as an immunogen and is induced at 20 ℃ with IPTG at a final concentration of 0.25mmol/L for 16 h.
Example 2:
a preparation method of an apostichopus japonicus AjAIF-1 polyclonal antibody comprises the following steps:
s1, carrying out codon optimization on the AjAIF-1 gene according to the apostichopus japonicus genomic database information;
s2, constructing an expression vector, realizing the recombinant expression of the AjAIF-1 prokaryotic cell, and transforming the expression vector into an escherichia coli competent cell;
s3, carrying out amplification culture, induction expression, thallus collection, thallus crushing and protein purification on the transformed escherichia coli in sequence to obtain target protein expressed by AjAIF-1 gene;
s4, diluting the purified AjAIF-1 protein serving as an antigen with physiological saline, performing secondary immunization on an animal serving as an immunogen, and collecting serum of the immunized animal;
s5, ELISA titer detection is carried out on the serum containing the AjAIF-1 antibody, and an antibody with high titer is selected.
Preferably, in S2, the Escherichia coli susceptible strain is Escherichia coli BL21 susceptible strain.
Preferably, in S3, protein purification is performed using a His-tag protein purification kit in the protein purification process.
Preferably, in S4, the animal is selected from 6-8 weeks old Kunming mice.
Preferably, in S4, the AjAIF-1 protein as an antigen requires immunization of an animal twice, wherein the first immunization: the immune amount of each mouse is 20 mug of AjAIF-1 antibody protein, and an adjuvant is prepared by using a five-week standard mouse monoclonal antibody/polyclonal antibody; and (3) second immunization: on day 21, the immunization amount of each mouse was 20 μ g of AjAIF-1 antibody protein, and the adjuvant was prepared using five weeks of standard mouse monoclonal/polyclonal antibody.
Preferably, in S4, serum samples are collected after immunization of mice on day 35.
Preferably, in S5, the antibody is used as an immunogen and is induced at 20 ℃ with IPTG at a final concentration of 0.25mmol/L for 18 h.
Example 3:
a preparation method of an apostichopus japonicus AjAIF-1 polyclonal antibody comprises the following steps:
s1, carrying out codon optimization on the AjAIF-1 gene according to the apostichopus japonicus genomic database information;
s2, constructing an expression vector, realizing the recombinant expression of the AjAIF-1 prokaryotic cell, and transforming the expression vector into an escherichia coli competent cell;
s3, carrying out amplification culture, induction expression, thallus collection, thallus crushing and protein purification on the transformed escherichia coli in sequence to obtain target protein expressed by AjAIF-1 gene;
s4, diluting the purified AjAIF-1 protein serving as an antigen with physiological saline, performing secondary immunization on an animal serving as an immunogen, and collecting serum of the immunized animal;
s5, ELISA titer detection is carried out on the serum containing the AjAIF-1 antibody, and an antibody with high titer is selected.
Preferably, in S2, the Escherichia coli susceptible strain is Escherichia coli BL21 susceptible strain.
Preferably, in S3, protein purification is performed using a His-tag protein purification kit in the protein purification process.
Preferably, in S4, the animal is selected from 8 weeks of Kunming mice.
Preferably, in S4, the AjAIF-1 protein as an antigen requires immunization of an animal twice, wherein the first immunization: the immune amount of each mouse is 20 mug of AjAIF-1 antibody protein, and an adjuvant is prepared by using a five-week standard mouse monoclonal antibody/polyclonal antibody; and (3) second immunization: on day 21, the immunization amount of each mouse was 20 μ g of AjAIF-1 antibody protein, and the adjuvant was prepared using five weeks of standard mouse monoclonal/polyclonal antibody.
Preferably, in S4, serum samples are collected after immunization of mice on day 35.
Preferably, in S5, the antibody is used as an immunogen and is induced at 20 ℃ with IPTG at a final concentration of 0.25mmol/L for 20 h.
And (4) conclusion:
the invention realizes the recombinant expression of the prokaryotic cell of AjAIF-1 by carrying out codon optimization on the AjAIF-1 gene, converts an expression vector into an escherichia coli competent cell, obtains a target protein expressed by the AjAIF-1 gene by the processes of amplification culture, induction expression, thallus collection, thallus crushing and protein purification, takes the obtained AjAIF-1 protein as an antigen to immunize an animal twice, extracts a serum sample after the animal is immunized, carries out ELISA titer detection, selects an antibody with high titer to obtain the AjAIF-1 polyclonal antibody, has simple and clear preparation method operation, has high specificity and high sensitivity, has certain specificity and high titer, provides a high-quality antibody for the detection of the AjAIF-1 protein, provides a new method and a research direction for the preparation of the apostichopus japonicus antibody, and further plays an important role in reducing economic loss caused by apostichopus japonicus diseases and promoting the development of the apostichopus japonicus breeding industry.
Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that changes, modifications, substitutions and alterations can be made in these embodiments without departing from the principles and spirit of the invention, the scope of which is defined in the appended claims and their equivalents.
Claims (6)
1. A preparation method of an apostichopus japonicus AjAIF-1 polyclonal antibody is characterized by comprising the following preparation steps:
s1, carrying out codon optimization on the AjAIF-1 gene according to the apostichopus japonicus genomic database information;
s2, constructing an expression vector, realizing the recombinant expression of the AjAIF-1 prokaryotic cell, and transforming the expression vector into an escherichia coli competent cell;
s3, carrying out amplification culture, induction expression, thallus collection, thallus crushing and protein purification on the transformed escherichia coli in sequence to obtain target protein expressed by AjAIF-1 gene;
s4, diluting the purified AjAIF-1 protein serving as an antigen with physiological saline, performing secondary immunization on an animal serving as an immunogen, and collecting serum of the immunized animal;
s5, ELISA titer detection is carried out on the serum containing the AjAIF-1 antibody, and an antibody with high titer is selected.
2. The method for preparing the AjAIF-1 polyclonal antibody against Apostichopus japonicus according to claim 1, wherein in S2, the Escherichia coli competent bacteria is Escherichia coli BL21 competent bacteria.
3. The method for preparing the AjAIF-1 polyclonal antibody against Apostichopus japonicus according to claim 1, wherein in S3, the protein is purified by using a His-tag protein purification kit during the protein purification process.
4. The method of claim 1, wherein the animal is selected from Kunming mice at 6-8 weeks in S4.
5. The method for preparing AjAIF-1 polyclonal antibody against Apostichopus japonicus according to claim 1, wherein S4, the AjAIF-1 protein as antigen needs to be immunized into the animal twice, wherein the first immunization: the immune amount of each mouse is 20 mug of AjAIF-1 antibody protein, and an adjuvant is prepared by using a five-week standard mouse monoclonal antibody/polyclonal antibody; and (3) second immunization: on day 21, the immunization amount of each mouse was 20 μ g of AjAIF-1 antibody protein, and the adjuvant was prepared using five weeks of standard mouse monoclonal/polyclonal antibody.
6. The method for preparing the AjAIF-1 polyclonal antibody against Apostichopus japonicus according to claim 1, wherein the serum sample of the immunized mouse is collected at day 35 in S4.
The method for preparing the AjAIF-1 polyclonal antibody of Apostichopus japonicus selenka as claimed in claim 1, wherein the antibody in S5 is used as an immunogen, and is induced at 20 ℃ with IPTG (isopropyl thiogalactoside) at a final concentration of 0.25mmol/L for 16-20 h.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112778415A (en) * | 2021-03-01 | 2021-05-11 | 大连工业大学 | Preparation method of apostichopus japonicus AjCYTB polyclonal antibody |
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2019
- 2019-11-05 CN CN201911070382.4A patent/CN110669131A/en active Pending
Non-Patent Citations (3)
| Title |
|---|
| FRANCESCO DRAGO: "Microglia of Medicinal Leech (Hirudo medicinalis) Express a Specific Activation Marker Homologous to Vertebrate Ionized Calcium-Binding Adapter Molecule 1 (Iba1/alias Aif-1)", 《DEVELOPMENTAL NEUROBIOLOGY》 * |
| NANJING JI: "Cloning and gene expression of allograft inflammatory factor-1 (AIF-1) provide new insights into injury and bacteria response of the sea cucumber Apostichopus japonicus (Selenka, 1867)", 《FISH & SHELLFISH IMMUNOLOGY》 * |
| TILO SCHORN: "Homolog of allograft inflammatory factor-1 induces macrophage migration during innate immune response in leech", 《CELL TISSUE RES》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112778415A (en) * | 2021-03-01 | 2021-05-11 | 大连工业大学 | Preparation method of apostichopus japonicus AjCYTB polyclonal antibody |
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Application publication date: 20200110 |