CN115028718A - Preparation method and application of USF3 rabbit polyclonal antibody - Google Patents

Abstract

The invention discloses a preparation method and application of a USF3 rabbit polyclonal antibody, wherein 1-180aa is selected by using a USF3 protein sequence and a plasmid template, and pET-28a-SUMO and prokaryotic expression immunogen are constructed by whole gene synthesis; then, the USF3 recombinant protein fragment is used as an antigen to immunize two experimental-grade white rabbits from big ears of Japan, and then, the antibodies after affinity purification are collected; then carrying out titer detection; and finally, carrying out polyclonal antibody affinity purification and western verification. The polyclonal antibody prepared by the invention lays a foundation for realizing qualitative and quantitative detection of the transcription factor USF3 in different tissues, organs and cells.

Description

A kind of preparation method and application of USF3 rabbit polyclonal antibody

technical field

The invention relates to the field of bioengineering, in particular to a preparation method of a USF3 rabbit polyclonal antibody.

Background technique

Upstream Transcription Factor Family Member 3 (USF3), consisting of 2221 amino acids, is widely expressed in different tissues and cells. Structural analysis of the protein indicated that USF3 functions by forming protein dimers, a bHLH domain responsible for transcriptional regulation, and serine- and glutamine-rich regions. Existing literature shows that gene deletion in the glutamine-rich region of the USF3 gene is associated with susceptibility to thyroid cancer and negatively regulates epithelial-to-mesenchymal transition (EMT). Therefore, as a transcription factor, USF3 is likely to be involved in the occurrence and development of tumors.

However, due to the very large molecular weight of USF3 protein, there are currently no immunoprecipitated antibodies designed against USF3 protein on the market, which greatly limits the study of the function of USF3 protein. Therefore, a specific preparation can be used for western blotting. It is particularly important to detect the protein of USF3.

SUMMARY OF THE INVENTION

In order to solve the problems of the prior art, the object of the present invention is to overcome the deficiencies of the prior art, provide a USF3 rabbit polyclonal antibody and a preparation method thereof, and provide a polyclonal antibody and its detection in cytology and zoology. The application lays the foundation for the quantitative detection of USF3 protein and the final study of the function of USF3.

In order to achieve the above-mentioned purpose of invention and creation, the present invention adopts the following technical solutions:

A preparation method of USF3 rabbit polyclonal antibody, comprising the following steps:

a) Antigen preparation:

Through prokaryotic expression, the USF3 recombinant protein fragment was obtained;

b) Immunized animals:

Using USF3 recombinant protein fragments as antigens, immunized white rabbits;

c) titer detection:

The tested antibody samples were collected from the immunized white rabbits, and the indirect ELISA method was used to dilute the antibody in a gradient manner, and to detect the titer and sensitivity of the antibody;

d) Post-processing of polyclonal antibody purification:

Antigen affinity purification method was used to obtain polyclonal antibodies.

Preferably, in the step a), the USF3 recombinant protein sequence and plasmid template are selected from 1-180aa, constructed into pET-28a-SUMO by total gene synthesis, and the immunogen is expressed prokaryotically.

Further preferably, in the step a), software is used to analyze the conserved domains and antigenic epitopes of the USF3 protein, and a region with good sequence specificity and a complete domain is selected as the antigenic region, and the region is cloned into pET-28a-SUMO vector.

Preferably, in the step b), the white rabbit immunized with the USF3 recombinant protein fragment is a Japanese white rabbit.

Further preferably, the titer of the produced polyclonal antibody is 1:128000 by indirect ELISA method.

Preferably, in the step c), a biological sample is collected from the immunized white rabbit, and after affinity purification, a sample of the antibody to be tested is obtained; then the indirect ELISA method is used to carry out gradient dilution of the antibody, and the titer of the antibody is detected and sensitivity.

Preferably, in the step d), a protein G affinity chromatography purification method is used to obtain a polyclonal antibody.

The application of a polyclonal antibody in detecting USF3 protein, the polyclonal antibody is the polyclonal antibody prepared by the preparation method of the USF3 rabbit polyclonal antibody according to the present invention.

The application of a polyclonal antibody in the preparation of a reagent for detecting USF3 protein by ELISA method, the polyclonal antibody is the polyclonal antibody prepared by the preparation method of the USF3 rabbit polyclonal antibody according to the present invention.

Compared with the prior art, the present invention has the following obvious outstanding substantive features and significant advantages:

1. The method of the present invention analyzes the antigenicity of the full-length USF3 protein, selects highly hydrophobic fragments, uses the high-purity USF3 protein expressed and purified by the engineered strain as the antigen, and obtains antiserum by immunizing Japanese white rabbits. The obtained antibody has an indirect ELISA titer of 1:128000. The polyclonal antibody can specifically recognize the recombinant USF3 protein expressed in prokaryotic cells, and can detect endogenous USF3 protein in C57BL/6J mouse tissues and different cells, and rabbits with USF3 protein. The construction of polyclonal antibodies provides material and technical support for the detection of the protein in animal and cellular levels;

2. The polyclonal antibody of the present invention lays a foundation for realizing the qualitative and quantitative detection of the transcription factor USF3 in different tissues, organs and cells.

Description of drawings

Fig. 1 is a diagram showing the analysis result of the conserved structural domain of Mus musculus USF3 according to a preferred embodiment of the present invention.

Fig. 2 is the analysis diagram of the Mus musculus USF3 antigenic epitope according to the preferred embodiment of the present invention.

Figure 3 is a graph showing the results of SDS-PAGE electrophoresis of USF3 recombinant protein fragments according to a preferred embodiment of the present invention.

FIG. 4 is a graph showing the result of SDS-PAGE electrophoresis after purification of the polyclonal antibody according to the preferred embodiment of the present invention.

FIG. 5 is a graph showing the results of polyclonal antibody titer according to the preferred embodiment of the present invention.

Figure 6 is a diagram showing the Western blot results of the specific detection of USF3 expression in C57BL/6J mouse tissues, organs and different cell lines by the polyclonal antibody according to the preferred embodiment of the present invention.

Detailed ways

The specific embodiments of the present invention will be described in detail below with reference to the accompanying drawings, but it should be understood that the protection scope of the present invention is not limited by the specific embodiments.

The experimental methods used in the following examples are conventional methods unless otherwise specified.

Materials, reagents, etc. used in the following examples can be purchased from commercial sources unless otherwise specified.

USF3 is a transcription factor. Its structural domain analysis is shown in Figure 1, and its antigenic epitope analysis is shown in Figure 2. The 1-180aa segment is selected. This segment has better sequence specificity, contains a complete structural domain, and has good antigenic epitope prediction properties. , has a certain secondary structure, which is conducive to protein expression.

The above scheme will be further described below in conjunction with specific embodiments, and preferred embodiments of the present invention are described in detail as follows:

In the present embodiment, a preparation method of USF3 rabbit polyclonal antibody, comprising the following steps:

1. Preparation of immunizing antigens:

(1) Construction of recombinant protein USF3 fragment expression strain, bacterial culture and lysis

USF3 recombinant protein fragment amino acid sequence:

MPEMTEHETPTKKQHRKKNRETHNAVERHRKKKINAGINRIGELIPCSPALKQSKNMILDQAFKYITELKRQNDELLLNGGSSEQAEEIKKLRKQLEEIQKENGRYIELLKANDICLYDDPTIHWKGNLKTSKVSVVIPSDQVQKNIIVYSNGSQPGGNSQGTAVQGITFNVGHGLQKQT

High-fidelity Fastpfu was used for PCR amplification. After the amplified PCR product was identified by (2.0%) agarose gel electrophoresis, the target band was cut into gel and recovered, and recovered with Universal DNA purification and recovery kit (Tiangen). The product was digested and gel-recovered and the pET28a plasmid fragment subjected to the same enzyme-digestion and gel recovery treatment was ligated at 4°C overnight, transformed into Trans10 competent cells, clones were picked, colony PCR identification was performed, and positive clones were sequenced.

The correctly identified recombinant expression vector pET28a-USF3 was transformed into E. coli Rosetta competent cells, and the bacterial solution was spread on LB plates containing kanamycin (50 μg/mL) and cultured at 37°C overnight. The next day, pick a single clone on the plate and inoculate it in liquid LB medium containing the same concentration of kanamycin for overnight culture at 37°C. The next day, it was inoculated with 1% of the inoculum, cultured at 37°C to an OD ₆₀₀ of 0.6, and added 0.8mM IPTG at 37°C for 4 hours to induce protein expression. After induction, the bacterial solution was dissolved with urea, and the supernatant was collected.

(2) Purification of recombinant protein

The supernatant of the bacterial lysate was purified, and the protein peak fractions were collected and the purity of the protein samples was detected by SDS-PAGE. Figure 3 is a graph showing the results of SDS-PAGE electrophoresis of USF3 recombinant protein fragments. Each lane in Figure 3 is 10ng, 5ng, 1ng, and 500pg antigen respectively; the antibody dilution ratio is 1:1000. As can be seen from Figure 3, electrophoresis-pure USF3 protein was finally obtained through gel filtration, with a molecular weight of about 35KDa.

2. Immunized animals:

Two 4-month-old SPF Japanese white rabbits were immunized with the above quality-controlled USF3 recombinant protein fragments as antigens, with equal volumes of complete Freund's adjuvant (the first immunization) and incomplete Freund's adjuvant ( Booster immunization) mixed and emulsified, fully mixed to the water-in-oil state for subcutaneous immunization, 3 booster immunizations, each immunization interval of 2-3 weeks, then blood was collected for titer detection, the specific immunization times and immunization dose are shown in Table 1 shown:

Table 1. Immunization frequency and immunization dose table

3. Polyclonal antibody purification:

The collected antiserum was purified by antigen affinity with pET-28a-SUMO-USF3 protein, and the concentrated antibody was tested for purity by SDS-PAGE. Figure 4 is a graph showing the results of SDS-PAGE electrophoresis after purification of the polyclonal antibody. The dilution ratio used and the measured E7879 antibody titer table are shown in Table 2:

Table 2. The results of serial dilution of antibodies and detection of antibody titers

dilution ratio E7879 antibody titer 1:1000 1.4692 1:4000 1.339 1:8000 1.269 1:16000 1.0544 1:32000 0.87 1:64000 0.6794 1:12800 0.4416 1:25600 0.2826 1:51200 0.2003 Blank 0.0368

The test results are shown in Table 2 and Figure 4. Store the antibody at -20°C to avoid freezing.

4. Determination of the titer of polyclonal antibodies:

The titer of the purified polyclonal antibody was detected by indirect ELISA with recombinant protein USF3 as antigen. Figure 5 is a graph showing the results of polyclonal antibody titers. The polyclonal antibody titer determination corresponding to the first inset of Figure 5:

Lane 1: C57 mouse spleen tissue protein extract 30ug, incubated with E7879 10ug/ml;

Lane 2: C57 mouse liver tissue protein extract 30ug, incubated with E7879 10ug/ml;

Lane 3: C57 mouse brain tissue protein extract 30ug, incubated with E7879 10ug/ml;

Lane 4: C57 mouse intestinal tissue protein extract 30ug, incubated with E7879 10ug/ml;

Lane 5: C57 mouse kidney tissue protein extract 30ug, incubated with E7879 10ug/ml.

The polyclonal antibody titer determination corresponding to the second inset of Figure 5:

Lane 1: SW480 intestinal cancer cell protein extract 30ug, incubated with E7879 10ug/ml;

Lane 2: U251 glioma cell protein extract 30ug, incubated with E7879 10ug/ml;

Lane 3: 293T cell protein extract 30ug, incubated with E7879 10ug/ml.

As can be seen from Figure 5, the anti-titer of the purified polyclonal antibody was 1:128000 as determined by ELISA.

5. Polyclonal antibody specific detection:

The proteins of C57BL/6J mouse spleen, liver, brain, intestine, kidney and intestinal cancer cells SW480, glioma U251 cells and 293T cells were extracted, run on SDS-PAGE gel, and transferred to membrane with purified polyclonal antibody as primary antibody , HRP Goat Anti-Rabbit IgG (H+L) was used as the secondary antibody, and the western detection results were analyzed by the Odyssey CLx infrared laser two-color image analysis system. As shown in Figure 6, the purified polyclonal antibody can specifically recognize USF3 in endogenous samples.

The foregoing descriptions of specific exemplary embodiments of the present invention have been presented for purposes of illustration and description. These descriptions are not intended to limit the invention to the precise form disclosed, and obviously many changes and modifications are possible in light of the above teachings. The exemplary embodiments were chosen and described for the purpose of explaining certain principles of the invention and their practical applications, to thereby enable others skilled in the art to make and utilize various exemplary embodiments and various different aspects of the invention. Choose and change. The scope of the invention is intended to be defined by the claims and their equivalents.

The above embodiments of the present invention relate to a method for preparing a USF3 rabbit polyclonal antibody in the field of bioengineering technology. The method for preparing the polyclonal antibody, USF3 protein sequence and plasmid template, select 1-180aa, and construct by whole gene synthesis To pET-28a-SUMO, prokaryotic expression of the immunogen; then the USF3 recombinant protein fragment was used as an antigen to immunize two experimental Japanese white rabbits, and then the affinity-purified antibodies were collected; then the titer was tested; finally, polyclonal Antibody affinity purification and western verification. The polyclonal antibodies prepared in the above embodiments of the present invention lay a foundation for realizing the qualitative and quantitative detection of the transcription factor USF3 in different tissues, organs and cells.

The embodiments of the present invention have been described above in conjunction with the accompanying drawings, but the present invention is not limited to the above-mentioned embodiments, and various changes can also be made according to the purpose of the invention and creation of the present invention. Changes, modifications, substitutions, combinations or simplifications should be equivalent substitution methods, as long as they meet the purpose of the present invention, as long as they do not deviate from the technical principles and inventive concepts of the present invention, all belong to the protection scope of the present invention.

Claims (9)

1. A preparation method of a USF3 rabbit polyclonal antibody is characterized by comprising the following steps:

a) antigen preparation:

obtaining a USF3 recombinant protein fragment through prokaryotic expression;

b) immunizing animals:

immunizing a white rabbit by using the USF3 recombinant protein fragment as an antigen;

c) and (3) potency detection:

collecting a sample of an antibody to be detected from an immune rabbit body, performing gradient dilution on the antibody by adopting an indirect ELISA method, and detecting the titer and the sensitivity of the antibody;

d) purification post-treatment of polyclonal antibody:

and (3) obtaining the polyclonal antibody by adopting an antigen affinity purification method.

2. The method for preparing rabbit polyclonal antibody USF3 according to claim 1, wherein the method comprises the following steps: in the step a), 1-180aa is selected from a USF3 recombinant protein sequence and a plasmid template, and pET-28a-SUMO is constructed through whole gene synthesis, and the immunogen is expressed in a prokaryotic way.

3. The method for preparing rabbit polyclonal antibody USF3 according to claim 2, wherein the method comprises the following steps: in the step a), the conserved structural domain and the epitope of the USF3 protein are analyzed by software, a region with good sequence specificity and containing the complete structural domain is selected as an antigenic region, and the region is cloned to a pET-28a-SUMO vector.

4. The method for preparing rabbit polyclonal antibody USF3 according to claim 1, wherein the method comprises the following steps: in the step b), the white rabbit immunized by the USF3 recombinant protein fragment is a Japanese big ear white rabbit.

5. The method for preparing rabbit polyclonal antibody USF3 according to claim 4, wherein the method comprises the following steps: the generated polyclonal antibody is detected by an indirect ELISA method, and the titer is 1: 128000.

6. The method for preparing rabbit polyclonal antibody USF3 according to claim 1, wherein the method comprises the following steps: in the step c), biological samples are collected from the bodies of the immunized white rabbits, and after affinity purification, a detected antibody sample is obtained; and then, performing gradient dilution on the antibody by adopting an indirect ELISA method, and detecting the titer and the sensitivity of the antibody.

7. The method for preparing rabbit polyclonal antibody USF3 according to claim 1, wherein the method comprises the following steps: in the step d), a Protein G affinity chromatography purification method is adopted to obtain the polyclonal antibody.

8. The application of the polyclonal antibody in detecting the USF3 protein is characterized in that: the polyclonal antibody is prepared by the preparation method of the USF3 rabbit polyclonal antibody according to claim 1.

9. The application of the polyclonal antibody in preparing the reagent for detecting the USF3 protein by the ELISA method is characterized in that: the polyclonal antibody is prepared by the preparation method of the USF3 rabbit polyclonal antibody according to claim 1.

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