CN114167065A

CN114167065A - Expression and purification of peanut allergenic protein Ara h1 and IgE antibody detection kit prepared from peanut allergenic protein Ara h1

Info

Publication number: CN114167065A
Application number: CN202111683002.1A
Authority: CN
Inventors: 徐珂; 张哲蓉; 陆浩; 张国家
Original assignee: Orient Immunoassay Suzhou Medical Technology Co ltd
Current assignee: Orient Immunoassay Suzhou Medical Technology Co ltd
Priority date: 2021-12-31
Filing date: 2021-12-31
Publication date: 2022-03-11

Abstract

The invention discloses expression and purification of peanut allergenic protein Ara h1 and an IgE antibody detection kit prepared by the same. The IgE antibody detection kit for the peanut allergenic protein Ara h1 comprises a peanut allergenic protein Ara h1 marked with a first substance, wherein the first substance and a second substance are a pair of substances with specific affinity, and the peanut allergenic protein Ara h1 is prepared by the following steps: determining an amino acid sequence of peanut allergenic protein Ara h1 to be expressed, performing codon optimization on the amino acid sequence to obtain a nucleotide sequence of peanut allergenic protein Ara h1 in prokaryotic bacteria, inserting the nucleotide sequence into a pET28b vector to obtain a pET28b-Arah1 vector, and transforming the pET28b-Arah1 vector into prokaryotic expression competent cells for expression to obtain peanut allergenic protein Ara h 1. The IgE antibody detection kit disclosed by the invention can be used for quickly and accurately detecting the IgE antibody of peanut allergenic protein Ara h 1.

Description

Expression and purification of peanut allergenic protein Ara h1 and IgE antibody detection kit prepared from peanut allergenic protein Ara h1

Technical Field

The invention belongs to the technical field of biological detection, and relates to an IgE antibody detection kit for peanut allergenic protein Ara h1 and a preparation method of peanut allergenic protein Ara h1 used by the kit.

Background

Adverse reactions to food fall into two categories, the first being non-immune system mediated and the second being immune system mediated. Food intolerance reactions represented by lactose intolerance and the like are mediated by a non-immune system; whereas food allergies are mediated by the immune system. In recent years, food allergy has brought about an increasing risk to human health. Food allergy refers to an adverse reaction generated after a person takes a certain food, belongs to one of allergic reactions in medicine, and can cause strong reactions in some tissues and organs of the body and even the whole body. Food allergies can present with a variety of symptoms including discomfort to the skin, digestive tract, respiratory tract, and cardiovascular system. Any food may become an allergen.

A person's allergy to a certain food is caused by various factors, including genetic inheritance, personal constitution and living environment. The allergic reaction is classified into four types, i.e., I, II, III, and IV according to the mechanism, and more than 90% of the food allergic reactions belong to IgE-mediated type I allergic reactions. At present, the incidence of food allergy in western countries is 2-8% in children and 1-2% in adults. WHO/FAO certified foods of the 8 major groups of allergies, including peanuts, fish, shellfish, milk, eggs, soybeans, nuts, and wheat, elicit about 90% of food allergies. In the united states, nuts such as peanuts are the main "murder" of the allergic shock that causes death. Peanut allergies are investigated to be relatively common worldwide, with peanut allergies reaching 1.4%, 1.7%, 1.9% and 0.2% in the united states, canada, uk, israel, respectively, of children. 17 kinds of allergenic proteins are found in peanuts, wherein Ara h1 and Ara h2 are considered as two most major allergens, and Ara h1 accounts for 12-16% of the total peanut protein and is the allergenic protein with the highest content in peanuts. Research shows that Ara h1 full-length protein contains 23 linear antigen epitopes, and has strong heat stability and enzymolysis resistance, so that dendritic antigen presenting cells can be activated to cause allergic reaction of organisms.

The main 2 types of methods for detecting the allergen components of peanuts in food are as follows: methods based on allergen gene residue detection, such as Polymerase Chain Reaction (PCR); methods based on the detection of allergenic proteins, such as enzyme linked immunosorbent assay (ELISA), Surface Plasmon Resonance (SPR), mass spectrometry. (1) PCR is a method based on DNA/RNA detection, which is earlier applied to allergen detection, while Chenjiajie and the like in China detect the main peanut allergen Ara h1 gene components in food by adopting two methods of nested PCR and fluorescent real-time quantitative PCR, thereby qualitatively detecting whether the food contains peanut allergen components. However, the PCR technique is a DNA-based detection technique, and does not directly detect the sensitized protein. The modern food industry technology can completely separate protein from other substances such as DNA and the like, thus bringing insurmountable difficulty to the PCR method. (2) The ELISA method utilizes the specific reaction between antibody and antigen to directly detect the sensitized protein. It is one of the common methods for detecting peanut allergens. At present, the detection method for peanut allergen comprises a double-antibody sandwich ELISA method and a competitive ELISA method. However, in practical application, the two methods have the defects of long detection time, high requirements on detection instruments and equipment, need of professional operation and the like; in addition, although it is possible to determine whether or not a peanut is allergic, it is not possible to accurately determine which peanut protein is allergic.

Disclosure of Invention

Aiming at the problems, the invention provides the IgE antibody detection kit for the peanut allergenic protein Ara h1, which can quickly and accurately detect the IgE antibody of the peanut allergenic protein Ara h 1. The invention also provides a preparation method of the peanut allergenic protein Ara h1, which provides a raw material support for the specificity detection of peanut allergenic protein Ara h1 allergy.

According to the first aspect of the invention, an IgE antibody detection kit for peanut allergenic protein Ara h1 comprises peanut allergenic protein Ara h1 marked with a first substance, wherein the first substance and a second substance are a pair of substances with specific affinity, and the peanut allergenic protein Ara h1 is prepared by the following steps: determining an amino acid sequence of peanut allergenic protein Ara h1 to be expressed, performing codon optimization on the amino acid sequence to obtain a nucleotide sequence of peanut allergenic protein Ara h1 in prokaryotic bacteria, inserting the nucleotide sequence into a pET28b vector to obtain a pET28b-Arah1 vector, and transforming the pET28b-Arah1 vector into prokaryotic expression competent cells for expression to obtain peanut allergenic protein Ara h 1.

In one embodiment, the IgE antibody detection kit further comprises a solid phase carrier and an enzyme-labeled anti-human IgE antibody, wherein the solid phase carrier comprises a carrier film, and the second substance is coated on the carrier film.

In a preferred embodiment, the first substance and the second substance are biotin and an anti-biotin antibody, or biotin and streptavidin, respectively.

In a preferred embodiment, the enzyme-labeled anti-human IgE antibody is an HRP-labeled murine anti-human IgE monoclonal antibody, an ALP-labeled murine anti-human IgE monoclonal antibody, or an acridinium ester-labeled murine anti-human IgE monoclonal antibody.

In one embodiment, the amino acid sequence of the peanut allergenic protein Ara h1 to be expressed is shown in SEQ ID NO. 2.

In one embodiment, the nucleotide sequence is set forth in SEQ ID NO. 3.

In one example, NdeI and EcoRI are added at both ends of the nucleotide sequence, GS Linker, 6 XHis and thrombin sites are fused at the N-terminal, and Avi Tag is fused at the C-terminal, and the nucleotide sequence is inserted into a pET28b vector.

According to the second aspect of the invention, the preparation method of the peanut allergenic protein Ara h1 comprises the steps of determining an amino acid sequence of peanut allergenic protein Ara h1 to be expressed, carrying out codon optimization on the amino acid sequence to obtain a nucleotide sequence of the peanut allergenic protein Ara h1 in prokaryotic bacteria, inserting the nucleotide sequence into a pET28b vector to obtain a pET28b-Arah1 vector, transforming the pET28b-Arah1 vector into prokaryotic expression competent cells, and carrying out expression to obtain the peanut allergenic protein Arah 1.

In one embodiment, the amino acid sequence of the peanut allergenic protein Ara h1 is expressed after the amino acid sequence of the peanut allergenic protein Ara h1 is removed from the N-terminal part of the amino acid sequence; and/or the amino acid sequence of the peanut sensitizing protein Ara h1 to be expressed is shown as SEQ ID NO. 2.

In one embodiment, the nucleotide sequence is set forth in SEQ ID NO. 3.

In one embodiment, NdeI and EcoRI are added at two ends of the nucleotide sequence, GS Linker, 6X His and thrombin sites are fused at the N end, Avi Tag is fused at the C end, the nucleotide sequence is inserted into a pET28b vector to obtain a pET28b-Arah1 vector, the pET28b-Arah1 vector is transformed into prokaryotic expression competence BL21(DE3) to prepare a bacterial liquid, the bacterial liquid is added into a culture medium, and an inducer is added to induce expression.

In a specific and preferred embodiment, the peanut allergenic protein Ara h1 is specifically prepared by the following steps:

transforming a pET28b-Arah1 vector into prokaryotic expression competent cells BL21(DE3) to prepare pET28b-Arah1(DE3) bacterial liquid, adding the bacterial liquid into a culture medium containing kanamycin, culturing at 37 ℃ and 200rpm overnight, inoculating the bacterial liquid into the culture medium containing kanamycin the next day according to the proportion of 1: 50, culturing until the light absorption value OD600 is about 0.6, adding IPTG (isopropyl thiogalactoside) until the concentration is 1mmol/L, carrying out induced expression, centrifuging, collecting supernatant, and purifying;

the purified supernatant was analyzed for protein expression by polyacrylamide gel electrophoresis.

In a specific and preferred embodiment, the electrophoresis conditions are such that the concentration of the concentration gel is 5% and the concentration of the separation gel is 12%. The protein staining is performed by a Coomassie brilliant blue staining method, wherein Coomassie brilliant blue R250 is stained for 3h, and an acetic acid-ethanol decolorizing solution is decolorized until the background is colorless.

Compared with the prior art, the technical scheme of the invention has the following beneficial effects:

the IgE antibody detection kit for the peanut allergenic protein Ara h1 can quickly and accurately detect whether the IgE antibody of the peanut allergenic protein Ara h1 exists in a sample, can judge whether peanut protein allergy exists or not, and can determine whether the allergy is caused by the peanut allergenic protein Ara h1 or not; the kit has the advantages of high sensitivity, strong specificity, high detection speed and simple and convenient operation, and is suitable for various occasions such as clinical detection, epidemiological investigation, site quarantine and the like. The method for preparing the peanut allergenic protein Ara h1 has the advantages of high flux, high immunogenicity of the obtained protein, no non-specific binding, concentrated epitope and the like.

Drawings

In order to more clearly illustrate the technical solution of the present invention, the drawings needed to be used in the description of the embodiments will be briefly introduced below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and it is obvious for those skilled in the art to obtain other drawings based on the drawings without creative efforts.

FIG. 1 is a molecular structure diagram of pET28b vector used in the examples of the present invention;

FIG. 2 is a diagram showing the result of SDS-PAGE electrophoresis in the example;

FIG. 3 is a graph of the correlation analysis of the kit of the example with the Thermofeisher peanut f13 allergen-specific IgE antibody detection kit.

Detailed Description

The following detailed description of the preferred embodiments of the invention is provided to enable those skilled in the art to more readily understand the advantages and features of the invention. It should be noted that the description of the embodiments is provided to help understanding of the present invention, but the present invention is not limited thereto. In addition, the technical features involved in the embodiments of the present invention described below may be combined with each other as long as they do not conflict with each other.

At present, no commercial detection kit capable of specifically detecting the Ara h1 peanut protein allergen exists. Although the Thermofish peanut f13 allergen-specific IgE antibody detection kit becomes the gold standard in the current peanut allergy detection, the kit cannot judge whether the peanut allergy is caused by peanut allergenic protein Arah 1. In view of this, the present embodiment provides a kit capable of specifically detecting an IgE antibody of peanut allergenic protein Ara h1, and a preparation method of peanut allergenic protein Ara h1, which can successfully solve the problem of raw material supply, can obtain a high-flux and high-concentration expression product at a time, and solve the problems of experiment result deviation and commercial antibody batch instability caused by the fact that the commercial antigen concentration does not meet the experiment requirement.

The IgE antibody detection kit for peanut sensitized protein Ara h1 in this embodiment includes peanut sensitized protein Ara h1 labeled with a first substance, a mouse anti-human IgE monoclonal antibody labeled with HRP, and a solid phase carrier, wherein the solid phase carrier includes a carrier membrane, and the second substance is coated on the carrier membrane. The first substance and the second substance are a pair of substances having specific affinity, and specifically in this example, the peanut sensitizing protein Ara h1 is labeled with biotin, and the carrier membrane is coated with an anti-biotin antibody. The solid phase carrier specifically comprises a shell with an inner cavity, a water absorption liner is arranged in the shell, and the carrier film is arranged on the upper surface of the water absorption liner. Further, the IgE antibody detection kit comprises a detection card, wherein a plurality of holes are formed in the detection card, and the peanut allergenic protein Ara h1 marked with biotin, the HRP-marked mouse anti-human IgE monoclonal antibody and the solid phase carrier are respectively and independently placed in the holes of the detection card and sealed by a sealing film. In use, the sealing membrane may be removed or punctured to allow removal of the reagents or solid support from the wells.

Peanut allergenic protein Ara Preparation of h1

The antigen raw material for preparing the kit by expression and purification of the peanut allergenic protein Ara h1, namely the peanut allergenic protein Ara h1, is specifically described below.

(1) Obtaining an amino acid sequence SEQ ID NO.1 of peanut allergenic protein Ara h1, which comprises the following steps:

(2) the amino acid sequence at the N-terminal part of the protein is removed to avoid influencing the activity of the protein, and the amino acid sequence SEQ ID NO.2 after removal is as follows:

(3) determination of host expression System

Through the analysis of an amino acid sequence modification model and the prediction of glycosylation sites, the peanut allergenic protein Ara h1 is self-made and adopts a prokaryotic expression system. Meanwhile, through amino acid sequence modification model analysis and glycosylation site analysis, the protein amino acid sequence has no special sequence modification site, so that a prokaryotic expression system is adopted for expression.

(4) Protein codon optimization

The nucleotide sequence SEQ ID NO.3 of the peanut allergenic protein Ara h1 in prokaryotic bacteria is obtained through a codon optimization website, and specifically comprises the following steps:

＞His-Arah1-Avi

(5) construction of prokaryotic expression recombinant vector pET28b-Ara h1

Through the DNA sequence analysis and enzyme cutting site selection, NdeI is added at two ends of the DNA sequence: 5 '-G ↓CTAGC-3' and EcoRI: 5 '-G ↓AATTC-3', and GS Linker, 6X His and thrombin sites are fused at the N end, and Avi Tag is fused at the C end. The DNA sequence is fused to obtain fused gene, and the whole gene is artificially synthesized. The gene is inserted into an expression vector, the expression vector is pET28b (the molecular structure is shown in figure 1), and the target gene is 1359bp (453 aa).

(6) Induced expression and purification of recombinant protein

The pET28b-Arah1 vector was transformed into prokaryotic expression competent cell BL21(DE3), 50. mu.L of pET28b-Arah1(DE3) bacterial solution was added to 5mL LB medium containing 100mg/mL kanamycin at 37 ℃ and 20 ℃Culturing at 0rpm overnight, inoculating into LB culture medium containing kanamycin at a ratio of 1: 50 the next day, and culturing to light absorption OD₆₀₀When the concentration is about 0.6, IPTG is added to the concentration of 1mmol/L, induction expression is carried out for 4 hours, centrifugation is carried out for 30 minutes at 5000rpm, and the supernatant is collected.

Passing the collected supernatant through Ni²⁺Affinity Chromatography (IMAC) was used for purification. Purifying and storing at 4 ℃. The purified supernatant was analyzed for protein expression by SDS-PAGE. Under electrophoresis conditions, the concentration of the concentrated gel is 5 percent, and the concentration of the separation gel is 12 percent. The protein staining is Coomassie brilliant blue staining method, Coomassie brilliant blue R₂₅₀And (5) dyeing for 3h, and decolorizing the acetic acid-ethanol decolorized solution until the background is colorless.

The SDS-PAGE electrophoresis result is shown in figure 2, the preparation method can obtain high-flux and high-concentration expression products once, and solves the problems that the commercial antigen concentration cannot meet the experimental requirement, the experimental result is deviated, the commercial antibody is unstable among batches and the like.

Preparation of the kit

The IgE antibody detection kit is prepared by the following method:

carrying out biotin labeling on the obtained floral sensitive protein Ara h1 by using a biotin rapid labeling kit produced by a commercial Found manufacturer to obtain biotinylated floral sensitive protein Ara h 1;

diluting biotinylated floral sensitive protein Ara h1 to a suitable concentration, and then sucking 20-30 μ L to a reagent hole site in a detection card (a single detection card containing a solid phase carrier coated with an anti-biotin antibody);

thirdly, diluting a commercial HRP-labeled mouse anti-human IgE monoclonal antibody of Medix to a proper concentration according to the dilution factor specified by the merchant, and absorbing 30-45 mu L of enzyme labeling solution added into the detection card;

and fourthly, the detection card is the IgE antibody rapid detection kit of the peanut allergenic protein Ara h 1.

Method of using kit

Contacting the liquid phase serum sample with biotinylated Ara h1 peanut allergenic protein to perform a first incubation reaction to form an antigen-antibody complex; transferring the incubated complex to a carrier membrane of a solid phase carrier for a second incubation reaction, wherein the biotinylated antigen-antibody complex is captured by an anti-biotin antibody coated on the membrane; and adding an HRP-labeled mouse anti-human IgE monoclonal antibody to perform a third incubation reaction on the carrier membrane to form an antigen-antibody-enzyme-labeled secondary antibody complex, cleaning to remove unbound free substances, adding a chromogenic substrate solution, catalyzing the substrate solution to generate a colored product, collecting diffuse reflection light intensity on the carrier membrane by using an integrating sphere detection system of Yino (Suzhou) eastern medicine science and technology Limited, calculating light intensity change, converting the light intensity change into concentration through a standard curve, wherein the faster the light intensity change, the higher the concentration of the specific IgE antibody of the peanut allergenic protein Arah1 in the sample, and the whole reaction process only needs 8 minutes. The whole detection process is completed in the MIA series full-automatic immunoassay analyzer of Yino (Suzhou) medical science and technology Limited, Oriental.

Evaluation of sensitivity and specificity to clinical samples

The kit of the embodiment and the Thermofeisher peanut f13 allergen-specific IgE antibody detection kit are respectively adopted to test samples with uniformly distributed concentrations in a linear range so as to verify the detection effect of the kit. The coincidence rate analysis is carried out on the IgE antibody detection kit of the embodiment and the Thermofish peanut f13 allergen specificity IgE antibody detection kit (fluorescence immunoassay), and the correlation coefficient R²The correlation was good at 0.973, as shown in fig. 3.

The detection kit of the embodiment has the advantages of high detection speed and simple operation while ensuring high sensitivity and specificity, and can particularly judge whether the peanut protein allergy is caused by Ara h 1. The method for preparing the peanut allergenic protein Ara h1 expresses the protein in prokaryotic cells, has the advantages of high flux, high immunogenicity of the obtained protein, no non-specific binding, concentrated epitope and the like.

As used herein, the terms "comprises" and "comprising" are intended to be inclusive and mean that there may be additional steps or elements other than the listed steps or elements. As used herein, the term "and/or" includes any combination of one or more of the associated listed items.

The above embodiments are merely illustrative of the technical ideas and features of the present invention, and are preferred embodiments, which are intended to enable those skilled in the art to understand the contents of the present invention and implement the present invention, and not to limit the scope of the present invention. All equivalent changes or modifications made according to the spirit of the present invention should be covered within the protection scope of the present invention.

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Claims

1. The IgE antibody detection kit of peanut allergenic protein Ara h1 is characterized by comprising peanut allergenic protein Ara h1 marked with a first substance, wherein the first substance and a second substance are a pair of substances with specific affinity, and the peanut allergenic protein Ara h1 is prepared by the following steps: determining an amino acid sequence of peanut allergenic protein Ara h1 to be expressed, performing codon optimization on the amino acid sequence to obtain a nucleotide sequence of peanut allergenic protein Ara h1 in prokaryotic bacteria, inserting the nucleotide sequence into a pET28b vector to obtain a pET28b-Arah1 vector, and transforming the pET28b-Arah1 vector into prokaryotic expression competent cells for expression to obtain peanut allergenic protein Ara h 1.

2. The IgE antibody detection kit of claim 1, further comprising a solid phase carrier and an enzyme-labeled mouse anti-human IgE antibody, wherein the solid phase carrier comprises a carrier membrane, and the second substance is coated on the carrier membrane.

3. The IgE antibody detection kit of claim 1 or 2, wherein the first substance and the second substance are biotin and anti-biotin antibodies, respectively, or biotin and streptavidin; and/or the enzyme-labeled anti-human IgE antibody is an HRP-labeled mouse anti-human IgE monoclonal antibody, an ALP-labeled mouse anti-human IgE monoclonal antibody or an acridinium ester-labeled mouse anti-human IgE monoclonal antibody.

4. The IgE antibody detection kit according to claim 1, wherein the amino acid sequence of the peanut allergenic protein Ara h1 to be expressed is shown as SEQ ID No. 2; and/or the nucleotide sequence is shown as SEQ ID NO. 3.

5. The IgE antibody detection kit according to claim 1, wherein NdeI and EcoRI are added at two ends of the nucleotide sequence, a GS Linker, 6X His and thrombin site are fused at the N end, and Avi Tag is fused at the C end, and the kit is inserted into a pET28b vector.

6. The preparation method of the peanut allergenic protein Arah1 is characterized by determining an amino acid sequence of peanut allergenic protein Ara h1 to be expressed, carrying out codon optimization on the amino acid sequence to obtain a nucleotide sequence of the peanut allergenic protein Ara h1 in prokaryotic bacteria, inserting the nucleotide sequence into a pET28b vector to obtain a pET28b-Arah1 vector, and transforming the pET28b-Arah1 vector into prokaryotic expression competent cells for expression to obtain the peanut allergenic protein Ara h 1.

7. The preparation method according to claim 6, wherein the amino acid sequence of the peanut sensitizing protein Ara h1 is obtained by removing the N-terminal part of the amino acid sequence of the peanut sensitizing protein Ara h 1; and/or the amino acid sequence of the peanut sensitizing protein Ara h1 to be expressed is shown as SEQ ID NO. 2.

8. The method according to claim 6, wherein the nucleotide sequence is represented by SEQ ID No. 3.

9. The preparation method of claim 6, wherein NdeI and EcoRI are added at both ends of the nucleotide sequence, GS Linker, 6X His and thrombin sites are fused at the N end, Avi Tag is fused at the C end, the nucleotide sequence is inserted into pET28b vector to obtain pET28b-Arah1 vector, the pET28b-Arah1 vector is transformed into prokaryotic expression competence BL21(DE3) to prepare bacterial liquid, the bacterial liquid is added into culture medium, and inducer is added to induce expression.

10. The preparation method according to claim 6, wherein the peanut allergenic protein Ara h1 is specifically prepared by the following steps:

transforming a pET28b-Arah1 vector into prokaryotic expression competence BL21(DE3) to prepare pET28b-Arah1(DE3) bacterial liquid, adding the bacterial liquid into a culture medium containing kanamycin, culturing at 37 ℃ and 200rpm overnight, inoculating the bacterial liquid into the culture medium containing kanamycin in proportion the next day, culturing until the light absorption value OD600 is about 0.6, adding IPTG (isopropyl thiogalactoside) to the concentration of 1mmol/L, carrying out induced expression, centrifuging, collecting supernatant, and purifying;