CN109837285B - Citrus allergen Cit 3.01 antigen expression purification, polyclonal antibody preparation and application - Google Patents

Abstract

The invention discloses citrus allergen Cit 3.01 antigen expression purification, polyclonal antibody preparation and application, and particularly discloses a codon-optimized rutaceae plant allergen Cit 3.01 gene, which is characterized by comprising a sequence shown as SEQ ID NO. 2. According to the invention, a prokaryotic expression system of allergen Cit s3.01 is constructed by optimizing a Cit s3.01 gene prokaryotic expression sequence and gene synthesis, a rabbit polyclonal antibody Anti-Cit 3.01 is prepared by using expressed and purified rCit s3.01 protein, the content of the allergen Cit s3.01 in citrus peel and fruit pulp is detected, and the potential allergenicity of the allergen Cit s3.01 in edible parts of citrus fruits can be evaluated by using a method of measuring the intensity of Western blot by using a light density value.

Description

Citrus allergen Cit 3.01 antigen expression purification, polyclonal antibody preparation and application

Technical Field

The invention belongs to the crossing field of plant molecular biology, immunology and food safety detection technology, and more particularly relates to citrus allergen Cit 3.01 antigen expression purification, polyclonal antibody preparation and application, and provides citrus allergen Cit 3.01 antigen expression, protein purification, polyclonal antibody preparation methods and application thereof, namely allergen Cit 3.01 gene prokaryotic expression vector construction, recombinant expression, protein purification, mass spectrum identification and polyclonal antibody preparation, and application thereof in detecting allergen Cit 3.01 protein content in rutaceae plants (especially citrus plants).

Background

According to incomplete statistics, about 4000 to 5000 ten thousand people are allergic to food in China. The symptoms of food allergy are generally local pathological conditions of the oral cavity, in addition to other symptoms such as gastrointestinal symptoms (nausea, vomiting, diarrhea, gastrointestinal colic, etc.), skin symptoms (measles, dermatitis, eczema, angioneurotic edema, cutaneous pruritus, etc.), respiratory symptoms (rhinitis, asthma, sore throat, etc.) and cardiovascular symptoms (anaphylactic shock, etc.) [1]. The best treatment for patients is to try to eliminate food allergens in the diet or to completely avoid the types of food allergies that can be caused, but there are many difficulties in practical practice, so identifying and selecting some foods that are free of allergens or hypoallergenic to allergic patients is a better solution.

The orange is a fruit which is popular with people, is considered as a health food at home and abroad, and contains rich nutrient substances. Besides the important edible value of the pulp of the citrus, the peel, the leaf and the pollen all have edible value. The orange peel has higher processing value, is rich in essential oil, pigment, pectin, hesperidin and dietary fiber, and can be used for carrying out microbial fermentation, such as fruit vinegar production, lactic acid beverage production, high-protein feed production and edible fungus cultivation [2]. Orange leaves have high utilization value in traditional Chinese medicines, and have the efficacies of soothing liver, promoting qi circulation, reducing phlegm, killing insects and the like [3]. The orange flower is pungent and warm, has the effects of guiding qi downward, widening diaphragm, harmonizing stomach, removing flatulence, relieving cough and reducing sputum, and has a certain medicinal value, and the orange flower tea scented by the orange flower also has a health-care effect [4]. The processed citrus products have special functions and provide abundant nutrition for people. Despite the benefits of citrus eating on humans, many people are allergic to citrus foods. Research shows that the positive rate of citrus allergen in the Skin Prick Test (SPT) positive dietary allergen of allergic disease children in Dalian province in China is 7.4% [5]. Of the enrolled patients allergic to hay fever pollen, 39% showed cross-allergic symptoms to citrus diet and all patients were positive for IgE-mediated immune response mechanisms by SPT [6]. In addition, as citrus fruits are becoming more abundant, the phenomenon of citrus allergy is also becoming more and more of a concern. How to evaluate the potential sensitization of the existing citrus fruits is developed as an emerging research direction and content in the field of food safety.

It has been shown that Cit s 1, cit s 2 and Cit s3 are the three major citrus allergens [7-10]. Cit s3 is a lipid transporter, a member of the pan-allergen family, and is one of the three major citrus allergens [9,10]. Therefore, the detection of the content of the Cit s3.01 allergen protein in edible value parts (such as fruits, flowers, tender leaves and the like) in the citrus is one of the indexes for evaluating whether the citrus is sensitized or not. At present, the international research on allergen involves little detection of the content of citrus allergen, and a method for directly detecting the content of allergen Cit 3.01 protein is lacked. Compared with an evaluation method for detecting Cit s3.01 gene mRNA level by Real-time fluorescent Quantitative PCR (Quantitative Real-time PCR), the method for directly detecting allergen Cit s3.01 by Western Blot can reflect the content of natural allergens in citrus fruits and can be used for evaluating potential allergenicity of citrus.

Reference documents:

[1] bai Qingyun, the problem of allergies in certain foods chinese food and nutrition, 2005, 7.

[2] Qiao Haiou, ding Xiaowen, zhang Qingzhou comprehensive utilization of citrus peel, zhejiang agricultural sciences, 2003,2 (03): 31-35.

[3] Cui Guojing, liu Fang, he Qiang properties and medicinal values of tangerine leaves, tangerine pith and tangerine seeds, first medicine, 2013,05.

[4] Li Yun. Research on scenting technique of citrus scented tea, modern agriculture technology, 2013,24,278-278.

[5] Qin Peng analysis of skin prick test results of 121 allergic disease children in Dalian region [ D ]. Dalian university of medicine 2012

[6]Rosa Anna Iorio,Stefano Del Duca,et al.Citrus Allergy from Pollen to Clinical Symptoms.PLOS ONE,2013,8(1):e53680.

[7]M.D.

J.Sastre,M.M.San Ireneo,M.T.Laso,D.Barber,M.Lombardero.Different patterns of allergen recognition in children allergic to orange.Journal of Allergy and Clinical Immunology,2004(113):175-177.

[8]O.Ahrazem,M.D.

G.López-Torrejón,R.Sánchez-Monge,J.Sastre,Lombardero,D.Barber,G.Salcedo.Orange germin-like glycoprotein Cit s1:an equivocal allergen.International archives of allergy and immunology,2006(139):96-103.

[9]G.López‐Torrejón,M.Ibanez,O.Ahrazem,R.Sánchez‐Monge,J.Sastre,M.Lombardero,D.Barber,G.Salcedo.Isolation,cloning and allergenic reactivity of natural profilin Cit s 2,a major orange allergen.Allergy,2005(60):1424-1429.

[10]J.F.Crespo,M.Retzek,K.Foetisch,E.Sierra-Maestro,A.B.Cid-Sanchez,C.Y.Pascual,A.Conti,A.Feliu,J.Rodriguez,S.Vieths,S.Scheurer.Germin-like protein Cit s 1and profilin Cit s 2are major allergens in orange(Citrus sinensis)fruits.Molecular Nutrition&Food Research,2006(50):282-290.

Disclosure of Invention

Aiming at the defects or the improvement requirements of the prior art, the invention aims to provide an antigen expression purification method, a polyclonal antibody preparation method and an application of citrus allergen Cit s3.01, wherein a prokaryotic expression system of the allergen Cit s3.01 is constructed by optimizing a Cit s3.01 gene prokaryotic expression sequence and gene synthesis, a rabbit polyclonal antibody Anti-Cit 3.01 is prepared by using expressed and purified rCit s3.01 protein, the content of the allergen Cit s3.01 in citrus peel and citrus pulp is detected, and the potential allergenicity of the allergen Cit s3.01 in edible parts of citrus fruits such as citrus fruits can be evaluated by a method of measuring the strength of Western blot by using a light density value. Compared with the prior art, the method can sensitively and quantitatively detect the content of the citrus allergen Cit 3.01, is very suitable for detecting the rutaceae plant allergen Cit 3.01, is used for evaluating the potential sensitization capability of rutaceae plant citrus fruits, and has important significance for genetic breeding improvement and food safety of rutaceae plants.

To achieve the above objects, according to one aspect of the present invention, there is provided a codon-optimized rutaceae plant allergen Cit 3.01 gene, characterized by comprising the sequence shown in SEQ ID No. 2.

According to another aspect of the invention, the invention provides a primer for constructing a prokaryotic expression vector of the Cit s3.01 gene, which is characterized by comprising a forward primer and a reverse primer, wherein,

the forward primer comprises a sequence shown by SEQ ID NO. 3 or a sequence shown by SEQ ID NO. 5;

the reverse primer comprises a sequence shown by SEQ ID NO. 4 or a sequence shown by SEQ ID NO. 6.

According to a further aspect of the invention there is provided a recombinant protein of rCit s3.01, characterised in that the protein is:

i. 1, a protein consisting of an amino acid sequence shown in SEQ ID NO;

a protein having from 80% to 100% homology with the amino acid sequence defined in SEQ ID No.1, which encodes an amino acid sequence of a protein having the same function;

and iii.a derivative protein with equivalent activity obtained by adding, deleting or replacing one or more amino acids in the amino acid sequence shown in SEQ ID No. 1.

According to a further aspect of the invention there is provided a method for inducible expression of a recombinant protein of rCit s3.01 comprising the steps of: adding IPTG (isopropyl-beta-D-thiogalactoside) with the final concentration of 0.01-1 mmol/L into the recombinant strain in the logarithmic growth phase, and inducing the recombinant strain at the temperature of 12-37 ℃ for 4-24 h to generate rCit s3.01 recombinant protein; preferably, the recombinant strain is added with IPTG (isopropyl thiogalactoside) with the final concentration of 0.1mmol/L in the logarithmic growth phase and induced at the temperature of 20 ℃ for 16h to generate rCit s3.01 recombinant protein;

wherein the recombinant bacterium is escherichia coli which is transferred into a cis 3.01 gene prokaryotic expression vector; the cis 3.01 gene prokaryotic expression vector is specifically constructed by taking the codon-optimized rutaceae plant allergen cis 3.01 gene as a template and utilizing the primer for constructing the cis 3.01 gene prokaryotic expression vector as defined in claim 2 through PCR reaction.

As a further optimization of the invention, the generated rCit s3.01 recombinant protein is further subjected to purification treatment, wherein the purification treatment specifically comprises the steps of collecting the induced recombinant bacteria, washing the recombinant bacteria with PBS, centrifuging, resuspending the recombinant bacteria in an ice-precooled lysis buffer solution, and ultrasonically breaking the bacteria, centrifuging to collect supernatant, filtering the supernatant with a filter membrane, purifying and eluting the obtained filtrate with an HIS nickel column purification system, and collecting eluent to obtain the rCit s3.01 recombinant protein.

According to a further aspect of the invention, there is provided a method of identifying recombinant rCit s3.01 protein, by identifying the protein to be identified and the Cit s3.01 protein by MALDI-TOF/TOF mass spectrometry, and if a valid specific peptide fragment matching the Cit s3.01 protein is identified from the protein to be identified, the protein to be identified belongs to recombinant rCit s3.01 protein; otherwise, the protein to be identified does not belong to rCit s3.01 recombinant protein;

wherein the Cit s3.01 protein has a sequence shown as SEQ ID NO 1.

According to another aspect of the invention, the invention provides a method for preparing Anti-Cit 3.01 rabbit polyclonal antibody, which is characterized in that the method comprises the steps of emulsifying purified rCit 3.01 recombinant protein and injecting the emulsified recombinant protein into the back of a rabbit subcutaneously for multiple times of immunization; then, collecting and separating serum containing Anti-Cit s3.01 rabbit polyclonal antibody;

preferably, the injections are performed four times in total, wherein the first injection is emulsified with Freund's complete adjuvant and the second, third and fourth injections are emulsified with Freund's incomplete adjuvant.

According to yet another aspect of the invention, there is provided an Anti-Cit 3.01 rabbit polyclonal antibody, characterized in that the antibody titer is 1:1 x 10 ³ ～1:1×10 ⁸ The method can specifically detect the Cit s3.01 protein in a sample to be detected;

the Cit s3.01 protein is:

i. 1, a protein consisting of an amino acid sequence shown in SEQ ID NO;

a protein consisting of an amino acid sequence having 80% to 100% homology with the amino acid sequence defined in SEQ ID No.1, which encodes a protein having the same function;

and iii.a derivative protein with equivalent activity obtained by adding, deleting or replacing one or more amino acids in the amino acid sequence shown in SEQ ID No. 1.

According to another aspect of the invention, the invention provides a method for measuring the intensity of Western blot blotting to detect the content of Cit s3.01 protein based on optical density values, which is characterized by comprising the following steps: treating a sample to be detected by using the Anti-Cit s3.01 rabbit polyclonal antibody or the Anti-Cit s3.01 rabbit polyclonal antibody prepared by the method for preparing the Anti-Cit s3.01 rabbit polyclonal antibody, detecting by using Western blot to obtain a blot, performing optical density analysis on the blot obtained by detecting the Western blot by using ImagJ software to obtain a blot strength and weakness value, and counting optical density values to obtain the content of the protein Cit s3.01 in the sample to be detected;

the Cit s3.01 protein is:

i. a protein consisting of an amino acid sequence shown in SEQ ID NO. 1;

a protein having from 80% to 100% homology with the amino acid sequence defined in SEQ ID No.1, which encodes an amino acid sequence of a protein having the same function;

or iii, a derivative protein having equivalent activity by adding, deleting or substituting one or more amino acids in the amino acid sequence shown in SEQ ID No. 1.

According to another aspect of the invention, the invention provides the codon-optimized rutaceae plant allergen cis 3.01 gene, the primer for constructing a cis 3.01 gene prokaryotic expression vector, the rCit s3.01 recombinant protein, the method for inducing expression of the rCit s3.01 recombinant protein, the method for identifying the rCit s3.01 recombinant protein, the method for preparing the Anti-Cit s3.01 rabbit polyclonal antibody, or the application of the method for detecting the content of the cis 3.01 protein based on the intensity of Western blot in detection of the rutaceae plant allergen cis 3.01 protein;

the Cit s3.01 protein is:

i. 1, a protein consisting of an amino acid sequence shown in SEQ ID NO;

a protein having from 80% to 100% homology with the amino acid sequence defined in SEQ ID No.1, which encodes an amino acid sequence of a protein having the same function;

or iii, a derivative protein having equivalent activity by adding, deleting or substituting one or more amino acids in the amino acid sequence shown in SEQ ID No. 1.

Compared with the prior art, the method for detecting the allergen Cit s3.01 of the rutaceae plant (such as citrus plants and the like) through the codon optimized rutaceae plant allergen Cit s3.01 gene, the primer for constructing a prokaryotic expression vector of the Cit s3.01 gene, the rCit s3.01 recombinant protein, the method for inducing and expressing the rCit s3.01 recombinant protein, the method for identifying the rCit s3.01 recombinant protein, the method for preparing the Anti-Cit s3.01 rabbit polyclonal antibody, the method for detecting the content of the protein of the rut s3.01 based on the light density value measurement Western blot strength and the like is provided, and the method for detecting the allergen Cit s3.01 of the rutaceae plant (such as citrus plants and the like) is filled in the blank for detecting the allergen Cit s3.01 of the citrus. People can use the method to detect the allergen Cit s3.01 of the citrus varieties of the rutaceae plants (particularly, western blot is used for detecting the allergen Cit s3.01 of the rutaceae plants), so that the potential allergenicity of the citrus varieties Cit s3.01 of different citrus varieties is evaluated, and the method has important significance for breeding low-allergenicity citrus varieties and selecting low-allergenicity citrus varieties for special consumers and improving food safety.

The invention particularly optimizes the process conditions and parameters of the steps of induction expression and purification of rCit s3.01 recombinant protein, adds the recombinant bacteria into the recombinant bacteria at the logarithmic growth phase with the final concentration of 0.01-1 mmol/L IPTG, and induces the recombinant bacteria at the temperature of 12-37 ℃ for 4-24 h (particularly, adds the recombinant bacteria into the recombinant bacteria at the logarithmic growth phase with the final concentration of 0.1mmol/L IPTG, induces the recombinant bacteria at the temperature of 20 ℃ for 16 h), so that the rCit s3.01 recombinant protein is induced and generated; and for the generated rCit s3.01 recombinant protein, the invention also utilizes a purification process to treat, namely, the induced recombinant bacteria are collected firstly, then PBS washing, centrifugation, heavy suspension in an ice-precooled lysis buffer solution and ultrasonic bacteria breaking treatment are carried out on the recombinant bacteria, then the supernatant is collected by centrifugation, then the supernatant is filtered by a filter membrane, and the obtained filtrate is purified and eluted by an HIS nickel column purification system and then the eluent is collected to obtain the rCit s3.01 recombinant protein. The invention controls the whole process flow adopted by the steps of induction expression and purification of the recombinant protein and the specific process conditions and parameters (such as the types and the proportion of reagents, the treatment temperature and time, the centrifugal treatment time and the centrifugal force and the like) related to each step, so that the rCit s3.01 recombinant protein has high yield and high purity.

The nucleotide sequence comprises a codon optimized rutaceae plant allergen Cit s3.01 gene of a sequence shown in SEQ ID NO. 2, namely the codon optimized nucleotide sequence of the Cit s3.01 gene, and is particularly suitable for escherichia coli expression; when the rCit s3.01 protein is induced and expressed, escherichia coli which is transferred into a Cit s3.01 gene prokaryotic expression vector is used as a recombinant strain (the Cit s3.01 gene prokaryotic expression vector is specifically obtained by taking the codon-optimized rutaceae plant allergen Cit s3.01 gene as a template and constructing through PCR reaction by using the primer for constructing the Cit s3.01 gene prokaryotic expression vector), so that the rCit s3.01 can be expressed in a large amount and in a soluble way; the bacteria after induction expression can be purified to obtain recombinant protein, and the purity of the purified protein is high (the molecular weight of the protein is about 40kDa, for example, 53.9% or more of the total length of the protein of Cit s3.01 can be obtained by identifying the protein through MALDI-TOF/TOF mass spectrum). On the other hand, anti-Cit s3.01 rabbit polyclonal antibody can be prepared, and the titer of the polyclonal antibody is 1:1 x 10 ³ ～1:1×10 ⁸ (e.g. 1 ⁷ Above) has high titer, and can specifically detect the protein Cit s3.01 in the citrus sample. In addition, the invention can also use the optical density value to measure the Western blot blotting strength, for example, the ImagJ software is used to carry out optical density analysis on the blotting strip obtained by Western blot detection to obtain a specific blotting strength value, and the Western blot result can be subjected to significance analysis by counting the optical density value.

Drawings

FIG. 1 is a technical flow chart of citrus allergen Cit 3.01 antigen expression, protein purification, polyclonal antibody preparation and application thereof.

FIG. 2 is a diagram of prokaryotic expression vector construction. (a) a schematic diagram of the constructed prokaryotic expression vector. (b), lane M: DNA Ladder; lane 1: the constructed vector pET-32a (+) - (Cit s 3.01) x 2, namely pET-32a (+)/Trx-His- (Cit s 3.01) x 2; lane 2, double digestion of pET-32a (+)/Trx-His- (Cit s 3.01). Times.2 plasmid.

FIG. 3 shows Trx-His- (Cit s 3.01). Times.2 protein expression and purification. M, protein molecular mass standard; s, inducing the recombinant bacteria by IPTG; p, cracking the supernatant of the recombinant bacteria induced by IPTG; e, purified Trx-His- (Cit s 3.01). Times.2 protein.

FIG. 4 is MALDI-TOF/TOF identification of Cit s3.01 protein. The peptide fragments that were identified efficiently are shown in bold.

FIG. 5 is the Anti-Cit s3.01 antibody-specific detection. The band in lane shows Cit s3.01 (9.46 kDa).

FIG. 6 shows the detection of 21 citrus variety allergens Cit s3.01 by Western blot and statistical analysis. (a) results of Western blot blotting; (b) Western blot banding blots were counted by optical density values for intensity and letters showing different significance differences (P.ltoreq.0.05, n = 3.

Detailed Description

In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is described in further detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and do not limit the invention. In addition, the technical features involved in the respective embodiments of the present invention described below may be combined with each other as long as they do not conflict with each other.

Example 1: allergen Cit 3.01 gene sequence retrieval, codon optimization, and whole-gene synthesis

1.1 allergen protein search

Known allergen information in Citrus is available at the website of the professional Committee for allergen nomenclature of the genus Union of International Immunology (http:// www.allergen.org /), as shown in Table 1.

TABLE 1 Citrus allergen information

Through the amino acid sequence of the allergen Protein Cit 3.01 (GenBank Protein CAI 23765) in the published article, the corresponding allergen gene was searched for and the gene sequence was obtained in the sweet orange genome (http:// citrus. Hzau. Edu. Cn/orange /) through sequence homology, as shown in Table 2.

TABLE 2 Citrus allergen Gene information

1.2 sequence codon optimization and full-gene synthesis of allergen gene Cit s3.01

There is codon bias as the usage frequency of synonymous codons differs between species of different species. Under the condition that the usage frequency of the synonymous codon of the exogenous gene is matched with that of an expression host, the expression level of the exogenous gene can be obviously improved. Therefore, the allergen gene Cit 3.01 of the present invention can be codon optimized using JCat tool (http:// www.jcat.de /), optimized amino acid and nucleotide sequences as shown in Table 3. The amino acid sequence encoded by the codon-optimized cis 3.01 gene is shown in Table 3. The whole gene synthesis is entrusted to Wuhan Jin Kairui bioengineering GmbH.

TABLE 3 codon-optimized nucleotide sequences

Note: cis 3.01 ^＊ Is an amino acid sequence, cit s3.01 is the original nucleotide sequence, cit s3.01 ^# For optimized nucleotideSequence of

Example 2 prokaryotic expression vector construction

2.1 primer design

The primers of the present invention were designed using Primer 5.0 software, and the nucleotide sequences of the primers are shown in Table 4. All primers were synthesized by Beijing Optimala New Biotechnology Ltd.

TABLE 4 primers

Note: in the table, P1 and P3 are forward primers, and P2 and P4 are reverse primers. The bold text 15bp indicates the linker used for vector construction. The linker sequence between the fusion proteins is underlined. The 5 'EcoRI and 3' XhoI cleavage sites are indicated in italics.

2.2 vector construction

The invention constructs a prokaryotic expression vector pET-32a (+)/Trx-His- (Cit 3.01) × 2 by using a method of homologous recombination one-step cloning multi-fragment kit (provided by Nanjing Novozam biotechnology limited). Specifically, the synthesized Cit s3.01 gene is used as a template, P1 and P2; p3, P4; the three pairs of primers P3 and P2 are respectively paired, and three PCR products are obtained through PCR reaction. PCR systems are shown in Table 5, and procedures are shown in Table 6, PCR Master Mix (2X) was purchased from Thermo Fisher Scientific (cat # K0171). Meanwhile, the pET-32a (+) vector was digested with EcoRI and XhoI restriction enzymes. The endonuclease system was referenced to Invitrogen. The three PCR products were mixed with the vector cleavage products according to the method of homologous recombination one-step cloning of the multi-fragment kit, the mixture was treated at 37 ℃ for 30 minutes, and immediately thereafter, it was incubated on ice for 5 minutes. According to the method of Beijing Quanji Biotechnology Limited, the obtained mixture is transformed into E.coli DH5 alpha, and under appropriate culture conditions, positive recombinants are screened and sent to Beijing Optimalaceae New Biotechnology Limited for DNA sequence determination. Taking the clone with complete correct sequence, using

AxyPrep plasmid DNA SmallRecombinant plasmids were extracted with the quantification kit and verified by double digestion with EcoRI and XhoI (fig. 2). The enzyme cutting fragment is 711bp found by agarose electrophoresis, so the invention successfully constructs a prokaryotic expression vector pET-32a (+) - (Cit s 3.01). Times.2.

TABLE 5 PCR System

TABLE 6 PCR reaction conditions

Example 3rCit s3.01 protein inducible expression, protein purification, mass Spectrometry identification

3.1rCit s3.01 protein induced expression

E.coli BL21 (DE 3) competent cells were transformed with the correctly sequenced positive plasmid, inoculated into 5mL LB medium containing 100. Mu.g/mL ampicillin, and shake-cultured overnight at 37 ℃. Transferring the culture solution into 50mL of the culture solution at a ratio of 1: 100 the next day, culturing at 37 deg.C to logarithmic phase (A600 = 0.4-0.6), adding IPTG with final concentration of 0.1mmol/L, inducing at 20 deg.C for 16h, centrifuging at 10000g for 5min, and collecting bacteria; washed with PBS, centrifuged, resuspended in ice-cold lysis buffer (50 mmol/L NaH) ₂ PO ₄ 300mmol/L NaCl, 0.1g/L lysozyme and 1mmol/L PMSF, pH 8.0), ultrasonic bacteria breaking, centrifugation at 10000g for 10min, collecting supernatant, and performing SDS-PAGE analysis (figure 3). According to the analysis of the results of the gel image, rCit s3.01 protein is expressed in the supernatant and is expressed in a soluble manner.

3.2rCit s3.01 protein purification

And (3) centrifuging 1L of bacteria liquid subjected to induced expression at 6000r/min to obtain thalli, adding 40mL of lysis buffer solution to fully resuspend the thalli, adding 1mmol/L protease inhibitor phenylmethylsulfonyl fluoride (PMSF) at the final concentration, and then carrying out ultrasonic lysis under the ice bath condition. The lysate was centrifuged at 12000r/min for 50min at 4 ℃ and the supernatant collected and filtered through a 0.22 μm filter for use. The HIS-labeled nickel-ion protein purification column was equilibrated with 5 column volumes of lysis buffer (50mM Tris,100mM NaCl,5% glycerol), added with lysis buffer, incubated with shaking at 4 ℃ for 4h, and the affinity medium was able to specifically adsorb the target protein, followed by elution of the target protein using imidazole eluent (50mM Tris,100mM NaCl and 5% glycerol,100-200mM imidazole). The eluate was collected and analyzed by SDS-PAGE. The fourth lane in FIG. 3 is the corresponding purified rCit s3.01 protein. The detection result shows that 1L of the bacteria can produce 9.6mg of recombinant protein.

3.3rCit s3.01 protein mass spectrometric identification

The gel strip corresponding to rCit s3.01 protein in the fourth lane of FIG. 3 was removed, and the gel strip was about 40kDa and sent to Xinshengshi Biotech, inc., shanghai for mass spectrometry. And (3) carrying out protein identification on the rCit s3.01 protein by using a MALDI-TOF/TOF protein identification technology. As shown in FIG. 4, the identified effective peptide fragment covers 53.9% of the complete amino acid sequence of the Cit s3.01 protein.

Example 4 preparation of polyclonal antibody and identification of antibody specificity by Western blot

4.1 preparation of polyclonal antibodies

Preparation of polyclonal antibodies was carried out by the firm of the Committee-Funecology (Wuhan) Co. A healthy New Zealand white rabbit was selected, the purified recombinant protein of Cit s3.01 obtained in example 3 (the concentration of the protein in the dispersion was 0.5 mg/ml) was mixed with an equal volume of Freund's complete adjuvant, and the mixture was sonicated to form a water-in-oil state, and injected subcutaneously into the back of the rabbit at a dose of 0.5mg/kg body weight at multiple points, each point being about 300-400. Mu.L. After 1 week re-immunization was performed, and the Cit s3.01 recombinant protein was mixed with an equal volume of incomplete Freund's adjuvant, sonicated, and injected subcutaneously in multiple spots in the back of rabbits at a dose of 0.25mg/kg body weight. The immunization was performed 3 times a week, 1 time a week. At 7d after the 3 rd booster immunization, the rabbit ear marginal vein blood sampling titer (ELISA endpoint method) reached 1.25 × 10 ⁷ Blood is taken from carotid artery, serum is separated, and sterile subpackaged and stored at-80 ℃. Of course, the titer ratio of the polyclonal antibody can be adjusted to satisfy 1:1 × 10 which is usually required for preparing polyclonal antibody ³ ～1:1×10 ⁸ Other specific titers within this range.

4.2Anti-Cit s3.01 Multi-antibody specificity detection

Extracting pulp protein of the Neuhel navel orange by a phenol extraction method. The BCA kit method is adopted for protein quantification, the powder is firstly resuspended in PBS buffer solution before protein quantification, and after full dissolution, the supernatant is centrifuged and then quantified according to the method provided in the kit. Performing SDS-PAGE on the pulp protein of the Newhall navel orange, then transferring the pulp protein to a membrane, taking a rabbit polyclonal serum antibody diluted by 1: 5000 as a primary antibody, and taking an HRP-labeled goat anti-rabbit antibody IgG (H + L) diluted by 1: 10000 as a secondary antibody, and performing Western blot analysis and color development analysis by using an ECL luminescence kit. As shown in FIG. 5, the rabbit polyclonal antibody recognized one band of about 10kDa with no other non-specific bands. The rabbit polyclonal antibody prepared by the invention has high titer and good specificity.

Example 5 application of Anti-Cit 3.01 Multi-antibodies in detection of 21 Citrus variety allergen Cit 3.01

5.1 sample preparation

5.1.1 sample Collection

In the present invention, 21 citrus varieties are used, which are Red Tangerine (RT), native early (BT), egan No. I (ET), criman Ding Ju (CT), wenzhou mandarin orange No. II (GNS), neholl Navel Orange (NNO), washington Navel Orange (WNO), red navel orange (CNO), red summer orange (RRVO), high Ban You (KPP), late white shaddock (WBP), satian Shaddock (STP), mixi honey shaddock (XP), cocktail Grapefruit (CG), star-Rainbo grapefruit (SRG), youke lemon (EL), mexican Leing (ML), rongan kumquat (MK), qing (KT), unknown fire (ST), qiu Hui (FT). The samples are picked in real time from the national orange breeding center of Huazhong agriculture university at mature fruit, and the samples are divided into peels and pulps, frozen by liquid nitrogen and stored in a refrigerator at the temperature of minus 80 ℃.

5.1.2 extraction of fruit proteins by phenol extraction

Preparation of protein extraction buffer (500 mL): sucrose, 119.8g; KCl,3.728g; trisbase;30.275g; EDTA,9.306g; adding about 450ml ddH2O, fully dissolving, and adjusting the pH value to 7.5 by using concentrated HCl; grinding fresh orange pulp, taking 4-5mL of homogenate, putting the homogenate into a 50mL centrifuge tube, and adding 10mL of extraction buffer; oscillating and mixing uniformly, adding 10ml of tris saturated phenol (taking down the lower layer), placing on ice, and oscillating and mixing uniformly for about 4 hours by using a shaking table;fourth, the mixture was centrifuged at 4 ℃ and 5000r/min for 15min. At the moment, the solution is divided into three layers, the upper layer is carefully sucked, and 10mL extraction buffer is added again; fifthly, continuing to put into ice, and oscillating and mixing for 1-2h by using a shaking table; sixthly, centrifuging the mixture for 15min at the temperature of 4 ℃ and at the speed of 5000 r/min. Taking the supernatant, adding 5 times of methanol containing 0.1mol/L ammonium acetate, and keeping the temperature at-20 ℃; after overnight, a clear white flocculent precipitate was visible without shaking. Centrifuging at 4 deg.C and 5000r/min for 15min, and removing supernatant; adding proper amount of acetone, standing at-20 deg.C for 1 hr, centrifuging at 4 deg.C for 15min, and removing supernatant; repeat step 8 twice, then use N ₂ And drying the precipitate. The extracted protein is dried by blowing and stored at-80 ℃. Protein quantification was performed by BCA kit method, powder was resuspended in PBS buffer, and supernatant was centrifuged after sufficient solubilization. Taking supernatant protein, adding 5 times of SDS-PAGE protein loading buffer solution, and keeping the temperature at 100 ℃ for 5min. 5-fold SDS-PAGE protein loading buffer formulation: 250mM Tris-HCl (pH 6.8), 10% (W/V) SDS,0.5% (W/V) BPB,50% (V/V) glycerol, 5% (W/V) beta-mercaptoethanol (2-ME).

5.2 detecting the content of allergen Cit s3.01 in 21 citrus varieties and comparing the differences

The content of Cit s3.01 in 21 citrus varieties is detected by Western blot by using the prepared Anti-Cit s3.01 polyclonal antibody. The citrus samples are collected from the mature period of the fruit and are divided into two parts, namely peel and pulp. The result of Western blot detection is shown in FIG. 6 (a), and β -actin is used as an internal reference. In order to facilitate the analysis of the detection result of the Western blot, the invention utilizes ImagJ software to carry out optical density analysis on the bands. The results are shown in FIG. 6 (b), where the highest content of allergen Cit s3.01 was found in citrus pulp such as Citrus grandis, citrus aurantium, citrus paradisi, citrus natsudai, citrus depressa, and Citrus grandis, and the lowest content of allergen Cit s3.01 was found in Citrus pulp such as Citrus grandis, citrus grandis Youl, citrus dulcis, and Citrus grandis; among these citrus peels, orange I, qing, crimeman Ding Ju, red summer orange, etc., had the highest allergen Cit 3.01 content, qiu Hui, mexican tomon, eulek lemon, late white grapefruit, high Ban You, etc., had the lowest. Therefore, the method can specifically, quantitatively and stably detect the content of the allergen Cit s3.01 in the sweet orange fruits, and further evaluate the potential sensitization of Cit s3.01 in different oranges. Therefore, the method provided by the invention is a reliable method for detecting the allergen Cit s3.01 of the citrus fruits, and has important application value.

In conclusion, the method for detecting the citrus allergen Cit 3.01 provided by the invention fills the blank of detecting the citrus allergen. The invention not only searches the allergen Cit 3.01 gene in the sweet orange genome, but also optimizes the codon in order to improve the expression level of the exogenous gene, synthesizes the nucleotide sequence suitable for prokaryotic expression, is particularly suitable for rutaceae plants, establishes a prokaryotic expression system of the allergen Cit 3.01, and can quickly obtain a large amount of rCit 3.01 protein. Meanwhile, the invention utilizes expressed and purified rCit s3.01 protein to prepare a rabbit polyclonal antibody Anti-Cit s3.01 with specificity and sensitivity. In addition, the content of the allergen Cit s3.01 in the peels and the pulps of 21 citrus varieties is detected, and the potential allergenicity of the allergen Cit 3.01 of the edible parts of the two citrus fruits is evaluated by a method for measuring the strength of Western blot based on an optical density value. By utilizing the method for detecting the citrus allergen Cit 3.01, developed by the invention, the allergen Cit 3.01 detection can be carried out on citrus varieties of rutaceae plants, so that the potential allergenicity of Cit 3.01 in different citrus varieties and different edible parts of the citrus varieties can be evaluated, and the method has important significance for breeding low-allergenicity citrus varieties and special consumers for selecting low-allergenicity citrus varieties and improving food safety.

The pET-32a (+) carrier adopted by the invention is provided with an HIS label which is a common affinity label and does not influence the function and immunogenicity of target protein. In order to improve the purification efficiency, the HIS tag of the vector is fully utilized, the termination codon of the target gene is deleted when a downstream primer is designed, and the T7 terminator of the vector is selected. The recombinant protein is produced by using the escherichia coli, and the method has the advantages of short period, easiness in purification and the like. The invention constructs a prokaryotic expression vector pET-32a (+)/Trx-His- (Cit s 3.01) × 2, successfully induces and expresses protein with the molecular weight of 40kDa after converting E.coli BL21 (DE 3) bacteria, and the protein mainly exists in a soluble form. In the protein purification process, the HIS-labeled nickel ion protein purification column is used for purifying the protein. The HIS nickel column purification system utilizes the principle that nickel chloride in a Ni column can be combined with HIs (histone) labeled protein, and the purification is carried out by imidazole elution. The purified rCit s3.01 protein is high in purity as identified by SDS-PAGE.

The Rutaceae plant in the invention comprises citrus (such as tangerine, mandarin orange, pomelo, orange, lemon and the like) and other common citrus (such as poncirus trifoliata, kumquat and the like). The polyclonal antibody for detecting rutaceae plant allergen Cit 3.01 is suitable for almost all citrus varieties.

The invention utilizes the codon optimization of allergen Cit s3.01, the construction of a prokaryotic expression vector, the recombinant expression, purification and identification of rCit s3.01 and the preparation of a rabbit polyclonal antibody, thereby realizing the application of measuring the strength of Western blot in the detection of rutaceae plant allergen Cit 3.01 through an optical density value. The method can be used for evaluating the potential sensitization capability of citrus edible part Cit 3.01 in rutaceae plants, and has important significance on genetic breeding improvement and food safety of the rutaceae plants.

It will be understood by those skilled in the art that the foregoing is only a preferred embodiment of the present invention, and is not intended to limit the invention, and that any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the scope of the present invention.

Sequence listing

<110> university of agriculture in Huazhong

<120> citrus allergen Cit 3.01 antigen expression purification, polyclonal antibody preparation and application

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<213> Citrus

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Ala Pro Val Thr Asn Ala Ile Thr Cys Gly Gln Val Thr Ala Ser Leu

20 25 30

Ala Pro Cys Ile Pro Phe Leu Arg Thr Gly Gly Arg Phe Pro Pro Pro

35 40 45

Pro Cys Cys Ser Gly Val Arg Ser Leu Asn Gly Ala Ala Arg Thr Thr

50 55 60

Pro Asp Arg Gln Ala Ala Cys Asn Cys Leu Lys Arg Ala Tyr Gly Thr

65 70 75 80

Ile Arg Gly Ile Lys Pro Asn Val Ala Ala Gly Leu Pro Ser Gln Cys

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Gly Val Arg Ile Pro Tyr Lys Ile Ser Pro Ser Thr Asp Cys Ser Arg

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Val Arg

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<213> Citrus

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accggtggtc gtttcccgcc gccgccgtgc tgctctggtg ttcgttctct gaacggtgcg 180

gcgcgtacca ccccggaccg tcaggcggcg tgcaactgcc tgaaacgtgc gtacggtacc 240

atccgtggta tcaaaccgaa cgttgcggcg ggtctgccgt ctcagtgcgg tgttcgtatc 300

ccgtacaaaa tctctccgtc taccgactgc tctcgtgttc gttaa 345

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<213> Artificial Sequence (Artificial Sequence)

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<211> 41

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

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agaaccagaa ccaccagaac ccagaccctg gtcgatcagg t 41

<210> 5

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<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 5

ggttctggtg gttctggttc ttggcagacc tacgttgacg a 41

<210> 6

<211> 41

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

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gtggtggtgg tggtgctcga gttaacgaac acgagagcag t 41

Claims (5)

1. A rutaceae plant allergen Cit 3.01 gene capable of being expressed in an protokaryon system is characterized in that the gene sequence is shown as SEQ ID NO. 2.

2. A primer for constructing the cis 3.01 gene prokaryotic expression vector of claim 1, comprising a forward primer and a reverse primer, wherein,

the sequence of the forward primer is shown as SEQ ID NO. 3 or SEQ ID NO. 5;

the reverse primer sequence is shown in SEQ ID NO. 4 or SEQ ID NO. 6.

3. A method for inducing expression of recombinant rCit s3.01 protein, comprising the following steps: adding IPTG (isopropyl thiogalactoside) with the final concentration of 0.01-1 mmol/L into the recombinant strain in the logarithmic growth phase, and inducing for 4-24 h at the temperature of 12-37 ℃ to generate rCit s3.01 recombinant protein;

wherein the recombinant bacterium is escherichia coli which is transferred into a cis 3.01 gene prokaryotic expression vector; the cis 3.01 gene prokaryotic expression vector is specifically constructed by taking the rutaceae plant allergen cis 3.01 gene which can be expressed in a prokaryotic nucleus system as a template according to claim 1 and utilizing the primers for constructing the cis 3.01 gene prokaryotic expression vector according to claim 1 as described in claim 2 through PCR reaction.

4. The method for inducible expression of rCit s3.01 recombinant protein according to claim 3, wherein the rCit s3.01 recombinant protein is induced to be produced by adding the recombinant bacterium to a final concentration of 0.1mmol/LIPTG during the logarithmic growth phase and inducing for 16h at 20 ℃.

5. The method for inducible expression of rCit s3.01 recombinant protein according to claim 3, wherein the generated rCit s3.01 recombinant protein is further subjected to purification treatment, wherein the purification treatment specifically comprises collecting recombinant bacteria after induction, then performing PBS washing, centrifugation, resuspension in an ice-precooled lysis buffer solution, and ultrasonic bacteria breaking treatment on the recombinant bacteria, then centrifugally collecting a supernatant, then filtering the supernatant with a filter membrane, purifying and eluting the obtained filtrate with a GST affinity column, and then collecting an eluent to obtain the rCit s3.01 recombinant protein.

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