CN103059109B - Mycoplasma pneumonia antigen, preparation method and immunodetection kit - Google Patents

Abstract

The invention provides a mycoplasma pneumonia (MP) antigen. The antigen fragment of the antigen is MP371 or MP661, wherein the locus of MP 371 is at an N terminal 371-480aa of MP adhesion protein P1, and the locus of MP661 is at an N terminal 661-772aa of MP adhesion protein P1. The invention also relates to a kit comprising any antigen or antigen composition and the application of the kit in detection of a mycoplasma pneumonia antibody. The mycoplasma pneumonia antigen is a recombinant antigen cloned and expressed by a codon-optimized gene and has stronger antigen specificity than that of the antigen cultivated and extracted by MP. The kit can be used for specific detection of an anti-MP-IgM (immunoglobulin m) antibody in clinical samples, thus identifying and diagnosing the infection of mycoplasma pneumonia at early stage.

Description

Mycoplasma pneumoniae antigen, preparation method and immunoassay kit

technical field

The present invention relates to mycoplasma pneumoniae (MP) adhesion protein 1 (P1) codon optimized gene, its antigen, its antigen composition, and also relates to the kit and application containing the antigen or antigen composition.

Background technique

Mycoplasma pneumonia (MP) infection clinically causes atypical pneumonia and bronchitis, and severe cases can lead to complications other than the respiratory tract, such as acute diffuse encephalomyelitis, damage to the blood system, motor system, etc., and the infected objects are mostly Children under 5 years old. The symptoms of pneumonia caused by MP are similar to those caused by other pathogens, but MP has no cell wall structure, and the clinical medicine mainly uses macrolide antibiotics that can penetrate into cells, such as roxithromycin, erythromycin, azithromycin, etc. It is not sensitive to antibiotics used in conventional pneumonia treatment, such as penicillin and cephalosporins (mainly drugs that inhibit the synthesis of pathogenic cell walls). Therefore, the differential diagnosis of MP has good guiding significance for clinical rational drug use.

At present, MP laboratory tests mainly include isolation and culture methods, serological tests, and nucleic acid tests. The isolation and culture method is the gold standard method for MP detection, but because MP grows slowly, the cycle is long, and the positive rate is low, it cannot be used as a routine clinical detection method. Nucleic acid detection is mainly aimed at samples such as patients' throat swabs, sputum, and alveolar lavage fluid. The templates obtained after washing and centrifugation are used for PCR amplification detection, which has the advantages of short time, high sensitivity and specificity, but requires operators to High, and the sample is easily contaminated, and the equipment is expensive, which limits its further application.

At present, the agglutination test developed in Japan is mainly used clinically. The main principle is to use the combination of gelatin particles and the extracted MP antigen to detect the specific antibody of MP in the patient's serum. This method is simple and practical, but it cannot distinguish between IgM and IgG antibodies. In fact, IgM antibodies appear earlier than IgG antibodies and are an important marker of acute MP infection. Although there are currently IgM-ELISA kits, due to the lack of in-depth research on MP epitopes, the sensitivity and specificity of the kits are poor, and it is difficult to meet the clinical detection standards.

The MP genome is a double-stranded circular DNA with a full length of 816394bp, encoding hundreds of proteins. Adhesion is a prerequisite for MP pathogenicity and infection. This process is mediated by the "adhesin complex" on the top of MP. Adhesin P1 not only plays an important role in MP infection and pathogenicity, but also plays an important role in the pathogenic process of MP. immunogen. Stimulating the body to produce a strong immune response, with good immunogen and antigenicity, is an important target for diagnostic antigen research. P1 protein is composed of 1627 amino acids, and its relative molecular weight is about 176.8kD. Chaudhry's research on the P1 epitope showed that: 1160-1521aa located at the C-terminus has better immunoreactivity, can react with 72.7% (24/33) of positive sera, and has a specificity of 100 with 32 negative sera %; while the 60-180aa at the N-terminus has no antigenic activity. Bioinformatics prediction was performed on the epitope of the C-terminus of P1 protein, and the 1154-1521aa segment antigen was screened and expressed. The sensitivity and specificity were 55.6% (20/36) and 100% (45/45), but the The epitopes of the N-terminus and the middle part of the P1 protein are not clear.

Different from other pathogens, MP uses a unique biased codon system. The universal stop codon UGA encodes tryptophan in MP. If the wild gene of MP is used directly, protein translation can be interrupted, which limits the ability of recombinant MP antigens. development. At present, the cloning and expression of recombinant MP antigens usually avoid UGA, and most of the obtained fragment antigens do not contain tryptophan, lack of effective prediction and screening of antigen epitopes, or directly use inactivated whole bacteria to extract antigens, which directly affects Development of later MP detection reagents.

Contents of the invention

In order to obtain a recombinant MP antigen with diagnostic significance, the present invention conducts bioinformatics epitope prediction on the less reported 90-1099aa antigen range of MP adhesion protein P1, and obtains the amino acid sequence of the antigen fragment that may immunoreact with the patient's serum .

In order to overcome the problem of wild MP gene codon bias, the present invention adopts Escherichia coli dominant codons to reverse-translate the amino acid sequence of the above antigen to obtain a brand new MP antigen gene composed of Escherichia coli dominant codons, the gene sequence of which is shown in the sequence listing Shown in the sequences 1-6, the modified MP gene is obtained by annealing extension PCR technology.

The above-mentioned MP gene was cloned and expressed by gene recombination technology to obtain highly expressed MP antigen. Based on the recombinant antigen, the IgM capture method MP-ELISA detection reagent was established, and its diagnostic significance in MP detection was evaluated.

The purpose of the present invention is to provide the position of the epitope in the 90-1099aa interval of the mycoplasma pneumoniae P1 adhesion protein. Another object of the present invention is to provide the above-mentioned antigen epitope gene composed of Escherichia coli dominant codons. The present invention provides the MP recombinant antigen obtained on the basis of the above-mentioned gene and its antigen composition. The present invention also provides a detection kit for mycoplasma pneumoniae P1-IgM antibody established on the basis of the above-mentioned antigen and its composition, which realizes the purpose of accurate detection in the early stage of mycoplasma pneumoniae infection.

Specifically, the present invention provides a Mycoplasma pneumoniae antigen, characterized in that: the antigen fragment is MP371 or MP661, the MP371 antigen is the N-terminal antigen of MP adhesion protein P1, and its site is located at 371-480aa; the MP661 The antigen is the N-terminal antigen of MP adhesion protein P1, and its site is located at 661-772aa.

Further, the structure of the MP371 antigen is shown in SEQ ID NO: 3, and the structure of the MP661 antigen is shown in SEQ ID NO: 4.

The present invention provides a codon-optimized gene of a Mycoplasma pneumoniae antigen, the nucleotide sequence of the MP371 antigen is shown in the list of SEQ ID NO: 9; the nucleotide sequence of the MP661 antigen is shown in the list of SEQ ID NO: 10.

The present invention also provides an application of the above-mentioned codon optimized gene in preparing a drug for detecting Mycoplasma pneumoniae, and the combined application of the MP371 antigen and MP661 antigen in preparing a drug for detecting Mycoplasma pneumoniae.

The present invention also provides a Mycoplasma pneumoniae antigen composition, which contains the above-mentioned MP371 antigen fragment and MP661 antigen fragment.

Further, the C-terminus of the MP371 antigen is connected to the N-terminus of the MP661 antigen.

The present invention also provides a kit for detecting Mycoplasma pneumoniae, wherein any one of the above-mentioned optimized genes or the above-mentioned composition of the kit is provided.

Further, the detection is carried out by using IgM antibody capture method.

The present invention also provides an application of the above kit in detecting IgM antibody of Mycoplasma pneumoniae.

Compared with the prior art, the present invention has the following characteristics:

1. The invention is based on the analysis of antigenic epitopes in the P1 (90-1099aa) interval of Mycoplasma pneumoniae: P1 is an important protein for MP-mediated adhesion and infection, and is also an important antigen that can cause the body’s immune response. Most of the studies on the epitope focus on its C-terminus, and the distribution of epitopes in other regions (90-1099aa) is not clear. In this study, bioinformatics was used to systematically analyze the epitopes and their activities in the above intervals, and the activity of antigens was verified and screened using MP hepatitis serum, so as to obtain new epitopes with diagnostic significance.

2. The invention is based on the establishment of Mycoplasma pneumoniae recombinant antigens obtained by codon optimization technology: At present, Mycoplasma pneumoniae detection reagents use two types of antigens, one is the antigen extracted from the inactivated MP whole bacteria, containing a variety of Membrane antigen components have high sensitivity, but poor specificity, and poor biological safety in the process of pathogen inactivation; the other is the recombinant antigen of Mycoplasma pneumoniae obtained through conventional genetic engineering techniques, because the wild-type Mycoplasma pneumoniae uses biased codons, The recombinant antigen obtained by this method is interrupted at tryptophan, and it is difficult to obtain the complete antigen, resulting in low sensitivity of the antigen; the present invention uses codon optimization technology to obtain the MP optimized gene that does not contain biased codons, and realizes the recombination of the complete MP antigen Expression, to overcome the defects of currently commonly used antigens.

3. The present invention realizes the clinical diagnosis of EV71 on the basis of the IgM capture method: the currently reported MP antigens are all coated with MP antigens and labeled with anti-IgM antibodies due to the limitations of the extraction method, expression level and subsequent antigen labeling process. Horseradish enzyme chromogenic indirect ELISA detection technology. In this study, anti-μ chain monoclonal antibody will be used to coat the enzyme-linked plate, horseradish enzyme will be used to label recombinant VP1 antigen, and the IgM capture method MP-ELISA detection technology will be established, which is comparable to the indirect ELISA technology. , with higher specificity.

Description of drawings

Fig. 1 is the prediction of MP-P1 epitope;

Fig. 2 is the acquisition of the gene of the main antigenic epitope of MP-P1.

Detailed ways

The principles and features of the present invention are described below in conjunction with the accompanying drawings, and the examples given are only used to explain the present invention, and are not intended to limit the scope of the present invention.

In order to achieve the above object, according to the amino acid sequence of Mycoplasma pneumoniae P1, the present invention uses bioinformatics technology to predict the position and corresponding sequence of the antigenic epitope in the P1 (90-1099aa) interval; adopts Escherichia coli dominant codons to reverse translate into nucleotide sequence ; Using annealing extension PCR technology to obtain the modified P1 epitope gene, after genetic engineering expression, a large number of recombinant P1 epitope antigens were purified, and horseradish enzyme-labeled to prepare enzyme-labeled secondary antibodies; IgM capture method was used to achieve Clinical detection of EV71-IgM.

The present invention is achieved through the following technical solutions:

Using the epitope prediction function of the bioinformatics BIOSUN software, predict the main antigenic epitope in the P1 (90-1099aa) interval and the corresponding amino acid sequence (such as 1-6 in the sequence table), and reverse-translate it into Nucleotide sequence (such as 7-12 in the sequence listing).

In order to obtain the above-mentioned genes, the present invention designs 4 pairs of primers for each P1 epitope gene, and performs annealing and extension PCR in 4 times; in order to facilitate gene cloning into the expression vector, two restriction enzymes, BamHI and EcoRI, are introduced at both ends of the tether site to fit the expression vector pGEX-4T-2. After double digestion, insert into the vector pGEX-4T-2 to construct the corresponding expression plasmid. The sequencing method proved that each gene fragment had been correctly inserted.

After the expression plasmid containing the above P1 antigen epitope gene was transformed into E.coliBL21, it was induced by IPTG, and the whole bacterial liquid was taken for SDS-PAGE identification, which proved that the plasmid had been expressed efficiently, and then purified by nickel column and gel filtration , Electrophoretic pure P1 antigen can be obtained. The sodium iodide method was used for horseradish enzyme labeling to prepare the enzyme-labeled antigen complex. The IgM capture method was used to detect the IgM in the serum of patients with Mycoplasma pneumoniae, and the sensitivity and specificity of the detection were evaluated.

Experimental result proves, MP93 provided by the present invention, MP236, MP371, MP661, MP874, MP987 detection rate is respectively 15%, 30%, 65%, 55%, 80%, 20% in 20 patients with mycoplasma pneumoniae ; Specificity is 100%, 80%, 100%, 100%, 30%, 90% in 50 parts of healthy human sera, wherein the MP371 and MP661 antigens obtained by the present invention have MP-specific immunological activity, and the two are used in combination The sensitivity is 80%, the specificity is 100%, and it has a certain application value in the research of diagnostic reagents.

The four aspects involved in the present invention are specifically described below.

1. Prediction of MP-P1 epitope

Using the epitope prediction function of bioinformatics BIOSUN software, it is predicted that the main epitopes in the P1 (90-1099aa) interval are located at 93-199aa, 236-346aa, 371-480aa, 661-772aa, 874-987aa, 987-1099aa, respectively, As shown in Fig. 1, they are respectively named MP93, MP236, MP371, MP661, MP874, and MP987, and the corresponding amino acid sequences are shown in 1-6 in the sequence listing.

2. Codon optimization and acquisition of the main epitope gene of MP-P1

Using bioinformatics software, the amino acid sequences of the above six antigens were reverse-translated into nucleotide sequences using the dominant codons of Escherichia coli, and the transformed six gene sequences are shown in sequences 7-12 in the sequence listing.

Considering the correct rate of domestic gene primer synthesis, the present invention synthesizes 8 primers for each gene design F1, R1, F2, R2, F3, R3, F4, and R4 respectively, and each primer has 16 nucleotides at the end The matching sequence of the acid, each synthetic primer sequence is shown in 13-60 in the sequence listing.

In order to describe the preparation method conveniently, the corresponding gene sequences are named, SEQ ID NO: 13 to 20 are respectively named as MP93F4, MP93F3, MP93F2, MP93F1, MP93R1, MP93R2, MP93R3, MP93R4; SEQ ID NO: 21 to 28 are respectively named as MP236F4, MP236F3, MP236F2, MP236F1, MP236R1, MP236R2, MP236R3, MP236R4; SEQ ID NO: 29 to 36 are named MP371F4, MP371F3, MP371F2, MP371F1, MP371R1, MP371R2, MP371R3, MP371IDR4 respectively; Named MP661F4, MP661F3, MP661F2, MP661F1, MP661R1, MP661R2, MP661R3, MP661R4; SEQ ID NO: 45 to 52 are named MP874F4, MP874F3, MP874F2, MP874F1, MP874R1, MP874R2, MP874R3, MP87; 60 are named MP987F4, MP987F3, MP987F2, MP987F1, MP987R1, MP987R2, MP987R3, MP987R4 respectively.

The method is as follows:

The synthesis of the above genes requires 3 rounds of annealing and extension PCR reactions. The first round of PCR reaction system consists of 25 μl of 2×PCR reaction solution from Biotech, 23 μl of double distilled water, 1 μl of XF1 and XR1 primers, and X represents MP93, MP236, MP371, For MP661, MP874, and MP987, the PCR reaction conditions are 94°C for 1 minute, 94°C for 1 minute, 55°C for 1 minute, 72°C for 1 minute, and 5 cycles; the subsequent n-round PCR reaction system is the 2×PCR reaction of Biotech Company solution 25 μl, double distilled water 22 μl, XFn and XRn primers 1 μl each, n-1 round PCR product 1 μl, n represents 2, 3, 4; the reaction conditions are PCR reaction conditions: 94°C for 3 minutes, 94°C for 1 minute, 55°C 1 minute at ℃, 1 minute at 72 ℃, and 30 cycles, and then extended at 72 ℃ for 5 minutes to obtain six genes, MP93, MP236, MP371, MP661, MP874, and MP987, as shown in FIG. 2 .

3. Cloning, expression and labeling of MP-P1 epitope gene

1. Construction of MP-P1 Major Antigen Epitope Gene Antigen Expression Plasmid

1.1PCR product and expression vector pGEX-4T-2 double digestion

Take 30 μl of each of the above 6 gene products and pGEX-4T-2 expression vector and place them in Eeppendorf centrifuge tubes, add 4 μl of 10×buffer (D), 1 μl of BamHI (10u/μl) and EcoRI (12u/μl), Add sterilized distilled water to 40 μl, and place in a 37°C water bath for enzyme digestion overnight.

Agarose gel electrophoresis purification and recovery of enzyme-cleaved products: PCR product and carrier pGEX-4T-2 are purified with 1.2% agarose gel after double digestion, and the specific method is according to "Molecular Cloning" (Science Press, second edition) method. The purified gene is then recovered with a small amount of gel recovery kit produced by Shanghai Huashun Bioengineering Co., Ltd.: cut out the agarose containing the plasmid and the target gene under ultraviolet light, put them in Eeppendorf centrifuge tubes, add S1 solution to each, Place in a water bath at 55°C for 10 minutes to dissolve the gel, add an equal amount of isopropanol, mix well, and incubate at 55°C for 1 minute, then transfer to adsorption columns, and purify according to the kit instructions.

1.2. Connection: Add 1 μl of the above enzyme-digested vector and target gene, 1 μl of 10×T4DNA Ligasebuffer, 1 μl of T4DNA Ligase (12u/ul) into a sterilized Eeppendorf centrifuge tube, add sterilized distilled water to 10 μl, and place at 16°C overnight .

1.3 Transformation: In the ultra-clean workbench, use a sterile tip to take 100 μl of competent cells (competent cells are carried out according to the method of "Molecular Cloning" (Science Press, second edition)) suspension in Eppendorf, and add the above connection Mix 5 μl by swirling gently, and place in ice bath for 30 minutes. Immediately transfer to a water bath at 42°C for 2 minutes, add 0.5ml LB medium (without antibiotics) to each tube, and after incubating on a shaker in a water bath at 30°C for 60 minutes, take 0.2ml each and smear them on LB agar medium plates (containing Antibiotics), after drying at room temperature, they were placed in an incubator at 37°C and cultured upside down overnight. Select several colonies, inoculate them in LB (5ml/tube), culture overnight, transfer 0.1ml each to LB (2ml/tube) the next day, incubate at 32°C for 3 hours, induce culture with 1mM IPTG for 8h, harvest the bacteria, Identify by SDS-PAGE, select the target gene to obtain high-expression strains, and sequence identification.

2. Antigen expression and purification

2.1 Cultivation of expression strains: Take 20 μl of expression strains stored at -70°C and inoculate them in LB medium (100ml LB/500ml Erlenmeyer flask), culture them on an air shaker at 30°C overnight, and transfer them to LB culture at a ratio of 5% the next day Base (same as above), culture on an air shaker at 30°C for about 3 hours, when the OD600 value reaches 0.7, add 1mM IPTG, induce culture for 8h, combine the bacterial liquid, centrifuge at 6000rpm for 20 minutes, discard the supernatant, and collect the precipitated part.

2.2 Extraction of inclusion bodies: weigh the wet weight of the precipitate, suspend the precipitate with 10 times the volume of 20mmol/L pH8.0TE buffer solution, add lysozyme (1mg/ml suspension), and stir magnetically at room temperature for 10 minutes. Sonicate the bacteria in an ice bath for 30 seconds each time, with an interval of 30 seconds, a total of 10 times. Centrifuge at 1,2000rpm at 8°C for 20 minutes, discard the supernatant, wash the precipitate once with 1mol/L NaCl (prepared with TE), wash twice with TE, and collect the precipitate. The precipitate was dissolved with 8M urea (prepared with PH8.0TE), and 1% β-mercaptoethanol was added. Then at 20°C, 1,2000 rpm, centrifuge for 10 minutes, remove the precipitate and get the supernatant.

2.3 Purification: pass the above-mentioned dissolved inclusion body solution through a nickel ion column, and equilibrately wash with adsorption buffer (pH8.0, 20mmol/L TE containing 6mol/L urea, 0.1% β-mercaptoethanol, 5mM imidazole, 0.25M NaCl) After sample loading, elute with elution buffer (pH8.0, 20mmol/L TE containing 6mol/L urea, 0.1% β-mercaptoethanol, 200mM imidazole), and collect the first elution peak. Pass through the Sephardex G-50 gel filtration column again, the buffer solution adopts pH8.0, 20mmol/L TE contains 0.1% SDS, collects the first elution peak. The eluted antigens were purified and identified by SDS-PAGE.

3. Horseradish enzyme (HRP) labeled MP-P1 epitope antigen

Dissolve 5 mg of HRP in 0.5 ml of 0.2 mol/L pH5.6 acetate buffer; add 0.25 ml of freshly prepared 0.1 mol/L Na IO; mix well. (The color of the solution should change from yellow-brown to dark green at this time)

30min at 4°C; add 0.5ml of 2.5% hexanediol and mix well. Place at room temperature for 30min. (The solution should return to yellow at this point)

Add 5-10mg of the antibody to be labeled, adjust the pH to 9.0 with 1.0mol/L PH9.5CBS, and mix well. Overnight at 4°C; add NaHB40.1ml (0.5mg) and mix well.

After 2 hours at 4°C, dialyze against 0.01mol/L PH7.4PBS, overnight at 4°C. After adding an appropriate amount of neutral glycerin, aliquot it in small quantities.

4. Establishment and application of MP-IgM antibody detection (capture method) technology based on recombinant P1 epitope antigen

1. Establishment of MP-IgM antibody detection (capture method) technology based on recombinant MP-P1 epitope antigen

Anti-IgM-μ chain monoclonal antibody coated enzyme-linked plate (diluted to 2 μg/ml in PBS), 100 μl per well, discarded after overnight at 4 °C, rinsed with distilled water 3 times, patted dry, added 100 μl of 1% BSA to each well, room temperature for 2 hours, Discard liquid. After adding 100 μl sample dilute solution to each well, add 10 μl serum of the sample to be tested respectively, discard the solution at 37°C for 30 minutes, wash the plate with washing solution for 5 times, discard the solution, pat dry, and add horseradish peroxidase-labeled MP- P1 antigen (1:1000) 100μl, incubate at 37°C for 20min, discard the solution and wash the plate 5 times and pat dry. Add TMB color development solution: 50 μl each of A and B solutions, develop color at 37°C in the dark for 10 minutes, add 50 μl of stop solution to each well, and read the OD at 450 nm with a microplate reader. OD>0.1 was judged as positive.

2. Clinical detection of MP-IgM antibody detection (capture method) technology

MP93 provided by the present invention, MP236, MP371, MP661, MP874, MP987 detection rate is respectively 17.5% (7/40), 32.5% (13/40), 65% (26/40) in 40 mycoplasma pneumoniae patients. 40), 55% (22/40), 77.5% (31/40), 17.5% (7/40); the specificity in 50 healthy human sera was 100% (50/50), 80% (40/50 ), 100% (50/50), 100% (50/50), 30% (15/50), 90% (45/50), wherein the MP371 and MP661 antigens obtained by the present invention have MP-specific immunological Activity, the specificity of the combination of the two is 100% (50/50), the sensitivity is 85% (34/40), significantly higher than the use of a single antigen (X ² =8.628; P<0.05); Dia-Maike II) detection sensitivity and specificity were 75% (15/20) and 98% (49/50), the two detection methods have good consistency (kappa = 0.800; P <0.01) It has certain application value in the research of diagnostic reagents.

The above descriptions are only preferred embodiments of the present invention, and are not intended to limit the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principles of the present invention shall be included in the protection of the present invention. within range.

Claims (5)

1. a mycoplasma pneumoniae antigen composition, is the combination of MP371 antigen and MP661 antigen, and described MP371 antigen is the N-terminal antigen of mycoplasma pneumoniae adhesion protein P1, and its site is positioned at 371-480aa, and concrete structure is shown in SEQ ID NO:3; Described MP661 antigen is the N-terminal antigen of mycoplasma pneumoniae adhesion protein P1, and its site is positioned at 661-772aa, and concrete structure is shown in SEQ ID NO:4.

2. mycoplasma pneumoniae antigen composition described in claim 1, is characterized in that: the Nucleotide of coding MP371 antigen is as shown in SEQ ID NO:9; The nucleotide sequence of coding MP661 antigen is as shown in SEQ ID NO:10.

3. described in claim 1, mycoplasma pneumoniae antigen composition detects the application in mycoplasma pneumoniae reagent in preparation.

4. a test kit that detects mycoplasma pneumoniae, is characterized in that, described test kit contains the composition described in claim 1.

5. test kit according to claim 4, is characterized in that, described detection is to utilize IgM antibodycapture to carry out.

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