阅读说明：本技术 一种肺炎支原体重组抗原的制备及应用 (Preparation and application of mycoplasma pneumoniae recombinant antigen ) 是由 吴素丽 段晓明 窦兰芳 付旭东 于 2018-12-29 设计创作，主要内容包括：本发明公开了在Genbank中搜集肺炎支原体基因的序列,建立肺炎支原体的基因数据,并且利用计算机软件对其进行分析,获得肺炎支原体粘附蛋白P1(GenBank:AF286371.1)和粘附蛋白P30(GenBank:EF614306.1)最具有检测活性的区段,P1基因的第252位氨基酸～第355位氨基酸(P1A),第898位氨基酸～第953位氨基酸(P1B),第1274位氨基酸～第1362位氨基酸(P1C),第1589位氨基酸～第1627位氨基酸(P1D),P30片段为肺炎支原体粘附蛋白P30的第174位氨基酸～第274位氨基酸,通过连接肽GSGSGS串联起来,串联顺序从C端到N端依次为P1A片段-第一连接肽-P1B片段-第二连接肽-P1C-第三连接肽-P1C-第四连接肽-P1D-第五连接肽--P30片段,对应的核酸序列通过化学合成的方法获得。(The invention discloses a method for collecting the sequence of Mycoplasma pneumoniae gene in Genbank, establishing the gene data of Mycoplasma pneumoniae, analyzing the gene data by computer software to obtain the section with the most detecting activity of Mycoplasma pneumoniae adhesive protein P1(GenBank: AF286371.1) and adhesive protein P30(GenBank: EF614306.1), the 252 rd to 355 th amino acids (P1A), the 898 th to 953 th amino acids (P1B), the 1274 th to 1362 th amino acids (P1C), the 1589 th to 1627 th amino acids (P1D), the P30 segment is the 174 th to the 274 th amino acids of Mycoplasma pneumoniae adhesive protein P45, connecting the P A segment-the first connecting peptide-P1B segment-the second connecting peptide-P1 segment-P37-the third connecting peptide-P961 to the fifth peptide-GS 8234 segment in sequence from C end to N end, the corresponding nucleic acid sequences are obtained by chemical synthesis.)

1. Preparation and application of a mycoplasma pneumoniae recombinant antigen, which is characterized in that: the amino acid sequence of the mycoplasma pneumoniae recombinant antigen is shown in SEQ ID NO. 1.

2. The preparation and use of the mycoplasma pneumoniae recombinant antigen according to claim 1, wherein the antigen comprises: the nucleotide sequence of the mycoplasma pneumoniae recombinant antigen is shown in SEQ ID NO. 2.

3. The method for producing a recombinant mycoplasma pneumoniae antigen according to claim 1, comprising the steps of:

firstly, constructing a gene expression sequence of the mycoplasma pneumoniae recombinant antigen, designing an upstream primer and a downstream primer according to the sequence, introducing a BamHI enzyme cutting site into the upstream primer, introducing a SalI enzyme cutting site into the downstream primer, wherein the specific nucleic acid sequence of the mycoplasma pneumoniae recombinant antigen is shown in NO1,

and secondly, performing PCR amplification by using upstream and downstream primers, recovering the PCR fragment obtained by amplification, and then connecting the PCR fragment into an expression vector pET28a to obtain a recombinant expression plasmid pET28 a-MP.

And thirdly, transforming the constructed recombinant plasmid pET-28a-MP into an escherichia coli competent cell BL21(DE3) to obtain an expression strain of the recombinant plasmid, cloning the strain to extract the plasmid, carrying out enzyme digestion after digestion by BamHI and SalI sites, carrying out induced expression and purification on the expression strain of the recombinant plasmid after sequencing verification, and obtaining the MP recombinant antigen.

4. The method for producing a recombinant mycoplasma pneumoniae antigen according to claim 3, wherein: the purification method of the third step: purifying the expressed protein by adopting a Ni-NTA affinity column purification mode to obtain the mycoplasma pneumoniae recombinant antigen with one type of antigen dominant epitope;

or purifying the expressed protein by adopting an electroelution purification mode to obtain the mycoplasma pneumoniae recombinant antigen with another type of antigen dominant epitope.

5. The method for producing a recombinant mycoplasma pneumoniae antigen according to claim 3, wherein: the PCR amplification primer sequence adopted in the second step is as follows:

an upstream primer: 5'-CGCGCGGATCCGATGAAAAACTCCAGGGCG-3'

A downstream primer: 5'-CGCGTCGACGTC GCGTTTTGGTGGAAAAGGC-3' are provided.

Technical Field

The invention relates to the technical field of bioengineering, in particular to preparation and application of a mycoplasma pneumoniae recombinant antigen.

Background

The mycoplasma pneumoniae is one of the main pathogens causing primary atypical pneumonia of human beings, is frequently generated in cold seasons in autumn and winter, and is infected by droplets in teenagers. Clinically, respiratory symptoms such as sore throat, fever, cough, low fever and the like are mainly seen, and cough is the most prominent manifestation. Patients with mycoplasma pneumonia have complicated lesions of the gastrointestinal system, the blood system, the nervous system and the cardiovascular system in addition to respiratory symptoms, and are drawing extensive attention.

The current diagnostic method applied to clinical mycoplasma pneumoniae mainly comprises the steps of separation culture of pathogens, indirect immunofluorescence assay, complement fixation assay, indirect hemagglutination ELISA detection, Polymerase Chain Reaction (PCR) and the like; the serological detection of mycoplasma pneumoniae mainly detects two antibodies, namely IgG and IgM, and the infection of the mycoplasma pneumoniae has a latent period, generally 2-3 weeks, IgM antibodies are generated about 1 week after the infection, and IgG antibodies are generated about 3 weeks. Therefore, positive IgM antibodies can be used as a diagnostic indicator of early infection. If the detected IgM antibody is negative, the infection of non-mycoplasma pneumoniae can not be judged, and then the detection of the IgG antibody is required; at present, the morbidity of mycoplasma pneumoniae in China is on the rise, particularly the morbidity of teenagers, the existing detection methods for etiology and serology are complex in operation and long in time consumption and are not beneficial to timely treatment of patients, the existing detection methods for serology use natural antigens mostly, the production process of the natural antigens is complex and expensive, the infection probability of experiment operators is increased, recombinant MP antigens with strong antigenicity and good sensitivity need to be developed, a foundation is laid for developing a quick and accurate detection reagent kit for mycoplasma pneumoniae IgM and IgG antibodies, and the preparation and application of the mycoplasma pneumoniae recombinant antigens are provided for people, so that the problems in the background technology are solved.

Disclosure of Invention

The invention aims to provide preparation and application of a mycoplasma pneumoniae recombinant antigen to solve the problems in the background technology.

In order to achieve the purpose, the invention provides the following technical scheme: preparation and application of a mycoplasma pneumoniae recombinant antigen, wherein the amino acid sequence of the mycoplasma pneumoniae recombinant antigen is shown in SEQ ID No. 1.

Preferably, the nucleotide sequence of the mycoplasma pneumoniae recombinant antigen is shown in SEQ ID NO. 2.

Preferably, the preparation method of the mycoplasma pneumoniae recombinant antigen provided by the invention is characterized by comprising the following steps:

firstly, constructing a gene expression sequence of the mycoplasma pneumoniae recombinant antigen, designing an upstream primer and a downstream primer according to the sequence, introducing a BamHI enzyme cutting site into the upstream primer, introducing a SalI enzyme cutting site into the downstream primer, wherein the specific nucleic acid sequence of the mycoplasma pneumoniae recombinant antigen is shown in NO1,

and secondly, performing PCR amplification by using upstream and downstream primers, recovering the PCR fragment obtained by amplification, and then connecting the PCR fragment into an expression vector pET28a to obtain a recombinant expression plasmid pET28 a-MP.

And thirdly, transforming the constructed recombinant plasmid pET-28a-MP into an escherichia coli competent cell BL21(DE3) to obtain an expression strain of the recombinant plasmid, cloning the strain to extract the plasmid, carrying out enzyme digestion after digestion by BamHI and SalI sites, carrying out induced expression and purification on the expression strain of the recombinant plasmid after sequencing verification, and obtaining the MP recombinant antigen.

Preferably, the purification method of step three: purifying the expressed protein by adopting a Ni-NTA affinity column purification mode to obtain the mycoplasma pneumoniae recombinant antigen with one type of antigen dominant epitope;

or purifying the expressed protein by adopting an electroelution purification mode to obtain the mycoplasma pneumoniae recombinant antigen with another type of antigen dominant epitope.

Preferably, the PCR amplification primer sequences used in step two are as follows:

an upstream primer: 5'-CGCGCGGATCCGATGAAAAACTCCAGGGCG-3'

A downstream primer: 5'-CGCGTCGACGTC GCGTTTTGGTGGAAAAGGC-3' are provided.

Compared with the prior art, the invention has the beneficial effects that:

1. the method is convenient and quick, and expensive instruments and equipment are not needed; the produced antigen has high sensitivity and strong specificity, and the prepared corresponding detection kit has good stability of the recombinant antigen, can be produced in large scale and is suitable for popularization and application;

2. the recombinant MP antigen with strong antigenicity and good sensitivity is developed, and a foundation is laid for the development of a rapid and accurate reagent kit for detecting the mycoplasma pneumoniae IgM and IgG antibodies.

Drawings

FIG. 1 is a result diagram of a recombinant plasmid for expressing Mp recombinant antigen in the present invention;

FIG. 2 is a graph showing the results of detection of antibodies of the present invention using Mycoplasma pneumoniae IgM and IgG antibodies, respectively.

Detailed Description

The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

As shown in fig. 1, the present invention provides a technical solution: firstly, gathering the sequence of Mycoplasma pneumoniae genes in Genbank, establishing gene data of Mycoplasma pneumoniae, analyzing the gene data by using computer software to obtain a section with the activity of detecting Mycoplasma pneumoniae adhesive protein P1(GenBank: AF286371.1) and adhesive protein P30(GenBank: EF614306.1), the 252 rd to 355 th amino acids (P1A), the 898 th to 953 th amino acids (P1B), the 1274 th to 1362 th amino acids (P1C), the 1589 th to 1627 th amino acids (P1D), a P30 fragment is the 174 th to 274 th amino acids of Mycoplasma pneumoniae adhesive protein P30, connecting the first connecting peptide-P1 fragment-second connecting peptide-P1-third connecting peptide-P1-P9634-fifth connecting peptide-P D in the serial sequence from the C end to the N end, the corresponding nucleic acid sequences are obtained by chemical synthesis. The upstream primer of the sequence introduces a BamHI site and adds a protective base CGC, the downstream primer introduces a SalI site and adds a protective base GTC, the specific Mp recombinant antigen amino acid sequence is shown in SEQ ID NO.1, and the nucleotide sequence is shown in SEQ ID NO. 2. After a target gene fragment is chemically synthesized, PCR amplification is carried out by using an upstream primer and a downstream primer (a primer sequence SEQ ID NO.3), the PCR fragment obtained by amplification is recovered (molecular biology extraction and recovery kits used by the invention are purchased from TAKARA bioengineering company, Inc.), then digestion is carried out by using BamHI and SalI (various enzymes used by the molecular biology used by the invention are purchased from TAKARA company, Inc.), the obtained product is connected to an expression vector pET-28a (purchased from Novagen company) digested by using BamHI and SalI, a recombinant plasmid pET-28a-MP is extracted, and the correct gene sequence is verified by digestion and sequencing, so that a recombinant plasmid pET-28a-MP, namely a recombinant plasmid for expressing MP recombinant antigen is obtained (figure 1). And (3) converting the positive recombinant plasmid pET-28a-MP into an expression strain E.coli BL2(DE3) competent cell to obtain an expression strain of the recombinant plasmid, and freezing for later use.

2. Prokaryotic expression of recombinant antigens from mycoplasma pneumoniae

Preparing Buffer A: 20mM Tris-HCl, 1mM EDTA, 50mM NaCl, 0.01% Triton X-100, pH 8.0.

The expression bacteria containing the recombinant plasmid are picked up and inoculated in 500mL LB culture medium containing 50 mug/mL kanamycin, the culture is carried out on a shaker at 37 ℃ and 170rpm for overnight culture until the concentration of the bacteria is 0.6-1.0 at OD600, IPTG (1 mM final concentration) is added for induction, the bacteria are induced at 170rpm and 37 ℃ for about 4 hours, 12,000g at 4 ℃ is centrifuged for 10 minutes, 5mL lysis buffer (50mM Tirs-HCl, pH8.0, 1mM EDTA, 100mM NaCl) is used for resuspension every 100mg of the obtained bacteria, ice bath ultrasonication is carried out, 12,000g at 4 ℃ is centrifuged for 20 minutes, supernatant and sediment are separated, after SDS-PAGE electrophoresis identification, most of target protein is expressed by inclusion bodies, and the sediment is reserved for subsequent treatment. The precipitate was washed 3 times with Buffer A at 4 ℃ for 12,000rpm 15min, the supernatant was removed, and the precipitate was dissolved in 8M urea + PBS (pH8.0) and purified.

3. Purification and renaturation of mycoplasma pneumoniae recombinant marker antigen (preparation MP-1)

Preparing a balance liquid: 20mM Tris-HCl (pH7.9), 0.5mM NaCl, 10mmol/L imidazole, 8M urea, pH 8.0;

preparation of an eluent: 20mM Tris-HCl (pH7.9), 0.5mM NaCl, 1500mmol/L imidazole, 8M urea, pH 8.0;

after the equilibration solution and the eluent are prepared, an ATKA purifier 10/UPC chromatograph (purchased from GE medical group) is used for cleaning and equilibrating a Ni-NTA affinity column (Qiagen company, product number 30210) by the equilibration solution, after the Ni-NTA column is equilibrated, a sample to be loaded on the column is combined on the column, after the combination is finished, the column is cleaned by the equilibration solution with 10 times of the column volume, then the eluent is used for gradually eluting, an elution peak is collected, the protein concentration is determined, and the renaturation is carried out.

The target protein obtained by the Ni-NTA affinity purification is diluted to the concentration of 0.5mg/mL by using an equilibrium solution, then dialyzed by using renaturation buffer solution (20mM PBS, 2mM reduced glutathione, 0.5mM oxidized glutathione, 5% glycerol, 1mM EDTA, pH8.0) containing 4M, 3M, 2M, 1M, 0.5M and 0M urea respectively at 4 ℃, the solution is changed once every 12 hours of gradient, finally the target protein is dialyzed into the buffer solution (20mM PBS, 1mM EDTA, pH8.0), a dialyzed sample is collected, centrifuged, concentrated by using an ultrafiltration concentration tube (purchased from Millipore company, the molecular weight of the trapped protein is 10kD), a concentrated sample is collected, namely the Mp-recombinant marker antigen MP-1, the protein concentration is measured, and the sample is placed at-20 ℃ for standby.

4. Purification and renaturation of mycoplasma pneumoniae recombinant envelope antigen (preparation of MP-2)

Preparing an electroelution buffer: 1/ten thousand SDS, PB solution with pH7.4;

performing gel electrophoresis by using 9% protein separation gel, performing sample loading on 3.5mL of crude antibody/plate gel at electrophoresis voltage of 100V at 4 ℃ overnight, performing electrophoresis at the distance of 2-4 cm from the bottom of the gel and the target protein band width of 1.2cm, cutting the target protein band, bagging 6 plates of target protein band adhesive tapes/bags, adding 6mL of electrophoretic eluent, fastening two ends of the opening of the bag, placing the dialysis bag in an electrophoresis tank, adding electrophoretic buffer solution in the electrophoresis tank until the buffer solution is over the dialysis bag filled with the adhesive tape, performing electrophoresis at 100V for 3h at 4 deg.C, stopping electrophoresis, squeezing out the liquid in the dialysis bag, filtering with 0.22 μm filter membrane, collecting filtrate, namely the mycoplasma pneumoniae recombinant envelope antigen MP-2, the protein concentration is determined, and the protein concentration is placed at-20 ℃ for standby.

Verification of Mycoplasma pneumoniae recombinant antigens

And (3) carrying out SDS-PAGE electrophoresis of 12% electrophoresis on the purified protein, transferring the protein to a PVDF membrane, and carrying out a protein immunoblotting test. Antibodies were detected using Mycoplasma pneumoniae IgM, IgG antibodies, respectively (see FIG. 2)