阅读说明：本技术 一种检测抗肺炎支原体抗体的天然抗原p1及其制备方法和应用 (Natural antigen P1 for detecting anti-mycoplasma pneumoniae antibody and preparation method and application thereof ) 是由 刘万建 刘向祎 杨帆 杜金芳 王婷 李林 于 2021-09-28 设计创作，主要内容包括：本发明属于生物学检测技术领域,具体涉及一种检测抗肺炎支原体抗体的天然抗原P1及其制备方法和应用,制备步骤为：取灭活的人肺炎支原体抗原培养液,离心得到肺炎支原体菌体,经支原体洗涤液洗涤后离心,弃上清,沉淀即为肺炎支原体；将其放入裂解液中,37℃水浴孵育,-20℃冻存后复融,离心后的沉淀用支原体洗涤液重悬,离心留沉淀；将沉淀重悬在裂解液中,超声后离心,上清液即为抗原粗品；粗品过滤后的上清进行离心超滤,收集膜上的蛋白,即为天然抗原P1。该方法步骤简单,适于推广大量生产,制得的天然抗原P1对肺炎支原体抗体有很好的特异性和敏感性,从而进一步提高肺炎支原体抗体在疾病诊断中的临床检测敏感性和特异性。(The invention belongs to the technical field of biological detection, and particularly relates to a natural antigen P1 for detecting mycoplasma pneumoniae-resisting antibodies, a preparation method and application thereof, wherein the preparation method comprises the following steps: taking inactivated mycoplasma pneumoniae antigen culture solution, centrifuging to obtain mycoplasma pneumoniae thalli, washing by using mycoplasma washing solution, centrifuging, removing supernatant, and precipitating to obtain mycoplasma pneumoniae; putting the cells into a lysis solution, incubating in a water bath at 37 ℃, freezing and preserving at-20 ℃, then re-melting, re-suspending the centrifuged precipitate with a mycoplasma washing solution, and centrifuging to leave the precipitate; resuspending the precipitate in a lysis solution, centrifuging after ultrasonic treatment, and obtaining supernate which is an antigen crude product; and (3) performing centrifugal ultrafiltration on the supernatant obtained after the crude product is filtered, and collecting the protein on the membrane, namely the natural antigen P1. The method has simple steps, is suitable for popularization and mass production, and the prepared natural antigen P1 has good specificity and sensitivity to the mycoplasma pneumoniae antibody, thereby further improving the clinical detection sensitivity and specificity of the mycoplasma pneumoniae antibody in disease diagnosis.)

1. A preparation method of a natural antigen P1 for detecting an anti-mycoplasma pneumoniae antibody is characterized by comprising the following steps:

(1) taking inactivated mycoplasma pneumoniae antigen culture solution, centrifuging at 4 ℃ to obtain mycoplasma pneumoniae thalli, washing by mycoplasma washing solution, centrifuging at 4 ℃, removing supernatant, and repeatedly washing to obtain precipitate, namely mycoplasma pneumoniae;

(2) putting the mycoplasma pneumoniae obtained in the step (1) into a lysate, uniformly mixing, incubating in a water bath at 37 ℃ for 0.5-2 h, freezing at-20 ℃ for 2-12 h, thawing again, centrifuging at 4 ℃, and removing a supernatant; resuspending the obtained precipitate with mycoplasma washing solution, mixing well and washing, centrifuging at 4 deg.C, leaving precipitate, and washing the precipitate repeatedly;

(3) resuspending the precipitate obtained in the step (2) in a lysate, performing ultrasonic treatment, and centrifuging at 4 ℃ to obtain a supernatant, namely a crude natural antigen of the mycoplasma pneumoniae antibody;

(4) and filtering the crude natural antigen, performing centrifugal ultrafiltration on the obtained supernatant by using an ultrafiltration tube, and collecting the protein on the membrane, namely the natural antigen P1.

2. The method according to claim 1, wherein the mycoplasma washing solution in steps (1) and (2) is 10 mM-20 mM PBS solution.

3. The method of claim 1, wherein the lysate obtained in steps (2) and (3) is formulated with 10 mM-20 mM PBS, 0.1-5 mM Tcep, 0.1-2.5 mM EDTA, 1-5% arginine at final concentration, and 1-4% trehalose at final concentration; the pH of the lysate was 7.6; and (3) carrying out ultrasound by using an ultrasonic crusher, wherein the ultrasound conditions are 70-110W, 1-2 s of ultrasound and 1-5 s of stopping, the time is 20-50 min, and the temperature is 2-8 ℃.

4. The preparation method according to claim 1, wherein the ultrafiltration tube in the step (4) has a molecular weight cut-off of 50-100 KD, and the ultrafiltration is carried out at a centrifugal speed of 6000-8000 rpm for 5-10 min.

5. The preparation method according to claim 1, wherein the centrifugation speed is 10000 rpm to 16000 rpm, and the centrifugation time is 0.5 to 2 hours; the number of washing times is 1-3.

6. A natural antigen P1 for detecting an anti-Mycoplasma pneumoniae antibody, wherein the natural antigen P1 is prepared by the preparation method of any one of claims 1 to 5.

7. Use of the natural antigen P1 obtained by the process according to any one of claims 1 to 5 in a diagnostic kit.

Technical Field

The invention belongs to the technical field of biological detection, and particularly relates to a natural antigen P1 for detecting an anti-mycoplasma pneumoniae antibody, and a preparation method and application thereof.

Background

Mycoplasma Pneumoniae (MP) is the causative agent of human mycoplasma pneumonia, accounting for over one third of acellular pneumonia. MP is mainly transmitted by oral and nasal secretions through air, respiratory tract infection is caused by droplets, the incubation period is 2-3 weeks, the number of attack groups is more in autumn and winter, children and young people are more, the clinical manifestations of the MP are cough, fever, headache, sore throat and myalgia, typical clinical symptoms do not exist, and the MP is not easily distinguished from bacterial pneumonia. However, mycoplasma pneumonia is very different from the treatment of bacterial pneumonia infection, and because mycoplasma pneumoniae is not sensitive to antibiotics such as penicillin and is very prone to generate drug resistance, early diagnosis of MP infection is important.

Laboratory methods for detecting MP include pathogen isolation culture and morphological detection, antigen detection, serological and molecular biological methods. MP has long growth period, long pathogen separation and culture time, and is not favorable for laboratory diagnosis, and various PCR, gene probes and other types in molecular diagnosis have high sensitivity and specificity, but the sample is easy to pollute and has higher requirements on equipment, so the method is not easy to popularize. The mycoplasma pneumoniae is negative in gram stain microscopic examination, the giemsa stain is light purple, no cell wall exists, the cell membrane consisting of three layers is arranged on the periphery of cytoplasm, the inner layer and the outer layer are protein and carbohydrate, and the middle layer is lipid; part of MP has a capsule layer outside cell membrane, and the main component is polysaccharide. The antigen substances of MP are mainly protein and glycolipid on cell membranes, and the glycolipid antigen can stimulate the organism to generate complement binding antibody, growth inhibition antibody and metabolism inhibition antibody, but the glycolipid antigen has common antigens with various other mycoplasma, human erythrocyte membrane type 1 antigen, streptococcus pneumoniae type 23, 32 and MG streptococcus, can cause cross reaction, and has poor specificity. The P1 membrane protein and mycoprotein have strong specificity, can stimulate the organism to generate durable high-efficiency antibody, and the P1 membrane protein is also the main type specific antigen of mycoplasma, so the antigen used for detecting the MP antibody is the P1 membrane protein.

In human infectious diseases, MP is more and more concerned by various scholars, and MP infection has a complex immunological mechanism which is not completely clear so far. Aiming at the specificity of MP treatment, a raw material with high specificity and sensitivity is urgently needed at present, and MP clinical diagnosis can be rapidly, simply and cheaply carried out. The codon for tryptophan in the Mycoplasma pneumoniae mRNA is UGA, not UGC. UGA is a stop codon in E.coli and most cellular expression systems. The nucleotide series of the P1 protein contains 21 UGAs, so the codon mutation is needed for in vitro expression, and the difficulty of recombination antigen is increased. MP is not invaded into cells after being absorbed to the mucous membrane, but grows between ciliated epithelium, is hidden in a cell crypt, and is adsorbed on a neuraminidase receptor of a cell membrane of the mucous epithelium through P1 protein. The antigenic site of P1 is mainly the spatial site of spatial structure, so in vitro expression must depend on cell expression, and the transfection efficiency of cell expression forward transfection reagent and mass culture are a great test; meanwhile, low protein expression and batch-to-batch instability are troublesome problems, and long time and high cost are also main reasons for limiting mass production due to stable cell expression.

Disclosure of Invention

The invention aims to solve the problems in the prior art and provides a natural antigen P1 for detecting anti-mycoplasma pneumoniae antibodies and a preparation method and application thereof. The method has simple steps, can be popularized and produced in large quantities, and has good specificity and sensitivity to the mycoplasma pneumoniae antibody, thereby further improving the clinical detection sensitivity and specificity of the mycoplasma pneumoniae antibody in disease diagnosis and providing an accurate treatment scheme for patients.

The technical scheme of the invention is as follows:

a preparation method of a natural antigen P1 for detecting an anti-mycoplasma pneumoniae antibody comprises the following steps:

(1) taking inactivated mycoplasma pneumoniae antigen culture solution, centrifuging at 4 ℃ to obtain mycoplasma pneumoniae thalli, washing by mycoplasma washing solution, centrifuging at 4 ℃, removing supernatant, and repeatedly washing to obtain precipitate, namely mycoplasma pneumoniae;

(2) putting the mycoplasma pneumoniae obtained in the step (1) into a lysate, uniformly mixing, incubating in a water bath at 37 ℃ for 0.5-2 h, freezing at-20 ℃ for 2-12 h, thawing again, centrifuging at 4 ℃, and removing a supernatant; resuspending the obtained precipitate with mycoplasma washing solution, mixing well and washing, centrifuging at 4 deg.C, leaving precipitate, and washing the precipitate repeatedly;

(3) resuspending the precipitate obtained in the step (2) in a lysate, performing ultrasonic treatment, and centrifuging at 4 ℃ to obtain a supernatant, namely a crude natural antigen of the mycoplasma pneumoniae antibody;

(4) filtering the crude product of the natural antigen by using a disposable filter with the diameter of 0.45 mu m, carrying out centrifugal ultrafiltration on the obtained supernatant by using an ultrafiltration tube, allowing the foreign protein to flow through, intercepting the natural antigen of the target protein mycoplasma pneumoniae antibody on a membrane, and collecting the protein on the membrane, namely the natural antigen P1.

Further, the mycoplasma washing solution in the steps (1) and (2) is 10 mM-20 mM PBS solution.

Further, the formula of the cracking liquid in the steps (2) and (3) comprises 10 mM-20 mM PBS, 0.1-5 mM Tcep, 0.1-2.5 mM EDTA, 1-5% arginine at the final concentration and 1-4% trehalose at the final concentration; the pH of the lysate was 7.6; and (3) carrying out ultrasound by using an ultrasonic crusher, wherein the ultrasound conditions are 70-110W, 1-2 s of ultrasound and 1-5 s of stopping, the time is 20-50 min, and the temperature is 2-8 ℃.

Furthermore, the molecular weight cut-off of the ultrafiltration tube in the step (4) is 50-100 KD, the centrifugal speed of ultrafiltration is 6000-8000 rpm, and the time is 5-10 min.

Further, the centrifugal speed is 10000 rpm-16000 rpm, and the centrifugal time is 0.5-2 h; the number of washing times is 1 to 3, preferably 1 to 2.

A natural antigen P1 for detecting an anti-mycoplasma pneumoniae antibody is prepared by the preparation method, wherein the natural antigen P1 is prepared by the preparation method.

Further, the natural antigen P1 is applied to a diagnostic kit.

The invention has the beneficial effects that:

(1) the invention selects the inventor to culture the thalli in large quantity (the specific culture method is explained by the patent application), takes the culture solution as the raw material directly, and prepares the natural antigen P1 of the mycoplasma pneumoniae antibody through four steps of centrifugal collection, freeze-thaw crushing treatment, crude product obtaining and purification; the preparation method has simple steps and easy operation, and is suitable for large-scale production and large-scale popularization.

(2) The invention adopts the alternative treatment at 37 ℃ and-20 ℃, can simply break the mycoplasma cell membrane and remove the cytoplasmic foreign protein component, and has simple operation and low cost; carrying out ultrasonic treatment by using a special lysate to obtain P1 protein and other antigens at the top of the mycoplasma pneumoniae; the operation is simple, and the production is easy to expand; the ultrafiltration tube is adopted for further purification, the steps are simple, the yield is high, and therefore the production and preparation cost of the mycoplasma pneumoniae antigen is reduced.

(3) The method does not need expensive instruments and equipment, does not need chemical substances harmful to the environment, has low preparation cost, high treatment speed, simple operation and stable process, and can realize mass production.

Detailed Description

The technical solutions will be described clearly and completely in the following with reference to 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 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.

For further understanding of the present invention, the present invention will be further described with reference to examples.

Example 1

The preparation method of the natural antigen P1 for detecting the mycoplasma pneumoniae antibody comprises the following steps:

(1) collection of mycoplasma pneumoniae: taking inactivated mycoplasma pneumoniae antigen culture solution, centrifuging at the temperature of 4 ℃ and the rpm of 16000 for 0.5 h to obtain mycoplasma pneumoniae thalli, washing with 10 mM PBS washing solution for 2 times, centrifuging at the temperature of 4 ℃ and the rpm of 16000 for 0.5 h, discarding supernatant, and repeatedly washing for 2 times to obtain precipitate, namely mycoplasma pneumoniae;

(2) freeze thawing and crushing treatment: firstly, preparing a lysate with the components of 10 mM PBS, 5 mM Tcep, 2.5 mM EDTA, 2% arginine at the final concentration and 2% trehalose at the final concentration, and adjusting the pH value of the lysate to 7.6; then putting the mycoplasma pneumoniae into the lysis solution, mixing uniformly, incubating in a water bath kettle at 37 ℃ for 0.5 h, freezing and storing at-20 ℃ for 12 h, then re-melting, centrifuging at 16000 rpm for 0.5 h at 4 ℃, removing the supernatant, re-suspending the precipitate in 10 mM PBS washing solution, mixing uniformly and washing for 2 times, centrifuging at 16000 rpm for 0.5 h at 4 ℃, leaving the precipitate, and repeatedly washing for 2 times;

(3) crude product: resuspending the precipitate in a lysate, performing ultrasound by using an ultrasonic pulverizer, setting the ultrasound conditions to be 110W, 2 s in excess, stopping for 3 s, performing ultrasound for 20 min at the temperature of 2 ℃, then performing centrifugation at 4 ℃, and performing centrifugation at 16000 rpm for 0.5 h to obtain supernatant, namely a crude natural antigen of the mycoplasma pneumoniae antibody;

(4) and (3) purification: filtering crude natural antigen of Mycoplasma pneumoniae antibody with 0.45 μm disposable filter, centrifuging and ultrafiltering supernatant with ultrafiltration tube with cut-off molecular weight of 100 KD at 8000 rpm for 5 min; at this time, the foreign protein flows through, the natural antigen P1 of the target protein Mycoplasma pneumoniae antibody is trapped on the membrane, and the protein on the membrane, namely the natural antigen P1 of the Mycoplasma pneumoniae antibody, is collected.

Example 2

The preparation method of the natural antigen P1 for detecting the mycoplasma pneumoniae antibody comprises the following steps:

(1) collection of mycoplasma pneumoniae: taking inactivated mycoplasma pneumoniae antigen culture solution, centrifuging at 10000 rpm for 2 h at 4 ℃ to obtain mycoplasma pneumoniae thalli, washing with 20mM PBS (phosphate buffer solution) for 3 times, centrifuging at 4 ℃, centrifuging at 10000 rpm for 2 h, removing supernatant, and repeatedly washing for 1 time to obtain precipitate, namely mycoplasma pneumoniae;

(2) freeze thawing and crushing treatment: firstly, preparing a lysate with the components of 20mM PBS, 0.5 mM Tcep, 0.5 mM EDTA, 1% arginine in final concentration and 1% trehalose in final concentration, and adjusting the pH value of the lysate to 7.6; then putting the mycoplasma pneumoniae into the lysate, mixing uniformly, incubating in a 37 ℃ water bath for 2 h, freezing at-20 ℃ for 2 h, then re-melting, centrifuging at 4 ℃ and 10000 rpm for 2 h, removing the supernatant, re-suspending the precipitate in 20mM PBS washing solution, mixing uniformly and washing for 3 times, centrifuging at 4 ℃ and 10000 rpm for 2 h, leaving the precipitate, and repeatedly washing for 1 time;

(3) crude product: resuspending the precipitate in a lysate, performing ultrasound by using an ultrasonic pulverizer, setting the ultrasound conditions to be 70W, 2 s in excess, stopping 2 s, performing ultrasound for 40 min at the temperature of 8 ℃, then performing centrifugation at 4 ℃ and 10000 rpm for 2 h to obtain a supernatant, namely a crude natural antigen of the mycoplasma pneumoniae antibody;

(4) and (3) purification: filtering crude natural antigen of Mycoplasma pneumoniae antibody with 0.45 μm disposable filter, centrifuging and ultrafiltering supernatant with ultrafiltration tube with cut-off molecular weight of 50 KD at 6000 rpm for 10 min; at this time, the foreign protein flows through, the natural antigen P1 of the target protein Mycoplasma pneumoniae antibody is trapped on the membrane, and the protein on the membrane, namely the natural antigen P1 of the Mycoplasma pneumoniae antibody, is collected.

Example 3

The preparation method of the natural antigen P1 for detecting the mycoplasma pneumoniae antibody comprises the following steps:

(1) collection of mycoplasma pneumoniae: taking inactivated mycoplasma pneumoniae antigen culture solution, centrifuging at the temperature of 4 ℃ and the rpm of 12000 for 1 h to obtain mycoplasma pneumoniae thalli, washing with 15 mM PBS (phosphate buffer solution) for 1 time, centrifuging at the temperature of 4 ℃ and the rpm of 12000 for 1 h, removing supernatant, and repeatedly washing for 2 times to obtain a precipitate, namely the mycoplasma pneumoniae;

(2) freeze thawing and crushing treatment: firstly, preparing a lysis solution with the components of 15 mM PBS, 1 mM Tcep, 1 mM EDTA, 5% arginine at the final concentration and 5% trehalose at the final concentration, and adjusting the pH value of the lysis solution to 7.6; then putting the mycoplasma pneumoniae into the lysis solution, mixing uniformly, incubating in a 37 ℃ water bath for 1 h, freezing and storing for 4 h at-20 ℃, re-melting, centrifuging at 12000 rpm for 1 h at the temperature of 4 ℃, removing the supernatant, re-suspending the precipitate in 15 mM PBS (phosphate buffer solution) washing solution, mixing uniformly and washing for 1 time, centrifuging at 12000 rpm for 1 h at the temperature of 4 ℃, leaving the precipitate, and repeatedly washing for 2 times;

(3) crude product: resuspending the precipitate in a lysate, performing ultrasound by using an ultrasonic pulverizer, setting the ultrasound conditions to be 90W, 1 s over, 3 s under, performing ultrasound at 4 ℃ for 50 min, then performing centrifugation at 4 ℃, and performing centrifugation at 12000 rpm for 1 h to obtain a supernatant, namely a crude natural antigen of the mycoplasma pneumoniae antibody;

(4) and (3) purification: filtering crude natural antigen of Mycoplasma pneumoniae antibody with 0.45 μm disposable filter, centrifuging and ultrafiltering supernatant with ultrafiltration tube with cut-off molecular weight of 100 KD at 8000 rpm for 6 min; at this time, the foreign protein flows through, the natural antigen P1 of the target protein Mycoplasma pneumoniae antibody is trapped on the membrane, and the protein on the membrane, namely the natural antigen P1 of the Mycoplasma pneumoniae antibody, is collected.

Example 4

The natural antigen P1 for detecting mycoplasma hyopneumoniae antibody was prepared by the preparation methods of examples 1 to 3 and applied to a diagnostic kit.

The kit adopts colloidal gold immunochromatography and indirect method principle to detect the anti-mycoplasma pneumoniae antibody in a sample, and specifically comprises the following steps: coating the mycoplasma pneumoniae antigen P1 on a nitrocellulose membrane to prepare a colloidal gold labeled immunochromatography detection kit, which comprises the following components:

(1) nitrocellulose membrane coated with mycoplasma pneumoniae antigen P1

(2) Colloidal gold labeled mouse anti-human IgG antibody

(3) Specimen dilution liquid

(4) Negative and positive control sera

Specificity = true negative/(true negative + false positive);

sensitivity = true positive detection/(true positive detection + false negative detection)

The kit detects 300 clinical positive samples and 1000 clinical negative samples, and the specific data are shown in the following table 1.

TABLE 1 detection data of Mycoplasma pneumoniae Natural antigen P1 kit

		Number of positive detections	Sensitivity of the composition	Number of negative detections	Specificity of
						Clinical positive sample	300	296	98.67%	/	/
Clinical negative sample	1000	/	/	1000	100%

As can be seen from the above table, the detection rate of the Mycoplasma pneumoniae natural antigen P1 of the invention for the clinical positive sample of the patient is more than 98%, and the specificity thereof is 100%, which indicates that the Mycoplasma pneumoniae natural antigen P1 of the invention has better sensitivity and specificity for diagnosing the clinical sample, and can be further applied to clinical detection.

Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art will understand that various changes, modifications and substitutions can be made without departing from the spirit and scope of the present invention. Any modification, equivalent replacement, or modification made within the spirit and principle of the present invention should be included in the protection scope of the present invention.