Preparation method of mycoplasma pneumoniae natural antigen

Abstract

The invention relates to a preparation method of a mycoplasma pneumoniae natural antigen, which belongs to the technical field of biological detection, and comprises the following steps: (1) concentrating 10L of mycoplasma pneumoniae culture solution to 100mL by using a hollow fiber column, performing liquid replacement by using the hollow fiber column after concentration, adding 4000 mL of replacement liquid in each replacement, and repeating the replacement for three times; (2) pretreatment of thalli; (3) ultrasonic treatment; and (4) adding the liquid obtained in the step (3) into isometric absolute ethyl alcohol, placing in a water bath kettle at 95 DEG C, and refluxing for 1h. The mycoplasma pneumoniae antigen with high purity, batch-to-batch stability and good activity can be obtained.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of biological detection, and particularly relates to a preparation method of a natural antigen of Mycoplasma pneumoniae. BACKGROUND

[0002] Mycoplasma pneumoniae (M. pneumoniae) is the pathogen of mycoplasma pneumonia, and is the smallest microorganism discovered by people that can grow on solid culture medium in vitro and survive without relying on living cells (as of 2023). Mycoplasma pneumoniae belongs to a class of mycoplasma, and has a special terminal structure at one end, which can make mycoplasma adhere to the surface of respiratory mucosa epithelial cells, causing lung infection, so it is called Mycoplasma pneumoniae.

[0003] Mycoplasma has no cell wall, and its antigenicity mainly depends on P1 protein, lipoprotein and glycolipid on the cell membrane. Some strains of Mycoplasma pneumoniae have a layer of capsule outside the cell membrane, and the main component is polysaccharide, which also has certain antigenicity.

[0004] At present, the main diagnosis methods of Mycoplasma pneumoniae include colloidal gold kit, ELISA kit, etc., and various detection kits need active, high-purity and batch-stable antigens. The common Mycoplasma pneumoniae antigens mainly include P1 protein antigen, whole bacteria antigen and recombinant protein antigen. Among them, the natural antigen has good activity, poor purity, large batch difference, and the stability of the antigen is difficult to guarantee; the antigen activity of the recombinant protein antigen is not as good as that of the natural antigen, the antigen quality is uneven, and the application in detection kits often has problems such as low sensitivity, poor specificity, low detection rate, etc. Therefore, it is particularly important to prepare a useful Mycoplasma pneumoniae antigen. SUMMARY

[0005] In view of the deficiencies of the prior art, the present application provides a preparation method of a natural antigen of Mycoplasma pneumoniae, which can obtain a Mycoplasma pneumoniae antigen with high purity, batch stability and good activity.

[0006] To solve the above technical problems, the technical scheme of the present application is as follows: a preparation method of a natural antigen of Mycoplasma pneumoniae, characterized by comprising the following steps: (1) Concentrate 10 L of Mycoplasma pneumoniae culture solution to 100 mL on a hollow fiber column, and perform liquid displacement on the hollow fiber column after concentration, adding 4000 mL of displacement liquid each time, and repeating the displacement for 3 times; Washing: Put the concentrated liquid after displacement into a centrifuge bottle, centrifuge at 12000 rpm for 30 min at 4 DEG C to obtain the precipitate, resuspend and mix evenly by adding 150 mL of PBS, centrifuge at 12000 rpm for 30 min at 4 DEG C to obtain the precipitate; repeat the above washing step 2 times, and dry the washed bacteria for 30 min.

[0007] (2) Pretreatment of bacterial cells: the bacterial cells obtained in step (1) were ground with PBS for 20 min, then suspended in PBS; the suspension was determined to have a final volume of 10 mL PBS per 1 g of bacterial cells.

[0008] (3) Ultrasonic treatment: the suspension obtained in step (2) was subjected to ice bath ultrasonic treatment: ultrasonic instrument ultrasonic treatment for 2 s and pause for 3 s, ultrasonic treatment time was 6 min, 10000 rpm, 20 min, 4℃ centrifugation, and the supernatant solution was taken. The obtained precipitate was suspended in an equal amount of PBS, and the obtained suspension was subjected to the above ice bath ultrasonic treatment step again. The obtained solution was combined with the above supernatant solution.

[0009] (4) The liquid obtained in step (3) was added to an equal volume of anhydrous ethanol, and placed in a 95℃ water bath for 1 h.

[0010] (5) The liquid obtained in step (4) was concentrated by 10 times, and the concentrated liquid was taken. Phenol was added according to the ratio of concentrated liquid: phenol = 9:11, 70℃ water bath oscillation for 1 h, 10000 rpm centrifugation for 20 min, the supernatant was taken and repeated centrifugation once, and the supernatant was taken and loaded into a dialysis bag for 48 h of pure water dialysis. The dialysis bag was taken out and placed in anhydrous alcohol for 4 h, and a precipitate appeared. The precipitate was suspended in a centrifuge tube with anhydrous ethanol, 10000 rpm, 5 min, 4℃ centrifugation, and the supernatant was discarded. The obtained precipitate was combined.

[0011] (6) 1 mL of pre-cooled acetone was added to the precipitate obtained in step (5), shaken for 1 min, 10000 rpm, 5 min, 4℃ centrifugation, and the supernatant was discarded. The precipitate was dried for 2 h.

[0012] (7) 0.2 mL of dissolving solution was added to the dried precipitate obtained in step (6) to obtain a final sample.

[0013] As an improved technical solution, the dissolving solution in step (7) is 1M NaAC and volume percentage 1% triton X-100, and the pH of the dissolving solution is 4.0.

[0014] As an improved technical solution, the displacement solution in step (1) is PBS.

[0015] As an improved technical solution, the ultrasonic instrument power in step (3) is set to 15# amplitude rod, 3% power.

[0016] As an improved technical solution, in step (5), the precipitate is suspended in a 1.5 mL centrifuge tube with anhydrous ethanol.

[0017] As an improved technical solution, in step (5), the obtained precipitate is combined into a 1.5 mL centrifuge tube.

[0018] The 2 times of ultrasonic in step (3) and the ethanol reflux after adding the precipitate can comprehensively extract the mycoplasma pneumoniae antigen components.

[0019] In step (5), the precipitate is first placed in a dialysis bag, and then suspended in a centrifugal tube with anhydrous ethanol, so that the amount of anhydrous ethanol can be saved.

[0020] After the above technical scheme is adopted, the application has the following advantages: 1. The purified mycoplasma pneumoniae antigen is a natural antigen, and has the advantage of good natural antigen activity. 2. The purification process is more perfect, the liquid volume is reduced by the hollow fiber column, and the liquid is replaced to realize the preliminary washing of the bacteria, and after the bacteria are collected by centrifugation, the bacteria are washed multiple times to further reduce the pollution of external proteins. 3. The bacteria are dried at room temperature, the humidity of the bacteria is controlled in the same time, the consistency of the bacteria amount of each batch of antigen samples is controlled, and then the consistency of the purified product is controlled, and the batch difference is reduced. 4. The bacteria and the ultrasonic crushing liquid are fully mixed by pre-grinding in a mortar, the ultrasonic crushing efficiency is improved, the bacteria crushing effect is improved by multiple ultrasonic, and then ethanol reflux is performed, the dissolution amount of the saccharides in the liquid is improved, and the yield is improved. 5. The heat phenol removes impurities, reduces the content of impurities in the antigen, and performs water extraction, alcohol precipitation and acetone degreasing at the same time, and multiple methods improve the purity of the saccharide substances in the antigen. 6. In the conventional water extraction and alcohol precipitation process, a large amount of ethanol is used, and then the centrifugal sample volume is suddenly increased, in the application, the dialysis alcohol precipitation reduces the amount of anhydrous ethanol and the cost, and also reduces the centrifugal sample volume. 7. By improving the dissolving liquid, an antigen dissolving liquid which can better play the antigenicity of the purified sample and has stable and non-fuzzy bands is obtained. 8. The prepared antigen is compared with the purchased antigen standard by colloidal gold platform, and the antigen activity is close to or better than the purchased antigen standard.

[0021] The application not only can improve the yield, but also can obtain the antigen components with high purity mainly composed of polysaccharides and glycolipids. BRIEF DESCRIPTION OF DRAWINGS

[0022] Figure 1 The protein electrophoresis diagram of the final sample of Comparative Example 5; Figure 2 The protein electrophoresis diagram of the final sample of Comparative Example 6. DETAILED DESCRIPTION

[0023] In order to make the purpose, technical scheme and advantages of the present application more clear, the present application will be further described in detail below with examples. It should be understood that the specific examples described herein are only used to explain the present application, and are not used to limit the present application. Example 1

[0024] A preparation method of a natural antigen of Mycoplasma pneumoniae, comprising the following steps: (1) Concentrate 10L of Mycoplasma pneumoniae culture solution to 100mL by using a hollow fiber column, and then perform liquid displacement by using the hollow fiber column, wherein the displacement liquid is PBS, 4000mL of displacement liquid is added each time, and the displacement is repeated for 3 times; Washing: the concentrated solution after displacement is loaded into a centrifuge bottle, and then centrifuged at 12000rpm for 30min at 4℃ to obtain a precipitate, 150mL of PBS is added to resuspend the precipitate, and then centrifuged at 12000rpm for 30min at 4℃ to obtain a precipitate; the above washing step is repeated for 2 times, and then the washed bacteria are dried for 30min and collected for weighing and recording.

[0025] (2) Bacteria pretreatment: the bacteria obtained in step (1) are transferred to a mortar, grinded with appropriate amount of PBS for 20min, and then suspended with appropriate amount of PBS; the suspension is transferred to a beaker, and the final volume is determined according to 1g of bacteria corresponding to 10mL of PBS.

[0026] (3) Ultrasonic treatment: the suspension obtained in step (2) is placed in ice water for ice bath ultrasonic treatment; the ultrasonic instrument used is purchased from Ningbo Xinzhi Biological Technology Co., Ltd., the model is JY98-IIIN, the power is set to 15# amplitude rod, 3% power, ultrasonic treatment for 2s and stop for 3s, the ultrasonic time is 6min, and then centrifuged at 10000rpm for 20min at 4℃ to obtain a supernatant solution; The obtained precipitate is suspended with an equal amount of PBS again, the obtained suspension is repeated with the above ice bath ultrasonic treatment step, and the obtained solution is combined with the above supernatant solution.

[0027] (4) The liquid obtained in step (3) is added with an equal volume of anhydrous ethanol, and then placed in a 95℃ water bath for 1h.

[0028] (5) The liquid obtained in step (4) is concentrated by 10 times, the concentrated liquid is taken, phenol is added according to the ratio of concentrated liquid: phenol = 9:11, 70℃ water bath is oscillated for 1h, centrifuged at 10000rpm for 20min, the supernatant solution is repeatedly centrifuged once in a 1mL centrifuge tube, the supernatant is loaded into a dialysis bag for water dialysis for 48h, the dialysis bag is taken out, stirred in anhydrous alcohol for 4h, a precipitate appears, the precipitate is suspended in a 1.5mL centrifuge tube with anhydrous ethanol, centrifuged at 10000rpm for 5min at 4℃, the supernatant is discarded, and the precipitate is combined into a 1.5mL centrifuge tube.

[0029] (6) To the precipitate obtained in step (5), 1 mL of pre-cooled acetone was added, and the vortex was shaken for 1 min, and then centrifuged at 10,000 rpm for 5 min at 4°C. The supernatant was discarded, and the precipitate was left. The 1.5 mL centrifuge tube containing the precipitate was placed in a fume hood and dried for 2 h.

[0030] (7) 0.2 mL of a dissolution solution was added to the dried precipitate obtained in step (6) to obtain a final sample.

[0031] The dissolution solution includes 1M NaAC and 1% volume percentage Triton X-100, and the pH of the dissolution solution is 4.0. Example 2

[0032] Example 1 was repeated for 3 batches in succession, and 3 final samples were obtained.

[0033] After verification by colloidal gold platform, a total of 300 random serum samples were detected, and the detection results of 3 samples were completely consistent, the titer fluctuation was low, and it met the requirements of colloidal gold raw materials. Example 3

[0034] The antigen sample of Example 2 was subjected to parallel colloidal gold platform experiments with a known standard purchased antigen with good activity, and the same 300 random serum samples were detected. The detection results of the antigen sample and the standard sample were consistent, the titer of the antigen sample was slightly better than that of the standard sample, and the detection results of some random serum samples were clearer, which indicated that the activity of the antigen prepared in Example 1 was close to or better than that of the standard sample, and the antigen could replace the purchased standard sample.

[0035] Comparative Example 1 (1) Washing: 10 L of Mycoplasma pneumoniae culture solution was placed in a centrifuge bottle, and centrifuged at 12,000 rpm for 30 min at 4°C to obtain a precipitate. 150 mL of PBS was added to resuspend and mix, and then centrifuged at 12,000 rpm for 30 min at 4°C to obtain a precipitate. The above steps were repeated twice. The washed bacteria were dried for 30 min and collected for weighing and recording.

[0036] (2) Bacterial pretreatment: The bacteria obtained in step (1) were transferred to a mortar, ground with an appropriate amount of PBS for 20 min, and then suspended with an appropriate amount of PBS. The suspension was transferred to a beaker, and the final volume was determined according to 1 g of bacteria corresponding to 10 mL of PBS.

[0037] (3) Ultrasonic treatment: The suspension obtained in step (2) was placed in ice water for ice bath ultrasonic treatment. The ultrasonic instrument used was purchased from Ningbo Xinzhi Biological Technology Co., Ltd., model JY98-IIIN, power setting 15# amplitude rod, 3% power, ultrasonic 2s stop 3s, ultrasonic time 6min, 10000rpm 20min 4℃ centrifugation to take the supernatant solution; The obtained precipitate was suspended again in an equal amount of PBS, the obtained suspension was subjected to the above-mentioned ice-bath ultrasonic treatment, and the obtained solution was combined with the above-mentioned supernatant solution.

[0038] (4) The liquid obtained in step (3) was added into an equal volume of anhydrous ethanol, and placed in a 95°C water bath for 1 h.

[0039] (5) The liquid obtained in step (4) was concentrated by 10 times, and the concentrated liquid was taken and added with phenol according to the ratio of concentrated liquid:phenol=9:11, and then subjected to oscillation in a 70°C water bath for 1 h, and centrifuged at 10,000 rpm for 20 min, and the supernatant was taken and repeatedly centrifuged once in a 1 mL centrifuge tube, and the supernatant was taken and loaded into a dialysis bag for dialysis in pure water for 48 h, and the dialysis bag was taken out and stirred in anhydrous alcohol for 4 h, and a precipitate appeared, and the precipitate was suspended in a 1.5 mL centrifuge tube with anhydrous ethanol, and centrifuged at 10,000 rpm for 5 min at 4°C, and the supernatant was discarded, and the precipitate was combined into a 1.5 mL centrifuge tube.

[0040] (6) 1 mL of pre-cooled acetone was added to the precipitate obtained in step (5), and the mixture was shaken for 1 min with a vortex instrument, and then centrifuged at 10,000 rpm for 5 min at 4°C, and the supernatant was discarded, and the 1.5 mL centrifuge tube containing the precipitate was placed in a fume hood and dried with the lid open for 2 h.

[0041] (7) 0.2 mL of a dissolving solution was added to the dried precipitate obtained in step (6) to obtain a final sample.

[0042] The dissolving solution comprises 1M NaAC and 1% volume percentage Triton X-100, and the pH of the dissolving solution is 4.0.

[0043] The difference between Comparative Example 1 and Example 1 is that the collection of the bacteria in step (1) is changed to centrifugal collection, and the false positive rate of Comparative Example 1 is obviously higher than that of Example 1, which indicates that the bacteria collected in Example 1 are cleaner.

[0044] Comparative Example 2 (1) 10 L of mycoplasma pneumoniae culture solution was concentrated to 100 mL by using a hollow fiber column, and after concentration, liquid displacement was performed by using the hollow fiber column, and the displacement liquid was PBS, 4000 mL of displacement liquid was added each time, and the displacement was repeated for 3 times. Washing: the concentrated solution after displacement was loaded into a centrifuge bottle, and centrifuged at 12,000 rpm for 30 min at 4°C to obtain a precipitate, and 150 mL of PBS was added to resuspend and mix, and then centrifuged at 12,000 rpm for 30 min at 4°C to obtain a precipitate; the above-mentioned washing step was repeated for 2 times, and the bacteria after washing were dried for 30 min and collected for weighing and recording.

[0045] (2) Bacterial cell pretreatment: The bacterial cell obtained in step (1) was transferred to a mortar and ground with an appropriate amount of PBS for 20 min, and then suspended with an appropriate amount of PBS; the suspension was transferred to a beaker, and the final volume was determined according to 1 g of bacterial cell corresponding to 10 mL of PBS.

[0046] (3) Ultrasonic treatment: the suspension obtained in step (2) was placed in ice water for ice bath ultrasonic treatment; the ultrasonic instrument used was purchased from Ningbo Xinzhi Biological Technology Co., Ltd., model JY98-IIIN, power setting 15# amplitude rod, 3% power, ultrasonic 2 s stop 3 s, ultrasonic time 6 min, 10000 rpm 20 min 4℃ centrifugation to take the supernatant solution; The obtained precipitate was suspended with an equal amount of PBS again, and the obtained suspension was repeated with the above ice bath ultrasonic treatment step, and the obtained solution was combined with the above supernatant solution.

[0047] (4) The liquid obtained in step (3) was added with an equal volume of anhydrous ethanol, and placed in a 95℃ water bath for 1 h.

[0048] (5) The liquid obtained in step (4) was concentrated by 10 times, and the concentrated liquid was taken, phenol was added according to the ratio of concentrated liquid: phenol = 9:11, 70℃ water bath oscillation for 1 h, 10000 rpm centrifugation for 20 min, the supernatant was repeatedly centrifuged once, and the supernatant was taken and loaded into a dialysis bag for 48 h of pure water dialysis, the dialysis bag was taken out and placed in anhydrous alcohol for stirring for 4 h, and a precipitate appeared, which was suspended with anhydrous ethanol into a 1.5 mL centrifuge tube, 10000 rpm 5 min 4℃ centrifugation, and the supernatant was discarded, and the precipitate was combined into a 1.5 mL centrifuge tube.

[0049] (6) 0.2 mL of dissolving solution was added to the precipitate obtained in step (5) to obtain a final sample.

[0050] The dissolving solution includes 1M NaAC and volume percentage 1% triton X-100, and the dissolving solution PH is 4.0.

[0051] The difference between Comparative Example 2 and Example 1 is that the step (6) acetone degreasing step is removed, and the gold colloid platform verification shows that the false positive of Comparative Example 2 is slightly higher than that of Example 1, which indicates that the step (6) acetone degreasing step of Example 1 can remove part of the irrelevant substances.

[0052] Comparative Example 3 (1) 10L of Mycoplasma pneumoniae culture solution was concentrated to 100mL by hollow fiber column, and after concentration, liquid displacement was carried out by hollow fiber column, and the displacement liquid was PBS, 4000mL of displacement liquid was added each time, and the displacement was repeated for 3 times; Washing: The replaced concentrated solution was loaded into a centrifuge bottle, and the precipitate was obtained by centrifugation at 12000 rpm for 30 min at 4 DEG C. 150 mL of PBS was added to resuspend the precipitate, and the precipitate was obtained by centrifugation at 12000 rpm for 30 min at 4 DEG C. The above washing step was repeated twice, and the washed bacteria were dried for 30 min and collected for weighing and recording.

[0053] (2) Bacterial pretreatment: The bacteria obtained in step (1) were transferred to a mortar, and a proper amount of PBS was added to grind for 20 min, and then the bacteria were suspended in a proper amount of PBS. The suspension was transferred to a beaker, and the final volume was determined according to 1 g of bacteria corresponding to 10 mL of PBS.

[0054] (3) Ultrasonic treatment: The suspension obtained in step (2) was placed in ice water for ice bath ultrasonic treatment. The ultrasonic instrument used was purchased from Ningbo Xinzhi Biological Technology Co., Ltd., model JY98-IIIN, power setting 15# amplitude rod, 3% power, ultrasonic 2 s stop 3 s, ultrasonic time 6 min, 10000 rpm 20 min 4 DEG C centrifugation to obtain the supernatant solution; the obtained precipitate was suspended in an equal amount of PBS, and the obtained suspension was repeated with the above ice bath ultrasonic treatment step, and the obtained solution was combined with the above supernatant solution.

[0055] (4) The liquid obtained in step (3) was added with an equal volume of anhydrous ethanol, and placed in a 95 DEG C water bath for 1 h.

[0056] (5) The suspension obtained in step (4) was centrifuged at 10000 rpm for 20 min at 4 DEG C, and the supernatant was added with anhydrous ethanol to a final concentration of 85%, and stirred for 4 h. The precipitate was obtained by centrifugation at 10000 rpm for 5 min at 4 DEG C, and the supernatant was discarded. All the precipitates were combined into one 1.5 mL centrifuge tube.

[0057] (6) 1 mL of pre-cooled acetone was added to the precipitate obtained in step (5), and the precipitate was obtained by centrifugation at 10000 rpm for 5 min at 4 DEG C. The 1.5 mL centrifuge tube containing the precipitate was placed in a fume hood for 2 h.

[0058] (7) 0.2 mL of a dissolving solution was added to the dried precipitate obtained in step (6) to obtain a final sample.

[0059] The dissolving solution comprises 1M NaAC and 1% volume percentage of triton X-100, and the pH of the dissolving solution is 4.0.

[0060] ​Comparative Example 3 differs from Example 1 in that the step (5) of removing the hot phenol impurities and water extraction and alcohol precipitation is removed and replaced with a single alcohol precipitation. The final sample inspection results are significantly higher than the false positive of Example 1, which shows that the step (5) of removing the hot phenol impurities and water extraction and alcohol precipitation in Example 1 can remove impurities and improve sample purity.

[0061] Comparative Example 4 (1) Concentrate 10 L of Mycoplasma pneumoniae culture solution to 100 mL on a hollow fiber column. After concentration, use the hollow fiber column for liquid displacement. The displacement fluid is PBS. Add 4000 mL of displacement fluid each time, and repeat the displacement for 3 times. Washing: Put the concentrated solution after displacement into a centrifuge bottle, centrifuge at 12000 rpm for 30 min at 4°C to obtain the precipitate, resuspend the precipitate with 150 mL of PBS, centrifuge at 12000 rpm for 30 min at 4°C to obtain the precipitate, and repeat the above washing steps for 2 times. Dry the washed bacteria for 30 min and collect and record the weight.

[0062] (2) Bacterial pretreatment: Transfer the bacteria obtained in step (1) to a mortar, grind with appropriate amount of PBS for 20 min, and then suspend with appropriate amount of PBS. Transfer the suspension to a beaker, and determine the final volume according to 1 g of bacteria corresponding to 10 mL of PBS.

[0063] (3) Ultrasonic treatment: Put the suspension obtained in step (2) in ice water for ice bath ultrasonic treatment. The ultrasonic instrument used is purchased from Ningbo Xinzhi Biological Technology Co., Ltd., model JY98-IIIN, power setting 15# amplitude rod, 3% power, ultrasonic 2s stop 3s, ultrasonic time 6min, centrifuge at 10000 rpm for 20 min at 4°C to obtain the supernatant.

[0064] (4) Add phenol to the supernatant solution obtained in step (3) according to the ratio of supernatant solution: phenol = 9:11, 70°C water bath oscillation for 1h, 10000rpm centrifugation for 20min. Take the supernatant and divide it into 1mL centrifuge tubes and repeat the centrifugation for 1 time. Take the supernatant and put it into a dialysis bag for 48h of pure water dialysis. Take out the dialysis bag and put it into anhydrous alcohol for 4h of stirring. A precipitate appears. Suspend the precipitate with anhydrous ethanol to 1.5mL centrifuge tubes. Centrifuge at 10000rpm for 5min at 4°C to discard the supernatant and leave the precipitate. Combine all the precipitates into 1 1.5mL centrifuge tube.

[0065] (5) Add 1mL pre-cooled acetone to the precipitate obtained in step (4), and vortex for 1min. Centrifuge at 10000rpm for 5min at 4°C to discard the supernatant and leave the precipitate. Put the 1.5mL centrifuge tube containing the precipitate into a fume hood and dry for 2h with the lid open.

[0066] (6) To the dried precipitate obtained in step (5), 0.2 mL of the dissolving solution was added to obtain the final sample.

[0067] The dissolving solution comprises 1M NaAC and 1% volume percentage Triton X-100, and the pH of the dissolving solution is 4.0.

[0068] The difference between Comparative Example 4 and Example 1 is that the secondary ultrasonic treatment in step (3) and the ethanol reflux in step (4) are removed. The specificities and sensitivities of the final sample of Comparative Example 4 do not change significantly compared with those of Example 1, and the titer slightly decreases. This indicates that the secondary ultrasonic treatment in step (3) and the ethanol reflux in step (4) can increase the yield of the antigen, and thus the antigen content in the final sample increases and the titer increases.

[0069] Comparative Example 5 (1) The 10 L mycoplasma pneumoniae culture solution was concentrated to 100 mL by using a hollow fiber column. After the concentration, liquid displacement was performed by using the hollow fiber column, and the displacement liquid was PBS. Each time, 4000 mL of the displacement liquid was added, and the displacement was repeated for 3 times. Washing: The concentrated solution after displacement was placed in a centrifuge bottle, and centrifugation was performed at 12000 rpm for 30 min at 4°C to obtain the precipitate. Then, 150 mL of PBS was added to resuspend and mix the precipitate, and centrifugation was performed at 12000 rpm for 30 min at 4°C to obtain the precipitate. The above washing step was repeated for 2 times. Then, the washed bacteria were dried for 30 min and collected to record the weight.

[0070] (2) Bacteria pretreatment: The bacteria obtained in step (1) were transferred to a mortar, and a proper amount of PBS was added to grind for 20 min. Then, a proper amount of PBS was added to suspend the bacteria. The suspension was transferred to a beaker, and the final volume was determined according to 1 g of bacteria corresponding to 10 mL of PBS.

[0071] (3) Ultrasonic treatment: The suspension obtained in step (2) was placed in ice water for ice bath ultrasonic treatment. The ultrasonic instrument used was purchased from Ningbo Xinzhi Biological Technology Co., Ltd., and the model was JY98-IIIN. The power was set to 15# amplitude rod and 3% power. Ultrasonic treatment was performed for 2 s and stopped for 3 s. The ultrasonic treatment time was 6 min. After centrifugation at 10000 rpm for 20 min at 4°C, the supernatant solution was obtained to obtain the final sample. The protein electrophoretogram of the final sample is shown in FIG. 2. Figure 1

[0072] The difference between Comparative Example 5 and Example 1 is that the secondary ultrasonic treatment and steps (4) to (7) are removed. The false positive of the supernatant solution obtained by ultrasonic centrifugation of Comparative Example 5 increases significantly. This indicates that the further purification of the supernatant solution obtained by ultrasonic centrifugation in Example 1 can improve the purity of the antigen.

[0073] Comparative Example 6 ​(1) Washing: 10 L of Mycoplasma pneumoniae culture solution was loaded into a centrifuge bottle, and the precipitate was obtained by centrifugation at 12,000 rpm for 30 min at 4°C. 150 mL of PBS was added to resuspend and mix, and the precipitate was obtained by centrifugation at 12,000 rpm for 30 min at 4°C. The above steps were repeated twice. The washed bacteria were dried for 30 min and collected for weighing and recording.

[0074] (2) Bacterial pretreatment: The bacteria obtained in step (1) were transferred to a mortar, and a suitable amount of PBS was added to grind for 20 min, and then a suitable amount of PBS was added to suspend. The suspension was transferred to a beaker, and the final volume was determined according to 1 g of bacteria corresponding to 10 mL of PBS.

[0075] (3) Ultrasonic treatment: The suspension obtained in step (2) was placed in ice water for ice bath ultrasonic treatment. The ultrasonic instrument used was purchased from Ningbo Xinzhi Biological Technology Co., Ltd., model JY98-IIIN, power setting 15# amplitude rod, 3% power, ultrasonic 2 s stop 3 s, ultrasonic time 6 min, 10,000 rpm 20 min 4°C centrifugation, and the supernatant solution was obtained. The protein electrophoretogram of the final sample is shown in Figure 2 .

[0076] The difference between Comparative Example 6 and Comparative Example 5 is that the collection method of the bacteria in step (1) is changed to centrifugal collection. The false positive rate of the supernatant solution obtained by ultrasonic centrifugation in Comparative Example 6 is higher than that in Comparative Example 5. This indicates that Comparative Examples 5 and 6 need to be further purified, and the further purification of the supernatant solution obtained by ultrasonic centrifugation in Example 1 can improve the purity of the antigen.

[0077] Experiment Mycoplasma pneumoniae natural antigen was prepared according to the preparation method of Mycoplasma pneumoniae natural antigen in Example 1, and the dissolving solution in step (7) was respectively: 1. 1M NaAC and volume percentage 1% triton X-100, PH4.0 of Example 1; 2. ultrapure water; 3. dilute hydrochloric acid PH2.6; 4. dilute NaOH PH10.6; 5. 1M NaAC, PH4.0; 6. PBS; 7. pure water, 0.14M NaCl; 8. pure water, 0.5M NaCl; 9. volume percentage 1% triton X115, 1M NaAC, PH4.0.

[0078] The prepared Mycoplasma pneumoniae natural antigens are verified by colloidal gold platform, and experiments prove that the Mycoplasma pneumoniae natural antigens prepared by the dissolving solution in Example 1 are the best, and the bands are stable and do not fade.

[0079] The above merely describes preferred embodiments of the present application but is not intended to limit the present application, and any modification, equivalent replacement and improvement made within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

1. A method for preparing natural antigen of Mycoplasma pneumoniae, characterized in that: Includes the following steps: (1) Concentrate 10L of Mycoplasma pneumoniae culture medium onto a hollow fiber column to 100mL. After concentration, use the hollow fiber column for liquid replacement. Add 4000mL of replacement solution each time and repeat the replacement 3 times. Washing: Transfer the replaced concentrate into a centrifuge bottle, centrifuge at 12000 rpm for 30 min at 4℃ to collect the precipitate, add 150 mL PBS to resuspend and mix well, centrifuge at 12000 rpm for 30 min at 4℃ to collect the precipitate; repeat the above washing steps twice, and dry the washed bacterial cells for 30 min. (2) Pretreatment of bacterial cells: The bacterial cells obtained in step (1) were ground with PBS for 20 min and then suspended in PBS; the final volume of the suspension was determined according to 10 mL PBS for 1 g of bacterial cells. (3) Ultrasonic treatment: The suspension obtained in step (2) is subjected to ice bath ultrasonication: ultrasonication for 2 seconds and pause for 3 seconds, ultrasonication time is 6 minutes, 10000 rpm for 20 minutes, centrifugation at 4℃, and then the supernatant solution is taken. The precipitate was resuspended in an equal volume of PBS, and the suspension was repeated with the ice bath sonication steps described above. The resulting solution was then combined with the supernatant solution described above. (4) Add an equal volume of anhydrous ethanol to the liquid obtained in step (3), place it in a 95°C water bath, and reflux for 1 hour; (5) Concentrate the liquid obtained in step (4) by 10 times, take the concentrate, add phenol at a ratio of concentrate:phenol = 9:11, shake in a water bath at 70°C for 1 hour, centrifuge at 10,000 rpm for 20 minutes, take the supernatant solution and repeat centrifugation once, take the supernatant and put it into a dialysis bag for pure water dialysis for 48 hours, take out the dialysis bag, put it in anhydrous alcohol and stir for 4 hours, a precipitate appears, suspend the precipitate in anhydrous ethanol into a centrifuge tube, centrifuge at 10,000 rpm for 5 minutes at 4°C and discard the supernatant, keep the precipitate, and combine the precipitates obtained; (6) Add 1 mL of pre-cooled acetone to the precipitate obtained in step (5), shake for 1 min, centrifuge at 10000 rpm for 5 min at 4℃, discard the supernatant, keep the precipitate, and dry the precipitate for 2 h; (7) Add 0.2 mL of dissolving solution to the dried precipitate obtained in step (6) to obtain the final sample.

2. The method for preparing a natural antigen of Mycoplasma pneumoniae according to claim 1, characterized in that: In step (7), the dissolving solution is 1M NaAC and 1% Triton X-100 by volume, and the pH of the dissolving solution is 4.

0.

3. The method for preparing natural antigen of Mycoplasma pneumoniae according to claim 2, characterized in that: The replacement solution in step (1) is PBS.

4. The method for preparing natural antigen of Mycoplasma pneumoniae according to claim 3, characterized in that: In step (3), the power of the ultrasonic instrument is set to 15# amplitude transformer and 3% power.

5. The method for preparing natural antigen of Mycoplasma pneumoniae according to claim 4, characterized in that: In step (5), the precipitate is suspended in anhydrous ethanol into a 1.5 mL centrifuge tube.

6. The method for preparing natural antigen of Mycoplasma pneumoniae according to claim 5, characterized in that: In step (5), the resulting precipitate is combined into a 1.5 mL centrifuge tube.

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