A highly efficient culture method for Chlamydia pneumoniae and a method for preparing its natural antigen and its application

Abstract

The invention discloses an efficient culture method of Chlamydia pneumoniae and a preparation method and application of its natural antigen. The efficient culture method of Chlamydia pneumoniae comprises the following steps: S1, Chlamydia pneumoniae release: Chlamydia pneumoniae is lysed and released from host cells; S2, PEG pretreatment host cells: 5-10% polyethylene glycol is added to the host cells, and pretreatment is performed for 1h; S3, Chlamydia pneumoniae infects host cells: Chlamydia pneumoniae is inoculated into host cells and incubated; 60-80r / min, shaker is slowly shaken for 1h; S4, culture of Chlamydia pneumoniae: after the mixed culture solution of the previous step is statically cultured for 2h, the culture medium is replaced with Chlamydia pneumoniae growth solution containing cycloheximide, and culture is continued for 3d-7d; S5, collection of Chlamydia pneumoniae: microscopic observation, collection of cell suspension, and obtaining Chlamydia pneumoniae intermediates. The present invention also provides a method for preparing natural antigens using Chlamydia pneumoniae intermediates. The Chlamydia pneumoniae prepared by the culture method has high yield and low cost, and is suitable for large-scale production.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technology, and in particular to an efficient culture method of Chlamydia pneumoniae and a preparation method and application of its natural antigen. Background Art

[0002] Chlamydia pneumoniae (C. Pneumoniae, referred to as Cpn) is an obligate intracellular parasitic prokaryotic microorganism that can only grow in cells and is mainly transmitted through the respiratory tract. It is one of the important pathogens that cause community-acquired pneumonia, bronchitis, pharyngitis and other human respiratory diseases. Chlamydia pneumoniae has a biphasic life cycle of extracellular infectious particles (also known as protosomes, EB) and intracellular replication forms (also known as reticulated bodies, RB), which is similar to Chlamydia trachomatis and Chlamydia psittaci.

[0003] There are two main detection methods for Chlamydia pneumoniae: pathogen detection method and serological detection method; pathogen detection method is divided into two types: Chlamydia pneumoniae culture and Chlamydia pneumoniae nucleic acid detection, among which Chlamydia pneumoniae culture is the gold standard, but Chlamydia pneumoniae needs to be cultured in a cell line derived from the respiratory tract, and the operation is cumbersome; nucleic acid detection mainly amplifies the target gene through specific probes and primers, but clinical samples are easily contaminated, the operation requirements are high, and it is not easy to promote; serological detection methods mainly use recombinant antigens to detect specific Chlamydia pneumoniae antibodies in serum, such as ELISA.

[0004] At present, since Chlamydia pneumoniae is difficult to culture, there is no perfect culture method. Its culture method is generally based on the culture method of Chlamydia trachomatis, and is improved in terms of cell lines, culture conditions and intervention measures. Previous studies have shown that in the process of Chlamydia pneumoniae culture, the addition of appropriate antibiotics can inhibit host cell metabolism, thereby providing more nutrients for the growth and reproduction of Cpn; multiple centrifugation of Cpn infected samples can significantly improve the effectiveness of culture; monolayer cells treated with right-rotating DEAE or Poly-L-lysine can also improve the sensitivity of culture, but the infection effect is not obvious.

[0005] Since it is difficult to cultivate Chlamydia pneumoniae, it is particularly important to establish a suitable in vitro culture method. In recent years, community-acquired pneumonia has occurred frequently, especially in children. A Chlamydia pneumoniae raw material with strong specificity and high sensitivity is urgently needed in clinical testing. At present, there are various problems in the existing Chlamydia pneumoniae culture methods, such as complex and time-consuming culture process, low infection rate, and difficulty in expanding culture, which greatly limit the rapid development of Chlamydia pneumoniae detection technology and related drugs. Therefore, the existing technology needs to be further improved. Summary of the invention

[0006] In response to the above problems, the present invention provides an efficient culture method for Chlamydia pneumoniae based on the characteristics of Chlamydia pneumoniae. The prepared Chlamydia pneumoniae has high yield and high infection rate. The method is simple to operate and easy to expand the culture. Based on this technology, the present invention further provides a preparation method for the corresponding natural antigen of Chlamydia pneumoniae and its application.

[0007] To solve the above problems, a large number of studies have been conducted on high-yield Chlamydia pneumoniae culture methods, and an improved culture scheme has been obtained: the yield of Chlamydia pneumoniae is increased by combining physical (slow shaking on a shaker) and chemical (PEG treatment) methods; at the same time, high-purity natural Chlamydia pneumoniae antigens are obtained by ultrahigh-speed centrifugation and applied to clinical testing of Chlamydia pneumoniae pneumonia.

[0008] The technical solution provided by the present invention is as follows:

[0009] In a first aspect, the present invention provides a method for efficiently culturing Chlamydia pneumoniae, comprising the following steps:

[0010] S1. Release of Chlamydia pneumoniae: The Chlamydia pneumoniae is lysed and released from the original host cells.

[0011] Preferably, the method for releasing Chlamydia pneumoniae in step S1 is specifically as follows: freezing and thawing the Chlamydia pneumoniae repeatedly at least three times under liquid nitrogen and 37°C water bath conditions, adding an appropriate amount of glass beads, and vortexing for 5-10 minutes to fully release the Chlamydia pneumoniae. The method of repeatedly freezing and thawing at least three times has the effect of destroying the cell membrane of the host cell, promoting the full release of intracellular Chlamydia pneumoniae, and having no effect on the activity of Chlamydia pneumoniae.

[0012] S2. PEG pretreatment of host cells: Add 5-10% polyethylene glycol to the cultured host cells for pretreatment for 1 hour. The conditions are preferably: 35°C, 5% CO2. PEG pretreatment changes the permeability of the cell membrane of the host cells, making it easier for Chlamydia pneumoniae (Cpn) to invade the host cells in the subsequent infection step, thereby improving the infection effect.

[0013] Preferably, the polyethylene glycol is PEG1500; in other embodiments, PEG with other molecular weights (such as PEG3350, PEG4500, PEG8000) is used to pretreat cells.

[0014] However, the experimental results showed that after the host cells (human laryngeal epidermoid carcinoma cells were used in the experiment) were pretreated with PEG1500, PEG3350, PEG4500, and PEG8000 in the same manner, and cultured for 3-7 days, microscopic observation showed that the cell shedding, cpn number, and infection rate of the three groups of PEG3350, PEG4500, and PEG8000 were all lower than those of the PEG1500 group. The host cells treated with PEG1500 in this method can improve the permeability of the cell membrane, and PEG with a small molecular weight has less damage to the cells.

[0015] S3. Chlamydia pneumoniae infects host cells: Chlamydia pneumoniae is inoculated into host cells and incubated at 35°C, 5% CO2 for 10 min; shake slowly at 35°C, 5% CO2, 60-80 r / min for 0.5-1.5 h to promote the contact between Cpn and the monolayer of host cells and ensure that Cpn infects a wider range.

[0016] Different from the conventional method of promoting the infection of Chlamydia pneumoniae with host cells by centrifugation, the present application adopts the method of slow shaking on a shaking table to "spread" Chlamydia pneumoniae between host cells to the greatest extent, promote the contact between Chlamydia pneumoniae and host cells, and facilitate the full infection of Chlamydia pneumoniae. In addition, the slow shaking method is easier to operate, has lower requirements for experimental instruments, is simple to operate, and is more conducive to mass production.

[0017] The combination of the aforementioned PEG pretreatment of host cells and the slow shaking assisted infection in this step can significantly increase the infection rate and substantially increase the yield of Chlamydia pneumoniae.

[0018] Preferably, during this step of inoculation, the number of Chlamydia pneumoniae AR-39 strains should reach 10 5 IFU / mL, inoculated according to the volume ratio of host cells: bacteria (1:50) to (1:100).

[0019] S4. Cultivation of Chlamydia pneumoniae: After culturing the mixed culture medium in the previous step for 1 to 3 hours, replace the culture medium with Chlamydia pneumoniae growth medium containing cycloheximide and culture for 3 days to 7 days.

[0020] Preferably, the Chlamydia pneumoniae growth solution is prepared with DMEM culture medium as solvent, and further comprises the following components: fetal bovine serum (1-10%), cycloheximide (1-10ug / ml). This preferred growth solution can reduce host cell metabolism, provide more nutrients for the growth of Chlamydia pneumoniae, and help increase its yield.

[0021] Furthermore, antibiotics are added to the Chlamydia pneumoniae growth solution; optionally, the Chlamydia pneumoniae growth solution also includes: gentamicin (10ug / ml) and vancomycin (25ug / ml). In other embodiments, the antibiotics can be replaced with other types of antibiotics or their concentrations can be adjusted.

[0022] Preferably, in the Chlamydia pneumoniae growth medium, the concentration of fetal bovine serum is 2%, and the concentration of cycloheximide is 2ug / ml. Experiments have shown that the infection rate and yield of Chlamydia pneumoniae cultured under these conditions are higher, which is more conducive to the reproduction of Chlamydia pneumoniae.

[0023] S5. Collection of Chlamydia pneumoniae: Under sterile conditions, scrape the cells (preferably with a scraper), mix them by blowing, and then collect the cell suspension to obtain the Chlamydia pneumoniae intermediate product, which is stored at low temperature.

[0024] After 3-7 days of culture, the infection rate of Cpn reached 100%, 90-100% of cells fell off, and Cpn was about 10 7 -10 8 IFU.

[0025] The Chlamydia pneumoniae intermediate is a mixture of host cells and a large number of proliferated Chlamydia pneumoniae. The Chlamydia pneumoniae can be fully released by lysing the cells for further application.

[0026] Preferably, in steps S1 to S5, the culture conditions of the host cells and Chlamydia pneumoniae are both 35° C. and 5% CO 2 .

[0027] Preferably, in the efficient culture method of Chlamydia pneumoniae, the host cell is: human laryngeal epidermoid carcinoma cell.

[0028] Preferably, the host cell culture method is as follows:

[0029] S01. Cell recovery: The host cells stored in liquid nitrogen were placed in a 37°C water bath to melt; centrifuged at 1000 rpm for 10-20 min, the supernatant was discarded, the precipitate was suspended in DMEM cell culture medium containing 10% fetal bovine serum, and cultured in a 25T cell flask; cultured at 35°C and 5% CO2 for 2-3 days;

[0030] S02. Cell passaging: Digest the cells with an appropriate amount of trypsin, and when gaps appear on the cell surface by naked eye, add DMEM cell culture medium containing 10% fetal bovine serum and mix well; centrifuge at 1000rpm for 10-20min, suspend the precipitate with DMEM cell culture medium containing 10% fetal bovine serum, plate and culture until the cell density of the cell culture dish reaches more than 90%.

[0031] In a second aspect, the present invention further provides a method for preparing a natural antigen of Chlamydia pneumoniae, comprising the following steps:

[0032] (1) Inactivating the Chlamydia pneumoniae intermediate prepared by the aforementioned culture method: adding formaldehyde solution to the Chlamydia pneumoniae intermediate and treating in a water bath for 20 to 30 hours.

[0033] Preferably, the inactivation treatment conditions are: to Cpn intermediate (about 10 8 -10 9 Formaldehyde was added to the cells at a final concentration of 0.1% and the cells were incubated in a 37°C water bath for 24 h.

[0034] (2) Ultrasonic disruption of inactivated Chlamydia pneumoniae.

[0035] Preferably, in the method for preparing the natural antigen of Chlamydia pneumoniae, the conditions for ultrasonic fragmentation are: power 180-240 W, fragmentation 2-3 s, pause 3-4 s, and working time 2-5 min.

[0036] (3) The liquid from the previous step is centrifuged at 4°C and 5000 rpm for 20-30 min to remove cell debris, collect the supernatant, and centrifuge the supernatant at 4°C and 30000 x g-50000 x g for 30 min to obtain a crude Chlamydia pneumoniae product.

[0037] (4) An equal volume of SPG buffer was added to the crude Chlamydia pneumoniae product, and the natural cpn antigen was obtained after ultrafiltration and concentration.

[0038] In a third aspect, the present invention also provides a use of the Chlamydia pneumoniae natural antigen prepared by the aforementioned preparation method in a clinical detection kit. This clinical detection kit can be widely used to detect whether a sample has Chlamydia pneumoniae antibodies, has high specificity, and the detection results are accurate and reliable.

[0039] The present invention has the following beneficial effects:

[0040] 1. The present invention provides an efficient culture method for Chlamydia pneumoniae, which not only optimizes the growth fluid of Chlamydia pneumoniae, reduces host cell metabolism, and provides more nutrients for Cpn growth; at the same time, it innovatively adopts a method of combining PEG pretreatment of host cells with slow shaking of a shaker to assist infection to increase the yield of Chlamydia pneumoniae.

[0041] On the one hand, the slow shaking of the shaker can "spread" Cpn between cells to the greatest extent, promoting full contact between Cpn and cells; on the other hand, PEG can change the permeability of the cell membrane and enhance adsorption infection to a certain extent. The organic combination of the two greatly increases the infection rate of Cpn, thereby obtaining a high yield of Chlamydia pneumoniae.

[0042] 2. This culture method also shortens the culture time of Chlamydia pneumoniae, reduces its culture cost, and is simple to operate and easy to expand the culture.

[0043] 3. The present invention also provides a natural Chlamydia pneumoniae antigen, which is obtained by ultra-high-speed centrifugation. The preparation process is simple, and the antigen has high purity, strong specificity and high sensitivity, which can further improve the clinical detection effect in the diagnosis of Chlamydia pneumoniae pneumonia.

[0044] 4. The method for culturing Chlamydia pneumoniae and the method for preparing natural antigens of the present invention involve few equipments, simple procedures, low costs, and will not produce harmful substances to the environment; the whole preparation process is stable and suitable for mass production and large-scale promotion. BRIEF DESCRIPTION OF THE DRAWINGS

[0045] Figure 1 This is a microscopic image of cells obtained by the culture method using only the shaker-assisted infection;

[0046] Figure 2 This is a microscopic image of cells obtained by using the PEG pretreatment cell and shaker-assisted infection culture method of the present application;

[0047] Figure 3 This is a microscopic image of cells obtained by centrifugation-assisted culture during the infection process;

[0048] Figure 4 This is a diagram illustrating the test results of colloidal gold test strips. DETAILED DESCRIPTION

[0049] The technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments in the present invention, all other embodiments obtained by those skilled in the art without creative work are within the scope of protection of the present invention. In the present invention, unless otherwise specified, the equipment and raw materials used can be purchased from the market or are commonly used in the art. The methods in the following embodiments are conventional methods in the art unless otherwise specified.

[0050] Example 1 Cultivation of Chlamydia pneumoniae

[0051] 1. Experimental materials:

[0052] Host cells: Human laryngeal epidermoid carcinoma Hep-2 cells.

[0053] Chlamydia pneumoniae: Chlamydia pneumoniae AR-39: ATCC 53592.

[0054] 2. This embodiment provides an efficient culture method for Chlamydia pneumoniae, which comprises the following steps:

[0055] S1. Cell recovery: human laryngeal epidermoid carcinoma cell Hep-2 stored in liquid nitrogen was thawed in a 37°C water bath; centrifuged at 1000 rpm for 10 min, the supernatant was discarded, the precipitate was suspended in DMEM cell culture medium containing 10% fetal bovine serum, and cultured in a 25T cell flask; cultured at 35°C, 5% CO2, after 3 days, the cells grew into a full monolayer and were ready for passaging.

[0056] S2. Cell passaging: Digest the cells with an appropriate amount of trypsin, and when gaps appear on the cell surface, add DMEM cell culture medium containing 10% fetal bovine serum and mix well; centrifuge at 1000 rpm for 10 min, suspend the precipitate with DMEM cell culture medium (containing 10% fetal bovine serum), and count under a microscope; count the cells at 3.5-4.5×10 5 Carry out plating; culture at 35°C, 5% CO2, so that the cell density of the cell culture dish reaches more than 90%.

[0057] S3. Release of Chlamydia pneumoniae: Chlamydia pneumoniae AR-39 from ATCC was repeatedly frozen and thawed four times in liquid nitrogen and 37°C water bath conditions, and an appropriate amount of glass beads was added and vortexed for 5 minutes to fully release Chlamydia pneumoniae.

[0058] S4. PEG pretreatment of Hep-2 cells: Add 200ul 5% PEG1500 to each cell well and incubate at 35°C, 5% CO2 for 1 hour to pretreat the cells. PEG is used to change the cell membrane of the host cells to facilitate the invasion of Chlamydia pneumoniae (Cpn) into the host cells.

[0059] S5. Cpn infection of host cells: Cpn was inoculated into Hep-2 cells at 200ul / ml and incubated at 35°C, 5% CO2 for 10min; the cells were shaken slowly at 60-80r / min for 1h to promote contact between Cpn and the monolayer of host cells and ensure a wider range of Cpn infection.

[0060] S7. After culturing at 35°C and 5% CO2 for 2 h, the culture medium was replaced with Cpn growth medium containing cycloheximide.

[0061] The preparation method of Cpn growth medium is as follows: 1-10% fetal bovine serum (FBS), 10ug / ml gentamicin, 25ug / ml vancomycin, and 1-10ug / ml cycloheximide are added to DMEM culture medium.

[0062] S8. Observation of Cpn: Incubate at 35°C, 5% CO2 for 72 h, prepare slides with Wright-Giemsa staining, and examine under a microscope.

[0063] Example 2 Optimization of Chlamydia pneumoniae Growth Fluid

[0064] 1. Experimental methods

[0065] According to the experimental method of Example 1, the following experimental groups were set up to culture Chlamydia pneumoniae, and the effects of the addition of FBS and cycloheximide in the Cpn growth medium on the growth of Cpn were detected. The experimental settings were as follows:

[0066] In group A, five groups A1, A2, A3, A4 and A5 were set up respectively. The volume percentage concentration of FBS added to the Cpn growth medium in each group was 0%, 1%, 2%, 5% and 10% respectively, and other components remained unchanged; the concentration of cycloheximide in the Cpn growth medium of each group in group A was 2ug / mL.

[0067] In group B, five groups, B1, B2, B3, B4 and B5, were set up. The concentrations of cycloheximide added to the Cpn growth medium in each group were: 0ug / mL, 1ug / mL, 2ug / mL, 5ug / mL, 10ug / mL, and other ingredients remained unchanged; the concentration of FBS in the Cpn growth medium of each group in group B was 2%.

[0068] 2. Detection method

[0069] Prepare slides by Swiss-Giemsa staining and examine under a microscope to observe and record cell density, cpn number, infection rate, cell shedding, etc.

[0070] 3. Experimental results and analysis

[0071] The test results of the products cultured in different experimental groups are shown in Tables 1 and 2:

[0072] (1) By comparing A1 to A5 in Table 1, it can be seen that the infection rate of Chlamydia pneumoniae prepared by group A3 with the addition of 2% fetal bovine serum is much higher than that of other groups, and its yield is also the highest; this shows that the addition of 2% fetal bovine serum can provide nutrients for Cpn proliferation without causing the host cells to grow too fast.

[0073] (2) By comparing B1 to B5 in Table 2, it can be seen that the infection rate of group B3 with 2ug / mL cycloheximide is much higher than that of other groups, and the yield of Chlamydia pneumoniae is also the highest, with better effect; 2ug / mL cycloheximide can inhibit the growth of host cells without being toxic to Cpn. The combination of 2% fetal bovine serum and 2ug / mL cycloheximide optimizes the Cpn growth medium and helps to improve the high infection rate of Cpn.

[0074] Table 1 Adding different concentrations of FBS (Group A)

[0075]

[0076] Table 2 Adding different concentrations of cycloheximide (Group B)

[0077]

[0078]

[0079] Example 3 Optimization of infection mode

[0080] 1. Group setting: In order to compare the effects of three infection methods, namely, traditional centrifugation, slow shaking on a shaker, and slow shaking on a shaker combined with PEG pretreatment of cells, on the culture effect of Chlamydia pneumoniae, three experimental groups, namely, Group C, Group D, and Group E, were set up in this example. The difference between the three experimental groups was only the difference in the treatment method of the infection process, as follows:

[0081] Group C: During the cpn passaging process, shaking table was added to assist the treatment;

[0082] In group D, host cells were pretreated with 5% PEG1500 for 1 h before cpn passage, and shaker-assisted treatment was added;

[0083] In group E, centrifugation was added to assist the passage of cpn.

[0084] 2. Experimental methods:

[0085] The experimental methods of the above three experimental groups are as follows:

[0086] (1) Group C: During the cpn passaging process, shaking table was added to assist the treatment;

[0087] S1 cell recovery: human laryngeal epidermoid carcinoma Hep-2 cells stored in liquid nitrogen were placed in a 37°C water bath to melt; centrifuged at 1000 rpm for 10 min, the supernatant was discarded, the precipitate was suspended in DMEM cell culture medium containing 10% fetal bovine serum, and cultured in a 25T cell flask; cultured at 35°C, 5% CO2, after 3 days, the cells grew into a monolayer and were ready for passage;

[0088] S2 cell passaging: Digest the cells with an appropriate amount of trypsin, and when gaps appear on the cell surface, add DMEM cell culture medium containing 10% fetal bovine serum and mix well; centrifuge at 1000 rpm for 10 min, suspend the precipitate with DMEM cell culture medium (containing 10% fetal bovine serum), and count under a microscope; count the cells according to 3.5-4.5*10 5 The cells were plated and cultured at 35°C with 5% CO2 until the cell density in the cell culture dish reached more than 90%.

[0089] S3 Chlamydia pneumoniae release: Chlamydia pneumoniae AR-39 from ATCC was repeatedly frozen and thawed 4 times in liquid nitrogen and 37°C water bath conditions, and an appropriate amount of glass beads was added and vortexed for 5 minutes to fully release Chlamydia pneumoniae;

[0090] S4 Cpn infection of host cells: Cpn was inoculated into Hep-2 cells at 200ul / ml and incubated at 35℃, 5% CO2 for 10min; the six-well cell plate was placed on a shaker and shaken slowly at room temperature for 1h;

[0091] S5 Cpn growth medium (1L) preparation: fetal bovine serum (2%), gentamicin (10ug / ml), vancomycin (25ug / ml), cycloheximide (2ug / ml), DMEM;

[0092] S6 Cpn culture: After culturing for 2 h at 35°C and 5% CO2, the culture medium was replaced with Cpn growth medium containing cycloheximide;

[0093] S7 Cpn observation: culture at 35℃, 5% CO2 for 72h, prepare slides with Wright-Giemsa staining, and examine under a microscope.

[0094] (2) In group D, host cells were pretreated with 5% PEG1500 for 1 h before cpn passage, and a shaking-assisted treatment was added;

[0095] S1 cell recovery: human laryngeal epidermoid carcinoma Hep-2 cells stored in liquid nitrogen were placed in a 37°C water bath to melt; centrifuged at 1000 rpm for 10 min, the supernatant was discarded, the precipitate was suspended in DMEM cell culture medium containing 10% fetal bovine serum, and cultured in a 25T cell flask; cultured at 35°C, 5% CO2, after 3 days, the cells grew into a monolayer and were ready for passage;

[0096] S2 cell passaging: Digest the cells with an appropriate amount of trypsin, and when gaps appear on the cell surface, add DMEM cell culture medium containing 10% fetal bovine serum and mix well; centrifuge at 1000 rpm for 10 min, suspend the precipitate with DMEM cell culture medium (containing 10% fetal bovine serum), and count under a microscope; count the cells according to 3.5-4.5*10 5 The cells were plated and cultured at 35°C with 5% CO2 until the cell density in each well reached more than 90%.

[0097] S3 Chlamydia pneumoniae release: Chlamydia pneumoniae AR-39 from ATCC was repeatedly frozen and thawed 4 times in liquid nitrogen and 37°C water bath conditions, and an appropriate amount of glass beads was added and vortexed for 5 minutes to fully release Chlamydia pneumoniae;

[0098] S4 PEG pretreatment of Hep-2 cells: Add 200ul 5% PEG1500 to each cell well and incubate at 35°C, 5% CO2 for 1 hour to pretreat the cells. PEG is used to change the cell membrane of the host cells, making it easier for Chlamydia pneumoniae (Cpn) to invade the host cells.

[0099] S5 Cpn infection of host cells: Cpn was inoculated into Hep-2 cells at 200ul / ml and incubated at 35℃, 5% CO2 for 10min; the six-well cell plate was placed on a shaker and shaken slowly at room temperature for 1h;

[0100] S6 Cpn growth medium (1L) preparation: fetal bovine serum (2%), gentamicin (10ug / ml), vancomycin (25ug / ml), cycloheximide (2ug / ml), DMEM;

[0101] S7 Cpn culture: After culturing at 35°C, 5% CO2 for 2 h, the culture medium was replaced with Cpn growth medium containing cycloheximide;

[0102] S8 Cpn observation: culture at 35℃, 5% CO2 for 72h, prepare slides with Wright-Giemsa staining, and examine under a microscope.

[0103] (3) Group E added centrifugation-assisted treatment during cpn passaging;

[0104] S1 cell recovery: human laryngeal epidermoid carcinoma Hep-2 cells stored in liquid nitrogen were placed in a 37°C water bath to melt; centrifuged at 1000 rpm for 10 min, the supernatant was discarded, the precipitate was suspended in DMEM cell culture medium containing 10% fetal bovine serum, and cultured in a 25T cell flask; cultured at 35°C, 5% CO2, after 3 days, the cells grew into a monolayer and were ready for passage;

[0105] S2 cell passaging: Digest the cells with an appropriate amount of trypsin, and when gaps appear on the cell surface, add DMEM cell culture medium containing 10% fetal bovine serum and mix well; centrifuge at 1000 rpm for 10 min, suspend the precipitate with DMEM cell culture medium (containing 10% fetal bovine serum), and count under a microscope; count the cells according to 3.5-4.5*10 5 The cells were plated and cultured at 35°C with 5% CO2 until the cell density in each well reached more than 90%.

[0106] S3 Chlamydia pneumoniae release: Chlamydia pneumoniae AR-39 from ATCC was repeatedly frozen and thawed 4 times in liquid nitrogen and 37°C water bath conditions, and an appropriate amount of glass beads was added and vortexed for 5 minutes to fully release Chlamydia pneumoniae;

[0107] S4 Cpn infection of host cells: Cpn was inoculated into Hep-2 cells at 200ul / ml, incubated at 35℃, 5% CO2 for 10min; centrifuged at 1000xg for 1h to make Cpn contact with the monolayer of host cells and press Cpn into the cells with the help of centrifugal force;

[0108] S5 Cpn growth medium (1L) preparation: fetal bovine serum (2%), gentamicin (10ug / ml), vancomycin (25ug / ml), cycloheximide (2ug / ml), DMEM;

[0109] S6 Cpn culture: After culturing at 35°C, 5% CO2 for 2 h, the culture medium was replaced with Cpn growth medium containing cycloheximide;

[0110] S7 Cpn observation: culture at 35℃, 5% CO2 for 72h, prepare slides with Wright-Giemsa staining, and examine under a microscope.

[0111] 3. Detection method: refer to Example 1.

[0112] 4. Experimental results and analysis

[0113] From the results in Table 3, it can be seen that the infection rate of group D using PEG pre-treated cells-shaker-assisted infection was the highest, reaching 100%, the cell density was the lowest, and the yield of Chlamydia pneumoniae was much higher than that of the other two groups.

[0114] The above experimental results prove that the improved culture process of Chlamydia pneumoniae of the present invention, which utilizes the infection method combining PEG pretreatment of host cells with shaker-assisted infection, can increase the infection rate of Chlamydia pneumoniae, shorten the culture time, and greatly increase the yield of Chlamydia pneumoniae; and it is simple to operate, easy to expand the culture, and reduce the culture cost.

[0115] Table 3 Comparison of different cpn passage methods (CE group)

[0116]

[0117] Example 4 Preparation of Chlamydia pneumoniae Natural Antigen

[0118] 1. Experimental methods:

[0119] The method for culturing Chlamydia pneumoniae of Group B3 in Example 2 was used to obtain 100 ml of Cpn intermediate, and the natural antigen was prepared according to the following steps, and the natural antigen was used in clinical testing:

[0120] A1. Inactivation of Cpn intermediate: Add 100 ml of Cpn intermediate (about 10 8 -10 9IFU) was inactivated by adding formaldehyde at a final concentration of 0.1% and incubating in a 37°C water bath for 24 h;

[0121] A2. Ultrasonic fragmentation of inactivated Chlamydia pneumoniae, ultrasonic conditions: power 180-240W, fragmentation 2-3s, pause 3-4s, working time 2-5min;

[0122] A3, 5000rpm, centrifuge for 20-30min, 4℃, remove cell debris; the supernatant is centrifuged at 30000×g-50000×g for 30min, 4℃ to obtain crude cpn;

[0123] A4. Add an equal volume of SPG buffer to the crude Cpn product, and concentrate it by ultrafiltration to obtain the natural Cpn antigen.

[0124] 2. Antigenicity test results

[0125] The Cpn immune serum (purchased from Shandong Shuojing Biotechnology Co., Ltd.) was diluted in series, and the cpn antigen sold on the market (purchased from Maidian Biotechnology Co., Ltd.) was used as a control. The titer of the Cpn natural antigen prepared by the present invention was detected by the ELISA indirect method, and the OD 450 The specific experimental results are shown in Table 4:

[0126] Table 4 Antigenicity test results

[0127] Cpn serum dilution multiple Self-produced Comparison 1 / 1k 1.556 0.669 1 / 1w 0.256 0.129

[0128] The experimental results show that the potency of the Cpn natural antigen prepared by the present invention is much higher than that of purchased Cpn under the two experimental conditions of 1 / 1k and 1 / 1w.

[0129] Example 5 Application of Chlamydia pneumoniae Natural Antigen in Colloidal Gold Chlamydia pneumoniae IgG Detection Kit 1. Application of Cpn Natural Antigen

[0130] (1) The Cpn natural antigen prepared in the present invention was diluted to a working concentration with a coating solution (20 mM PBS 5% sucrose pH 7.4) as a test line (T line) coating material, and the quality control line (C line) coating material (sheep anti-mouse IgG, purchased from Shandong Shuojing Biotechnology Co., Ltd.) was streaked on the NC membrane using a film streaker, and then baked at 37°C for 30 min to complete the solid phase of the raw materials.

[0131] (2) Assemble the solid-phased NC membrane with the sample pad, gold label pad (gold-labeled mouse anti-human IgG), PVC base plate, absorbent paper, and Marker of the Chlamydia pneumoniae IgG detection kit, and cut them into 3-4 mm test strips with a strip cutter for later use.

[0132] The sample pad, gold label pad, NC membrane, PVC base plate, absorbent paper and Mark sticker involved in the present invention were all purchased from Shandong Kanghua Biomedical Technology Co., Ltd.

[0133] 2. Performance testing

[0134] In this experiment, 72 serum samples were tested using the comparative test strips and the test strips prepared in this application to analyze the sensitivity, specificity and compliance rate of the raw materials.

[0135] (1) Detection method:

[0136] Comparison test strips: Chlamydia pneumoniae IgG antibody detection kit (colloidal gold method), purchased from Shandong Kanghua Biological, registration number: National Medical Device Registration No. 20153401655. Before use, restore the test strips and samples to room temperature.

[0137] Place the comparison test strip and the test strip prepared in this application flat on the table, and test the samples according to the following method: add 15ul of the serum sample to be tested, add 50-70ul of the sample diluent, and observe the experimental results for about 20 minutes. If only the T line is colored, the result is negative, and if both the T line and the C line are colored, the result is positive. If neither of them is colored, the test strip is invalid and the test result is invalid. See the colloidal gold test strip test result description for details. Figure 4 .

[0138] (2) Test results and analysis

[0139] The test results of 72 serum samples are shown in Tables 5 and 6. From the results in the tables, we can see that:

[0140] ① The Chlamydia pneumoniae IgG detection kit (test strip) prepared by using the Chlamydia pneumoniae natural antigen prepared in the present invention is compared with the control kit (test strip), and the positive color development can be improved, the sensitivity meets the requirements, the detection rate is higher than the control, and the specificity is better;

[0141] ②Compared with the control, the statistical coincidence rate results showed that the positive coincidence rate was 100%, the negative coincidence rate was 98.1%, and the total coincidence rate was 98.6%. This shows that the Chlamydia pneumoniae natural antigen of the present invention has good specificity, sensitivity and coincidence rate when used as a diagnostic raw material in the Chlamydia pneumoniae diagnostic reagent.

[0142] Table 5 Sample test results

[0143]

[0144]

[0145] Table 6 Compliance rate

[0146]

[0147] Note: The values ​​in the positive interval column in Table 5 and Table 6 represent the positive color intensity. The larger the value, the stronger the color. T≥1 is positive and T<1 is negative.

[0148] It is understandable that those skilled in the art can make equivalent substitutions or changes based on the technical solution and concept of the present invention, and all these changes or substitutions should fall within the protection scope of the claims attached to the present invention.

Claims

1. A method for culturing Chlamydia pneumoniae, characterized in that: The following steps are involved: S1. Release of Chlamydia pneumoniae: Chlamydia pneumoniae is lysed and released from the original host cells, specifically: Chlamydia pneumoniae is repeatedly frozen and thawed at least three times under liquid nitrogen and 37°C water bath conditions, and then an appropriate amount of glass beads are added, and vortexed for 5-10 minutes to fully release Chlamydia pneumoniae; the treatment method of repeatedly freezing and thawing at least three times has the effect of destroying the cell membrane of the host cells, promoting the full release of intracellular Chlamydia pneumoniae, and has no effect on the activity of Chlamydia pneumoniae; S2, PEG pretreatment of host cells: Add 5-10% PEG1500 to the cultured host cells and pretreat for 1 hour; S3. Chlamydia pneumoniae infects host cells: Chlamydia pneumoniae is inoculated into host cells and incubated; shake slowly at 60-80 r / min for 0.5-1.5 h; S4. Cultivation of Chlamydia pneumoniae: After the mixed culture medium in the previous step is statically cultured for 1-3 hours, the culture medium is replaced with Chlamydia pneumoniae growth medium containing cycloheximide, and the culture is continued statically for 3 days to 7 days; the Chlamydia pneumoniae growth medium is prepared with DMEM culture medium as a solvent, which also includes the following components: 2% fetal bovine serum, 2ug / ml cycloheximide, 10ug / ml gentamicin, and 25ug / ml vancomycin; S5. Collection of Chlamydia pneumoniae: Under sterile conditions, scrape the cells, blow and mix them, collect the cell suspension, obtain the Chlamydia pneumoniae intermediate, and store it at low temperature.

2. The method for culturing Chlamydia pneumoniae according to claim 1, characterized in that: Host cells: human laryngeal epidermoid carcinoma cells.

3. The method for culturing Chlamydia pneumoniae according to claim 1, characterized in that: The host cell culture method is: S01. Cell recovery: Thaw the host cells stored in liquid nitrogen; Centrifuge for 10-20 minutes, discard the supernatant, suspend the precipitate in DMEM cell culture medium containing 10% fetal bovine serum, and culture in a cell bottle; Culture at 35°C and 5% CO2 for 2-3 days; S02. Cell passaging: Digest the cells with an appropriate amount of trypsin, and when gaps appear on the cell surface by naked eye, add DMEM cell culture medium containing 10% fetal bovine serum and mix well; centrifuge at 1000rpm for 10-20min, suspend the precipitate with DMEM cell culture medium containing 10% fetal bovine serum, and then plate; culture at 35℃, 5% CO2, so that the cell density of the cell culture dish reaches more than 90%.

4. The method for culturing Chlamydia pneumoniae according to claim 1, characterized in that: In steps S1 to S5, the culture conditions of host cells and Chlamydia pneumoniae are both 35° C. and 5% CO 2 .

5. A method for preparing a natural antigen of Chlamydia pneumoniae, characterized in that: The following steps are involved: (1) Inactivating the Chlamydia pneumoniae intermediate prepared by the culture method of claim 1 by adding formaldehyde solution to the Chlamydia pneumoniae intermediate and treating in a water bath for 20 to 30 hours; (2) Ultrasonic fragmentation of inactivated Chlamydia pneumoniae; (3) The liquid in the previous step is centrifuged at 4°C and 5000 rpm for 20-30 min, the supernatant is collected, and the supernatant is centrifuged at 4°C and 30000 x g-50000 x g for 30 min to obtain a crude Chlamydia pneumoniae product; (4) Add an equal volume of SPG buffer to the crude Chlamydia pneumoniae product, and obtain the natural cpn antigen after ultrafiltration and concentration.

6. The method for preparing the Chlamydia pneumoniae natural antigen according to claim 5, characterized in that: The conditions for ultrasonic crushing are: power 180-240W, crushing 2-3s, pause 3-4s, and working time 2-5min.

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