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CN-122012394-A - Extracellular vesicle preparation method carrying mycobacterium tuberculosis specific antigen

CN122012394ACN 122012394 ACN122012394 ACN 122012394ACN-122012394-A

Abstract

The invention provides a preparation method of extracellular vesicles carrying mycobacterium tuberculosis specific antigens, which belongs to the technical field of extracellular vesicles, and comprises the steps of inducing THP-1 monocytes to differentiate into macrophages by phorbol esters, treating mycobacterium tuberculosis H37Rv standard strains by ultrasonic crushing and needle blowing to prepare monodisperse bacterial liquid, carrying out infection according to an infection complex value of 5, monitoring real-time infection rate every 12 hours in the culture process, dynamically adjusting the concentration of initial bacterial liquid according to the value to maintain the infection rate within a range of 45-55%, purifying extracellular vesicles by a differential centrifugation method, detecting the particle size, concentration and lipoarabinomannan content of vesicles, dividing qualified vesicle products into three concentration grades according to the antigen content, and adding dimethyl sulfoxide for freezing and storing, thereby solving the technical problem of poor antigen load stability among batches of extracellular vesicles carrying mycobacterium tuberculosis specific antigens.

Inventors

  • WANG RUIXUAN
  • WANG SHUOSHUO
  • PENG YONGYI
  • XU YANYAN
  • SUN MI

Assignees

  • 青岛瑞斯凯尔生物科技股份有限公司

Dates

Publication Date
20260512
Application Date
20260122

Claims (10)

  1. 1. A preparation method of extracellular vesicles carrying mycobacterium tuberculosis specific antigen is characterized by comprising the steps of culturing THP-1 mononuclear cells in RPMI-1640 culture medium containing fetal calf serum until the cell density reaches a preset density, adding phorbol ester to induce differentiation, discarding the culture medium, washing with phosphate buffer solution, and replacing with fresh culture medium containing fetal calf serum to continue culturing; collecting cultured adherent cells, detecting the expression level of a cell surface marker CD11b and the expression level of a cell surface marker CD14 by adopting a flow cytometry, judging differentiated qualified macrophages, placing a mycobacterium tuberculosis H37Rv standard strain suspension in an ultrasonic breaker, performing ultrasonic treatment, repeatedly blowing a bacterial liquid by using a needle, measuring and adjusting the bacterial liquid concentration to an initial bacterial liquid concentration by using an optical density value, adding the treated bacterial liquid into the differentiated qualified macrophages for infection and incubation, washing the cells by using a culture medium containing gentamicin to remove extracellular bacteria, replacing the cells with a maintenance culture medium containing gentamicin for continuous culture, monitoring the positive rate of fluorescent labeled mycobacterium tuberculosis in the infected cells by adopting the flow cytometry at preset time intervals, recording the positive rate as the real-time infection rate, dynamically adjusting the initial bacterial liquid concentration according to the real-time infection rate to ensure that the real-time infection rate among different batches is maintained in a target interval, collecting the cultured cell supernatant, separating extracellular vesicles by adopting a differential centrifugation method, detecting the particle size distribution and vesicle concentration of extracellular vesicles by adopting a nanoparticle tracking analysis technology, judging the vesicle concentration, quantitatively detecting the content of qualified vesicle by adopting an enzyme-linked immunosorbent assay (ELISA) and the content of the qualified carrier of the arabinan M, dividing qualified vesicle products into three concentration levels of high-concentration positive quality control products, medium-concentration positive quality control products and low-concentration positive quality control products according to the content of lipoarabinomannan, respectively subpackaging positive quality control products with different concentration levels into polypropylene freezing storage tubes, adding dimethyl sulfoxide as a freezing storage protective agent, and freezing and storing.
  2. 2. The method for preparing extracellular vesicles carrying a specific antigen of Mycobacterium tuberculosis according to claim 1, wherein THP-1 monocytes are cultured in RPMI-1640 medium containing fetal bovine serum to a predetermined cell density, and phorbol ester is added to induce differentiation, specifically, THP-1 monocytes are cultured in RPMI-1640 medium containing 10% fetal bovine serum to a cell density Phorbol ester was added at a concentration of 100ng/mL and differentiation was induced for 48 hours.
  3. 3. The method for preparing extracellular vesicles carrying a mycobacterium tuberculosis specific antigen according to claim 2, wherein the phorbol ester is phorbol ester-12-myristate-13-acetate, and the differentiation of THP-1 monocytes to adherent macrophages is induced by activating a protein kinase C signaling pathway.
  4. 4. The method for preparing extracellular vesicles carrying a specific antigen of mycobacterium tuberculosis according to claim 3, wherein the phosphate buffer solution is an aqueous solution prepared by mixing disodium hydrogen phosphate and potassium dihydrogen phosphate according to a mass ratio of 8:1, the pH value is 7.4, and the osmotic pressure is 300mOsm/kg.
  5. 5. The method for preparing extracellular vesicles carrying a specific antigen of mycobacterium tuberculosis according to claim 4, wherein the step of detecting the expression level of the cell surface marker CD11b and the expression level of the cell surface marker CD14 by flow cytometry and determining differentiated macrophages is performed, specifically, the differentiated macrophages are determined when the positive expression rate of the cell surface marker CD11b is >85% and the positive expression rate of the cell surface marker CD14 is >80%, and the secondary induced differentiation treatment is performed if the positive expression rate of the cell surface marker CD11b is not more than 85% or the positive expression rate of the cell surface marker CD14 is not more than 80%.
  6. 6. The method for preparing extracellular vesicles carrying a specific antigen of mycobacterium tuberculosis according to claim 5, wherein the secondary induction differentiation treatment means that after 48 hours of the first induction differentiation, if the positive expression rate corresponding to the expression level of the cell surface marker CD11b or the positive expression rate corresponding to the expression level of the cell surface marker CD14 is not standard, the fresh medium is replaced and the phorbol ester with a concentration of 50ng/mL is added again for further induction for 24 hours.
  7. 7. The method for preparing extracellular vesicles carrying a specific antigen of Mycobacterium tuberculosis according to claim 6, wherein the step of subjecting the suspension of Mycobacterium tuberculosis H37Rv standard strain to ultrasonic treatment in an ultrasonic breaker and repeatedly blowing the bacterial liquid with a needle, specifically, subjecting the suspension of Mycobacterium tuberculosis H37Rv standard strain to ultrasonic treatment in an ultrasonic breaker with a power of 150W for 5 seconds, intermittent 10 seconds and repeated 3 times, and then repeatedly blowing the bacterial liquid with a 27-gauge needle for 12 times.
  8. 8. The method for preparing extracellular vesicles carrying a specific antigen of Mycobacterium tuberculosis according to claim 7, wherein the step of adjusting the concentration of the bacterial liquid to the initial bacterial liquid concentration is carried out by measuring the optical density value, in particular, the step of adjusting the concentration of the bacterial liquid to the initial bacterial liquid concentration is carried out by measuring the optical density value CFU/mL。
  9. 9. The method for preparing extracellular vesicles carrying a specific antigen of Mycobacterium tuberculosis according to claim 8, wherein the step of adding the treated bacterial liquid to the differentiated qualified macrophages for infection and incubation is carried out by adding the bacterial liquid corresponding to the initial bacterial liquid concentration after treatment to the differentiated qualified macrophages for infection, wherein the infection complex value is set to 5, and the bacterial liquid is infected at 37 ℃ and 5% After 4 hours of incubation in the incubator, the cells were washed 3 times with medium containing 100. Mu.g/mL gentamicin to remove extracellular bacteria, and the culture was continued for 48 hours with replacement of the maintenance medium containing 10. Mu.g/mL gentamicin.
  10. 10. The method for preparing extracellular vesicles carrying a specific antigen of mycobacterium tuberculosis according to claim 9, wherein the step of dynamically adjusting the initial bacterial liquid concentration to maintain the real-time infection rate between different batches within a target interval according to the real-time infection rate, specifically monitoring the positive rate of fluorescent-labeled mycobacterium tuberculosis in the infected cells by flow cytometry every 12 hours and recording as the real-time infection rate, and increasing the initial bacterial liquid concentration to a value of <40% when the real-time infection rate is less than the real-time infection rate CFU/mL and re-infection of the next batch of cells, the initial bacterial liquid concentration is reduced to the value when the real-time infection rate is more than 60 percent CFU/mL, so that the real-time infection rate between different batches is E [45%,55% ].

Description

Extracellular vesicle preparation method carrying mycobacterium tuberculosis specific antigen Technical Field The invention belongs to the technical field of extracellular vesicles, and particularly relates to a preparation method of extracellular vesicles carrying mycobacterium tuberculosis specific antigens. Background In tuberculosis diagnosis, specific antigen of mycobacterium tuberculosis needs to be detected as a marker, and a traditional method is used for collecting extracellular vesicles secreted by macrophages after the macrophages are infected to obtain vesicle products carrying antigens such as lipoarabinomannan and the like, and the vesicle products are used for development of quality control products of immunodiagnosis reagents or research of vaccine carriers. However, the existing preparation method lacks dynamic monitoring on the infection process, and the infection efficiency of cells in different batches has remarkable fluctuation, so that the quantity of mycobacterium tuberculosis taken up by macrophages is unstable. Because the antigen load in the extracellular vesicles directly depends on the quantity of intracellular thalli, the batch-to-batch difference of the infection efficiency leads the content of the lipoarabinomannan in the finally obtained vesicle products to have great fluctuation, and the accurate division of the quality control product concentration grade is affected. That is, the prior art has a technical problem that the stability of the antigen load between batches of extracellular vesicle preparations carrying a specific antigen of mycobacterium tuberculosis is poor. Disclosure of Invention In view of the above, the present invention provides a method for preparing extracellular vesicles carrying mycobacterium tuberculosis specific antigens, which can solve the technical problem in the prior art that the extracellular vesicles carrying mycobacterium tuberculosis specific antigens have poor stability of antigen load between batches. Culturing THP-1 mononuclear cells in RPMI-1640 culture medium containing fetal bovine serum until the cell density reaches a preset density, adding phorbol ester to induce differentiation, discarding the culture medium, washing with phosphate buffer solution, and replacing with fresh culture medium containing fetal bovine serum to continue culturing; collecting cultured adherent cells, detecting the expression level of a cell surface marker CD11b and the expression level of a cell surface marker CD14 by adopting a flow cytometry, judging differentiated qualified macrophages, placing a mycobacterium tuberculosis H37Rv standard strain suspension in an ultrasonic breaker, performing ultrasonic treatment, repeatedly blowing a bacterial liquid by using a needle, measuring and adjusting the bacterial liquid concentration to an initial bacterial liquid concentration by using an optical density value, adding the treated bacterial liquid into the differentiated qualified macrophages for infection and incubation, washing the cells by using a culture medium containing gentamicin to remove extracellular bacteria, replacing the cells with a maintenance culture medium containing gentamicin for continuous culture, monitoring the positive rate of fluorescent labeled mycobacterium tuberculosis in the infected cells by adopting the flow cytometry at preset time intervals, recording the positive rate as the real-time infection rate, dynamically adjusting the initial bacterial liquid concentration according to the real-time infection rate to ensure that the real-time infection rate among different batches is maintained in a target interval, collecting the cultured cell supernatant, separating extracellular vesicles by adopting a differential centrifugation method, detecting the particle size distribution and vesicle concentration of extracellular vesicles by adopting a nanoparticle tracking analysis technology, judging the vesicle concentration, quantitatively detecting the content of qualified vesicle by adopting an enzyme-linked immunosorbent assay (ELISA) and the content of the qualified carrier of the arabinan M, dividing qualified vesicle products into three concentration levels of high-concentration positive quality control products, medium-concentration positive quality control products and low-concentration positive quality control products according to the content of lipoarabinomannan, respectively subpackaging positive quality control products with different concentration levels into polypropylene freezing storage tubes, adding dimethyl sulfoxide as a freezing storage protective agent, and freezing and storing. Wherein THP-1 mononuclear cells are cultured in RPMI-1640 medium containing fetal bovine serum until the cell density reaches a preset density, and phorbol ester is added for inducing differentiation, specifically, THP-1 mononuclear cells are cultured in RPMI-1640 medium containing 10% fetal bovine serum until the cell density reachesPhorbol este