A method for extracting gas vesicles and uses thereof

Abstract

This invention discloses a method for extracting gaseous vesicles and its application. The method includes: mixing microorganisms containing gaseous vesicles with an alkali to obtain a first mixture, and then dissolving the OH- in the first mixture. ‑ The final concentration is 0.0001–1.00 mol / L. The first mixture is mixed with acid to obtain a second mixture, and the pH of the second mixture is adjusted to 6.0–8.0. The second mixture is centrifuged once, and the supernatant is collected. The supernatant is mixed with isotonic buffer, centrifuged a second time, and the supernatant is collected to obtain the gas vesicles. This invention is the first to design a method for extracting gas vesicles (GVs) based on alkaline lysis. It can significantly shorten the extraction cycle while ensuring the ultrasound imaging capability of GVs. The process is simple to operate, requires low equipment, and is easy to scale up for production. The extraction rate of GVs is comparable to that of traditional methods, and it has good application potential and practical application value.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biotechnology, and relates to a method for extracting gas vesicles and application thereof. BACKGROUND

[0002] As a common means of clinical diagnosis and biological research, ultrasound imaging has the advantages of high spatiotemporal resolution, deep penetration and low cost. At present, nanoscale ultrasound contrast agents mainly include acoustic liposomes, fluorocarbon nanodroplets and nanobubbles, etc. The acoustic liposomes are mainly obtained by freeze-drying treatment of liposomes, and the gas component is obtained by atmospheric pressure penetration. Due to the different internal gas contents and easy leakage of acoustic liposomes and nanobubbles, the ultrasound imaging performance is poor. The fluorocarbon nanodroplets have good ultrasound imaging performance, but they need special external stimulation to improve the temperature of the nanodroplets. At present, nanoscale ultrasound contrast agents are mainly obtained by chemical synthesis, which has many problems such as uneven particle size, poor stability, large toxic and side effects, and environmental unfriendliness.

[0003] In 1895, Klebahn, a German microbiologist, discovered a gas vesicle structure in blue-green algae, and similar structures were also found in extreme halophilic archaea. With the gradual deepening of research, this structure was determined to be a nanoscale gas vesicle (GVs). GVs are a kind of hollow organelles composed of proteins, which are two sharp ends, a circular cylinder or a spindle in the middle. The width is between 45-250nm, and the length can reach 100-300nm, and the vesicle wall thickness is about 2nm. These gas vesicles can be used for ultrasound imaging, and have good ultrasound imaging effect in vitro and in vivo, and show great potential in ultrasound imaging.

[0004] At present, the method for extracting GVs from microorganisms such as halophilic archaea mainly includes: bacterial amplification → pear-shaped separatory funnel standing → low-osmotic buffer lysis of the last floating bacteria → centrifugation and concentration until all the bacteria are lysed. It takes about 30 days to extract 1L of bacterial solution, which is complicated, high in cost and time-consuming. To some extent, it limits the wide application of GVs.

[0005] In summary, it is of great significance to develop a new method for extracting gas vesicles to realize efficient, rapid and low-cost extraction of GVs with ultrasound imaging capability for the application field of GVs. SUMMARY

[0006] In view of the deficiencies of the prior art and actual needs, the present application provides a method for extracting gas vesicles and application thereof, so as to solve the problems of complicated operation, high cost and long time consumption in the current method for extracting gas vesicles.

[0007] To achieve the above purpose, the technical scheme adopted by the present application is as follows:

[0008] In a first aspect, the present application provides a method for extracting gas vesicles, the method comprising:

[0009] mixing the microorganism containing gas vesicles with an alkali to obtain a first mixed solution, so that the final concentration of OH- in the first mixed solution is 0.0001-1.00 mol / L, mixing the first mixed solution with an acid to obtain a second mixed solution, so that the pH of the second mixed solution is 6-8; centrifuging the second mixed solution once to collect the upper floating matter, mixing the upper floating matter with an isotonic buffer solution, centrifuging the mixture again to collect the upper floating matter, and obtaining the gas vesicles.

[0010] In the present application, the method for extracting gas vesicles (GVs) based on alkali lysis is creatively designed for the first time, the concentration of OH- is accurately controlled, the extraction period of GVs can be greatly shortened under the condition of ensuring the ultrasonic imaging ability of GVs, the process operation is simple, the equipment requirement is low, the production scale is easy to enlarge, the extraction rate of GVs is equivalent to that of the traditional method, and the application potential and practical application value are good.

[0011] In the present application, the final concentration of OH- in the first mixed solution is 0.0001-1.00 mol / L, including but not limited to 0.0002 mol / L, 0.0004 mol / L, 0.002 mol / L, 0.004 mol / L, 0.02 mol / L, 0.04 mol / L, 0.2 mol / L, 0.4 mol / L or 0.9 mol / L.

[0012] Preferably, the microorganism includes halophilic archaea and / or blue-green algae and other microorganisms containing gas vesicles.

[0013] Preferably, the halophilic archaea includes Halobacteria NRC-1 and other archaea living in high-salinity environments.

[0014] It can be understood that the alkali commonly used in the art is suitable for the present application, which can be selected according to the actual situation and will not significantly affect the effect of the present application.

[0015] Preferably, the alkali includes any one or a combination of at least two of sodium hydroxide, potassium hydroxide, calcium hydroxide, barium hydroxide, cesium hydroxide or lithium hydroxide.

[0016] It can be understood that the alkali commonly used in the art is suitable for the present application, which can be selected according to the actual situation and will not significantly affect the effect of the present application.

[0017] Preferably, the acid includes any one or a combination of at least two of hydrochloric acid, nitric acid, sulfuric acid, phosphoric acid, perchloric acid, hydroiodic acid or hydrobromic acid.

[0018] Preferably, the base can be in the form of a base solution.

[0019] Preferably, the acid can be in the form of an acid solution.

[0020] It can be understood that in the present application, the overall system is mainly controlled by the base and the acid, and the final concentration and pH of OH - and H - in the base solution and the acid solution used can be freely selected and will not significantly affect the effect of the present application. +

[0021] Preferably, the concentration of OH - in the base solution is 1.00 mol / L to 5.00 mol / L, including but not limited to 2 mol / L, 3 mol / L, or 4 mol / L.

[0022] Preferably, the concentration of H + in the acid solution is 1.00 mol / L to 5.00 mol / L, including but not limited to 2 mol / L, 3 mol / L, or 4 mol / L.

[0023] Preferably, the conditions of the first centrifugation and the second centrifugation are each independently 4 to 8°C (for example, it can be 4°C, 5°C, 6°C, or 7°C), 250 to 400 x g (for example, it can be 280, 300 x g, 320 x g, 350 x g, 360 x g, 380 x g, or 390 x g), and centrifugation for 1.5 to 4 h (for example, it can be 1.6 h, 1.7 h, 1.8 h, 1.9 h, 2 h, 3 h, 3.2 h, 3.5 h, 3.6 h, 3.8 h, or 3.9 h).

[0024] It can be understood that the isotonic buffer commonly used in the present application to provide mild conditions (such as maintaining pH, etc.) is suitable for the present application.

[0025] Preferably, the isotonic buffer includes any one or a combination of at least two of PBS buffer, DPBS buffer, HBSS buffer, or EBSS buffer.

[0026] As a preferred technical solution, the method for extracting gas vesicles includes the following steps:

[0027] (1) mixing microorganisms containing gas vesicles with a base solution to obtain a first mixed solution, so that the final concentration of OH in the first mixed solution is 0.0001 to 1.00 mol / L, and mixing the first mixed solution with an acid solution to obtain a second mixed solution, so that the pH of the second mixed solution is 6 to 8;

[0028] (2) centrifuging the second mixed solution at 4 to 8°C and 250 to 400 x g for 1.5 to 4 h, and collecting the upper floating matter; ​

[0029] (3) mixing the upper floating matter with isotonic buffer, centrifuging at 4-8℃, 250-400xg for 1.5-4h, and collecting the upper floating matter;

[0030] (4) repeating step (3) for 2-5 times to obtain the gas vesicles.

[0031] In a second aspect, the present application provides a gas vesicle, which is prepared by the method for extracting gas vesicles according to the first aspect.

[0032] In a third aspect, the present application provides application of the method for extracting gas vesicles according to the first aspect or the gas vesicle according to the second aspect in preparation of an ultrasound contrast agent.

[0033] In the present application, the GVs with ultrasound imaging capability can be quickly and efficiently extracted, and can be effectively applied in preparation of an ultrasound contrast agent.

[0034] Compared with the prior art, the present application has the following beneficial effects:

[0035] The present application first designs an alkali lysis-based method for extracting gas vesicles (GVs), which can greatly shorten the extraction period of GVs under the condition of guaranteeing the ultrasound imaging capability of GVs, is simple in process operation, low in equipment requirement, easy to scale up, has a GVs extraction rate comparable to that of a traditional method, and has good application potential and practical application value. BRIEF DESCRIPTION OF DRAWINGS

[0036] Figure 1 GVs sample prepared by the present application;

[0037] Figure 2 Picture of layering after centrifugation of Halobacteria NRC-1 bacterial solution after addition of low-osmotic buffer and NaOH solution, respectively;

[0038] Figure 3 Particle size comparison chart of GVs prepared by the present application and GVs obtained by a traditional method;

[0039] Figure 4 Zeta potential comparison chart of GVs prepared by the present application and GVs obtained by a traditional method;

[0040] Figure 5 Representative picture of in-vitro imaging effect of GVs prepared by the present application and GVs obtained by a traditional method;

[0041] Figure 6 Picture density quantification result chart of Figure 5

[0042] Figure 7 ​The liver imaging effect of GVs prepared by the present application and GVs obtained by traditional methods is represented by the following pictures;

[0043] Figure 8 For the purpose of Figure 7 The quantitative result picture of the density of the pictures. DETAILED DESCRIPTION

[0044] In order to further illustrate the technical means adopted by the present application and its effects, the present application is further described below in conjunction with the embodiments and the drawings. It can be understood that the specific embodiments described herein are only used to explain the present application, and not to limit the present application.

[0045] The specific techniques or conditions not specified in the embodiments are carried out according to the techniques or conditions described in the literature in the art, or according to the product instructions. The reagents or instruments not specified by the manufacturer are all conventional products that can be purchased through regular channels.

[0046] In the present application, the method of extracting gas vesicles based on alkaline lysis is first designed, and the characteristics of gas vesicle-containing microorganisms such as halophilic archaea are analyzed in depth. The alkaline lysis process is designed skillfully, and the extraction period is greatly shortened by precise control. The process is simple to operate, and the equipment requirement is low. In the specific embodiments of the present application, Halobacteria NRC-1 is used as an example to verify the method of extracting gas vesicles.

[0047] Example 1

[0048] Halobacteria NRC-1 was purchased from the American Type Culture Collection. 250.0 g of NaCl, 20.0 g of MgSO4·7H2O, 3.0 g of Trisodium citrate, 2.0 g of KCl, 5.0 g of Tryptone, and 3.0 g of Yeast extract were added to 1.0 L of pure water, and autoclaved at 121℃ for 15 min to obtain a basic medium; 1.32 g of ZnSO4·7H2O, 0.34 g of MnSO4·H2O, 0.82 g of Fe(NH4)2(SO4)2·6H2O, and 0.14 g of CuSO4·5H2O were added to 200.0 mL of pure water to obtain a trace element solution. 1.0 L of the basic medium was added with 0.1 mL of the trace element solution to prepare a complete medium for Halobacteria NRC-1. A 2.0 L conical flask was used, 1.0 L of the complete medium and 10.0 mL of the bacterial solution were added, and cultured in a constant temperature shaker for 13 days (conditions: 37℃, 220 rpm).

[0049] Take 40.0 mL Halobacteria NRC-1 bacteria solution in centrifuge tube, add NaOH solution with concentration of 2 mol / L, so that OH"concentration in the mixture is 0.5 mol / L; then add HCl solution, adjust pH of the mixture to 7.2, mix quickly; place the centrifuge tube in 4°C centrifuge for 3 h (300 x g), divide into three layers, discard the middle layer solution and the lower layer precipitate, collect the upper layer floating material; add 15 mL PBS buffer to resuspend the upper layer floating material, place the centrifuge tube in 4°C centrifuge for 3 h (250 x g), discard the middle layer solution and the lower layer precipitate, repeat this step for 3 times to obtain white floating GVs Figure 1 ); add 1 mL PBS buffer to resuspend the GVs, store in 4°C for use.

[0050] Example 2

[0051] Cultivate Halobacteria NRC-1 according to the method of Example 1.

[0052] Take 500 mL Halobacteria NRC-1 bacteria solution in centrifuge tube, use magnetic stirrer to stir (600 rpm) while adding KOH solution with concentration of 4 mol / L, so that OH - concentration in the mixture is 0.5 mol / L; then add H2SO4 solution, adjust pH of the mixture to 6.5; place the centrifuge tube in 4°C centrifuge for 3 h (300 x g), discard the middle layer solution and the lower layer precipitate; add 100 mL PBS buffer to resuspend the upper layer floating material, place the centrifuge tube in 4°C centrifuge for 3 h (250 x g), discard the middle layer solution and the lower layer precipitate, repeat this step for 4 times to obtain white floating GVs; add 10 mL PBS buffer to resuspend the GVs, store in 4°C for use.

[0053] Example 3

[0054] Cultivate Halobacteria NRC-1 according to the method of Example 1.

[0055] Take 40.0 mL Halobacteria NRC-1 bacteria solution in centrifuge tube, add Ca(OH)2 solution with concentration of 1 mol / L, so that OH -The concentration of NaOH solution was 5 mol / L, and the concentration of OH"was 1.00 mol / L. Then, HNO3 solution was added to adjust the pH of the mixture to 8, and the mixture was mixed rapidly. The centrifuge tube was centrifuged at 300 x g for 3 h at 8 °C, and the intermediate layer solution and the lower layer precipitate were discarded. Then, 15 mL of PBS buffer was added to resuspend the upper layer floating matter, and the centrifuge tube was centrifuged at 250 x g for 3 h at 8 °C. The intermediate layer solution and the lower layer precipitate were discarded, and this step was repeated 5 times to obtain white floating GVs. Finally, 1 mL of PBS buffer was added to resuspend the GVs, and the GVs were stored at 4 °C for use.

[0056] Example 4

[0057] Halobacteria NRC-1 was cultured according to the method of Example 1. The bacteria were transferred to a sterilized separatory funnel, and the opening of the separatory funnel was sealed with a breathable film. The separatory funnel was placed in a dark place at 25 °C for 10 days until floating bacteria layers appeared in the upper layer of the liquid in the separatory funnel. The lower layer of the bacterial culture solution was discarded, and 20.0 mL of the floating bacteria layer was collected in a centrifuge tube. Then, 20.0 mL of low-osmotic buffer TMC (Tris 10.0 mmol / L, MgCl2 2.5 mmol / L, CaCl2 2.0 mmol / L, pH = 7.4) was added to the separatory funnel to wash the floating bacteria adhering to the wall of the separatory funnel, and the floating bacteria were collected in the centrifuge tube. The centrifuge tube was centrifuged at 300 x g for 3 h at 4 °C, and the upper layer floating matter was collected. Then, 15 mL of PBS buffer was added to resuspend the upper layer floating matter, and the centrifuge tube was centrifuged at 250 x g for 3 h at 4 °C. The intermediate layer solution and the lower layer precipitate were discarded, and this step was repeated 5 times to obtain white floating GVs. Finally, 1 mL of PBS buffer was added to resuspend the GVs, and the GVs were stored at 4 °C for use.

[0058] Halobacteria NRC-1 was cultured according to the method of Example 1. The bacteria were transferred to a sterilized separatory funnel, and the opening of the separatory funnel was sealed with a breathable film. The separatory funnel was placed in a dark place at 25 °C for 10 days until floating bacteria layers appeared in the upper layer of the liquid in the separatory funnel. The lower layer of the bacterial culture solution was discarded, and 20.0 mL of the floating bacteria layer was collected in a centrifuge tube. Then, 20.0 mL of low-osmotic buffer TMC (Tris 10.0 mmol / L, MgCl2 2.5 mmol / L, CaCl2 2.0 mmol / L, pH = 7.4) was added to the separatory funnel to wash the floating bacteria adhering to the wall of the separatory funnel, and the floating bacteria were collected in the centrifuge tube. The centrifuge tube was centrifuged at 300 x g for 3 h at 4 °C, and the upper layer floating matter was collected. Then, 15 mL of PBS buffer was added to resuspend the upper layer floating matter, and the centrifuge tube was centrifuged at 250 x g for 3 h at 4 °C. The intermediate layer solution and the lower layer precipitate were discarded, and this step was repeated 5 times to obtain white floating GVs. Finally, 1 mL of PBS buffer was added to resuspend the GVs, and the GVs were stored at 4 °C for use.

[0059] Comparative Example 1

[0060] In this comparative example, the bacterial solution in Example 1 was extracted by a traditional method, which included the following steps:

[0061] Halobacteria NRC-1 was cultured according to the method of Example 1. The bacteria were transferred to a sterilized separatory funnel, and the opening of the separatory funnel was sealed with a breathable film. The separatory funnel was placed in a dark place at 25 °C for 10 days until floating bacteria layers appeared in the upper layer of the liquid in the separatory funnel. The lower layer of the bacterial culture solution was discarded, and 20.0 mL of the floating bacteria layer was collected in a centrifuge tube. Then, 20.0 mL of low-osmotic buffer TMC (Tris 10.0 mmol / L, MgCl2 2.5 mmol / L, CaCl2 2.0 mmol / L, pH = 7.4) was added to the separatory funnel to wash the floating bacteria adhering to the wall of the separatory funnel, and the floating bacteria were collected in the centrifuge tube. The centrifuge tube was centrifuged at 300 x g for 3 h at 4 °C, and the upper layer floating matter was collected. Then, 15 mL of PBS buffer was added to resuspend the upper layer floating matter, and the centrifuge tube was centrifuged at 250 x g for 3 h at 4 °C. The intermediate layer solution and the lower layer precipitate were discarded, and this step was repeated 5 times to obtain white floating GVs. Finally, 1 mL of PBS buffer was added to resuspend the GVs, and the GVs were stored at 4 °C for use.

[0062] Comparative Example 2

[0063] This comparative example is compared with Example 1, the only difference is that the OH - concentration is 0.00001 mol / L, and the rest is the same as Example 1.

[0064] Comparative Example 3

[0065] This comparative example is compared with Example 1, the only difference is that the OH - concentration is 1.5 mol / L, and the rest is the same as Example 1.

[0066] Test Example 1

[0067] The GVs extracted from Examples 1-5 and Comparative Examples 1-3 are subjected to particle size and zeta potential detection and in-vitro and in-vivo ultrasound imaging tests.

[0068] The particle size and zeta potential detection method comprises:

[0069] At 25°C, the GVs are placed in a quartz dish, and a Malvern particle size potential analyzer is used to analyze the particle size and zeta potential of the GVs.

[0070] The in-vitro ultrasound imaging test method comprises:

[0071] Preparation of agarose gel cavity phantom: 1% W / V agarose aqueous solution is heated and dissolved in a microwave oven to form a uniform and clear solution; a 1.5 mL centrifuge tube is inserted into a cavity template at a suitable distance to form a mold; the mold is inserted into the agarose gel solution until it solidifies, and then the mold is removed to form a sample addition hole. 120 μL of GVs with OD 500 = 1.5 are added, and a mindray R7 ultrasound imaging instrument is used to perform in-vitro ultrasound imaging with the mode set to contrast mode.

[0072] The in-vivo ultrasound imaging test method comprises:

[0073] BALB / c male mice (8 weeks old, 20-22 g) are anesthetized with isoflurane, and 100 μL of GVs with OD 500 = 1.5 are injected into the mice through the tail vein, and a mindray R7 ultrasound imaging instrument is used to perform ultrasound imaging of the liver with the mode set to contrast mode.

[0074] As can be seen from the comparison of the extraction results of Comparative Example 1 and the results of Comparative Example 1, compared with the traditional method, the operation of the present application is simple, the cycle is short, and the equipment requirement is low, such as Figure 2 As can be seen from the comparison of the extraction results of Comparative Example 1 and the results of Comparative Example 1, compared with the traditional method, the operation of the present application is simple, the cycle is short, and the equipment requirement is low, such as Figures 3-8As shown, Figure 3 Figure 4 is a particle size comparison chart of GVs, indicating that there is no obvious difference in particle size between GVs extracted by the traditional method and the method of the present application; Figure 4 Figure 5 is a zeta potential comparison chart of GVs, indicating that there is no obvious difference in zeta potential between GVs extracted by the traditional method and the method of the present application; Figures 5-6 Figure 6 is an effect chart and quantitative chart of ultrasonic in vitro imaging of GVs, indicating that GVs extracted by the method of the present application can achieve the in vitro imaging performance of GVs obtained by the traditional method; Figures 7-8 Figure 7 is an effect chart and quantitative chart of ultrasonic imaging of mouse liver, indicating that GVs extracted by the method of the present application can achieve the in vivo imaging performance of GVs obtained by the traditional method.

[0075] Comparing Example 1 with Comparative Examples 2 and 3, it can be seen that OH - When the concentration is too low, the lysis of bacterial wall is insufficient, and the content of obtained GVs is small; OH - When the concentration is too high, the structure of GVs will be damaged, and the content of obtained GVs is small.

[0076] In summary, the method for extracting gas vesicles based on alkaline lysis is designed for the first time, which can greatly shorten the extraction period of GVs under the condition of ensuring the ultrasonic imaging ability of GVs, i.e. 10-15h can complete the extraction, the process operation is simple, the equipment requirement is low, and the production scale is easy to enlarge. The extraction rate of GVs is equivalent to that of the traditional method, and the method has good application potential and practical application value.

[0077] The applicant declares that the detailed method of the present application is illustrated by the above examples, but the present application is not limited to the above detailed method, i.e. it does not mean that the present application must rely on the above detailed method to be implemented. It should be understood by those skilled in the art that any improvement of the present application, equivalent replacement of each raw material of the product of the present application, addition of auxiliary ingredients, selection of specific methods, etc. fall within the protection scope and disclosure scope of the present application.

Claims

1. A method for extracting gas vesicles, characterized in that, The method includes: (1) Mix halophilic archaea containing gas vesicles with alkali to obtain a first mixture, and make the OH in the first mixture... - The final concentration is 0.0001~1.00 mol / L. The first mixture is mixed with acid to obtain a second mixture, and the pH of the second mixture is adjusted to 6.0~8.0; the halophilic archaea is... Halobacteria NRC-1 The base is any one or a combination of at least two of sodium hydroxide, potassium hydroxide, or calcium hydroxide; the acid is any one or a combination of at least two of hydrochloric acid, sulfuric acid, perchloric acid, or nitric acid. (2) Centrifuge the second mixture at 4~8℃ and 250~400×g for 1.5~4 h, and collect the upper floating matter; (3) Mix the upper floating matter with isotonic buffer, centrifuge at 4~8℃ and 250~400×g for 1.5~4 h, and collect the upper floating matter; (4) Repeat step (3) 2 to 5 times to obtain the gas vesicles.

2. The method for extracting gas vesicles according to claim 1, characterized in that, The alkali is in the form of an alkaline solution.

3. The method for extracting gas vesicles according to claim 1, characterized in that, The acid is in the form of an acid solution.

4. The method for extracting gas vesicles according to claim 1, characterized in that, The isotonic buffer is any one or a combination of at least two of PBS buffer, DPBS buffer, HBSS buffer, or EBSS buffer.

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