KR100463415B1 - The preparation of LPS-free bacterial DNA - Google Patents

Abstract

The present invention relates to a method for removing lipopolysaccharides (LPS), which are difficult to remove and incorporated when separating and purifying DNA from bacteria. More specifically, the present invention relates to lipopolysaccharides and DNA according to changes in alcohol concentration. It relates to a method for removing lipopolysaccharide from bacterial DNA or RNA using differences in precipitation rates.

According to the present invention, lipopolysaccharide can be easily and efficiently recovered from bacterial DNA or RNA, overcoming the difficulty of separation from bacterial DNA or RNA due to the commonality of DNA or RNA with lipopolysaccharide as a polysaccharide. Can be removed.

Description

Method for removing lipopolysaccharide from DNA or RNA in bacteria {The preparation of LPS-free bacterial DNA}

The present invention relates to a method for removing lipopolysaccharide (LPS), which is difficult to remove and incorporated when separating and purifying DNA from bacteria, and more specifically, to lipopolysaccharide and DNA according to a change in alcohol concentration. It relates to a method for removing lipopolysaccharide from bacterial DNA or RNA using differences in precipitation rates.

Lipopolysaccharides consist of Lipid A, core polysaccharides, and O-specific polysaccharide side chains.

LPS is heat stable and exhibits strong antigenicity. Its molecular weight consists of several complexes with basic units of 10 kDa, varying in size up to 10,000 kDa, and chemically both affinity complexes.

Lipopolysaccharides cause inflammatory reactions in animals, most of which are present in the outermembrane of gram-negative bacteria such as Salmonella and E. coli . The lipopolysaccharide, the toxic part of the inflammatory response, is a lipid moiety called endotoxin. When endotoxin enters the human body, fever, changes in white blood cell count, vascular coagulation, tumor necrosis, hypotension, and shock occur, and endotoxin produces various kinds of cytokine such as IL-1, IL-6, IL-8, TNF, and immune system. Complement activation, blood coagulation activation and the like, polyclonal differentiation as mitogen in B cells, B cell proliferation, Ig M, Ig G antibody production is increased.

Lipopolysaccharides containing such endotoxins are incorporated when DNA or RNA is separated from bacteria. It will be essential to remove lipopolysaccharide from the DNA or RNA incorporating lipopolysaccharide at the present time when DNA or RNA has been clinically widely attempted as a treatment for disease.

The method of separating proteins from endotoxins has been reported in the academic field, such as ultracentrifugation method and chromatographic separation method. However, no method of effectively removing lipopolysaccharide from DNA or RNA has been reported. This is because the structure of DNA and RNA can be included in the category of polysaccharides (polymers of deoxyribose or ribose) in a broad range, and lipopolysaccharides also have similar properties as polysaccharides.

However, there is a method for removing endotoxin at the laboratory level, but a method using a detoxi gel column is only available (Tibtech. 1999. 4, 17, 169-174). In addition, the nucleic acid-containing preparation is passed through one or several times while contacting the semipermeable membrane, so that the nucleic acid molecules are retained by the membrane and a lower molecular weight material can pass through the membrane and / or be adsorbed onto the membrane, thereby essentially removing the endotoxin-free nucleic acid solution. A method for separating exotoxin from a nucleic acid agent characterized by obtaining has been reported (Patent Publication 2000-69943).

However, this method has many problems in purifying a large amount of DNA, and can be a factor of price increase in the production of the product from an economic point of view.

Accordingly, the present inventors have recognized that when applying bacterial DNA to the treatment of diseases, lipopolysaccharide mixed during the manufacture of DNA may affect the immune system of the mammal, and the main component of this endotoxin is lipopolysaka. Considering that it is a ride to try to remove it.

As a result, the conventionally known fact that the polysaccharide, DNA or RNA, is precipitated in alcohol (Molecular cloning, A laboratory manual, 2nd edition, E10 ~ E11) and lipopolysaccharide is also precipitated in alcohol. Recognizing the newly discovered fact by the present inventors, we found that the precipitation rate of these lipopolysaccharides and DNA or RNA depends on the concentration of alcohol, and thus a method for removing lipopolysaccharides from bacterial DNA. It was completed.

Therefore, the main object of the present invention is to provide a method for removing lipopolysaccharide from DNA or RNA using the difference in precipitation rate according to the change in alcohol concentration.

It is another object of the present invention to provide a method of using DNA or RNA from which lipopolysaccharide has been removed by the above method as a raw material of a pharmaceutical product.

Figure 1 is a schematic of the process of removing lipopolysaccharides according to the ethanol content from the DNA of the bacteria in A and B of Example 1.

Figure 2 shows the measurement of the content of lipopolysaccharide and DNA of the precipitate produced in each step of the process of removing lipopolysaccharide according to the content of ethanol from the DNA of bacteria in A and B of Example 1 One graph.

Figure 3 shows the purity of the DNA, its content and the presence of lipopolysaccharide measured at each step of the lipopolysaccharide removal process including the repeated process of Example 2.

In order to achieve the object of the present invention, the present invention provides a method for removing lipopolysaccharide from bacterial DNA or RNA using the difference in the precipitation rate of lipopolysaccharide and DNA or RNA according to the change in alcohol concentration.

The alcohol in the process is preferably methanol, ethanol, or isopropanol and most preferably ethanol.

In order to achieve another object of the present invention, the present invention provides a method of using the DNA or RNA from which lipopolysaccharide has been removed by the above method as a raw material of a pharmaceutical product.

The present invention is described in more detail below. Alcohols have the property of precipitating polynucleotides. Alcohols that precipitate polynucleotides also have the property of precipitation of lipopolysaccharides. Although alcohols have the property of precipitating both polynucleotides and lipopolysaccharides, each substance has its own precipitation constant.

The present invention provides a method for removing lipopolysaccharide from bacterial DNA or RNA according to the principle of differential precipitation using a difference in precipitation constant between nucleotides and lipopolysaccharides.

The alcohol here is preferably methanol, ethanol, isopropanol and most preferably ethanol.

The principle and method of application are very simple and convenient. Lipopolysaccharides, like DNA or RNA, have negative charges, vary in size, and are inseparable in aqueous solution, but are not separable, but show different precipitation rates from DNA depending on the concentration of ethanol.

If alcohol is the most preferred case of ethanol, DNA or RNA samples isolated from bacteria

Adding a ethanol to the sample to 1 to 30% by volume and centrifuging at 5,000 to 15,000 rpm for 1 second to 72 hours at 0 ^o C to 30 ^o C and then taking the supernatant;

Adding a ethanol to the supernatant taken in the first step to 31 to 50% by volume, followed by centrifugation under the same centrifugation conditions as step 1 and taking the supernatant;

A third step of adding ethanol to the supernatant taken in the second step so as to be 51-100% by volume, followed by centrifugation under the same range of centrifugation as step 1 and taking the supernatant;

Lithium from DNA or RNA is characterized in that it comprises a fourth step in which ethanol is added to the supernatant taken in the third step to 100 to 1000% by volume, followed by centrifugation under the same range of centrifugation as in step 1, and taking a precipitate. Separate polysaccharides.

It may be treated without centrifugation in the process of each step.

In this case, the precipitate removed by centrifugation in the first and second steps corresponds to the precipitate of lipopolysaccharide, and the precipitate removed by centrifugation in the third step is a mixture of lipopolysaccharide and DNA or RNA. The precipitate corresponds to the precipitate, and finally, the precipitate obtained by centrifugation in the fourth step is a precipitate in which only DNA or RNA is present.

In the above method, more preferably, the precipitate obtained in the third step may be dissolved and diluted, and the first to fourth steps may be repeatedly performed to increase the yield of DNA or RNA.

In the above method, before performing the first to fourth steps, first, chloroform, ether or phenol is added, and then left for 1 second to 72 hours in the temperature range of -70 ^o C to 30 ^o C and 1 to 5,000 to 15,000 rpm. It is preferable to further include a step of removing the chloroform layer, ether layer or phenol layer by centrifugation at 0 ^° C ~ 30 ^° C for 2 seconds to 72 hours. Lipopolysaccharide is dissolved in the chloroform layer, the ether layer, or the phenol layer that is not present, and thus, the lipopolysaccharide is dissolved to further remove the lipopolysaccharide. In the treatment step, the centrifugation step may be omitted.

In order to achieve another object of the present invention, the present invention provides a method of using DNA or RNA from which lipopolysaccharide has been removed as a raw material of a pharmaceutical product. Various diseases such as liver cancer, brain cancer, colon cancer, pancreatic cancer, lung cancer, liver cancer, gastric cancer, small intestine cancer, esophageal cancer, prostate cancer, breast cancer, blood cancer, lymph cancer, skin cancer, various tumors, angiogenesis suppression disease, spinal cord tumor, Drugs using bacterial DNA or RNA have been manufactured to treat all kinds of cancers, including uterine cancer, leukemia, thymic cancer, nerve tumors, esophageal cancer, anal cancer, ovarian cancer, lung cancer, etc. Or RNA can be used.

EMBODIMENT OF THE INVENTION Hereinafter, this invention is demonstrated more concretely based on an Example. However, these examples are only for the understanding of the present invention, and the scope of the present invention is not limited to the examples in any sense.

(Example)

Example 1

A. Bacterial Shredding and Gene Isolation

In order to isolate the bacterial DNA DNA, after incubating for 10 to 12 hours, the bacteria were recovered by centrifugation at 5,000 rpm and 4 ^o C for 20 minutes. The recovered bacteria (25 grams) were washed with 500 ml of TEN (10 mM Tris, 1 mM EDTA, 0.1 M NaCl) buffer, and recovered by centrifugation at 5,000 rpm and 4 ^° C. for 30 minutes. TE (10 mM Tris, 1 mM EDTA) buffer was added to the recovered bacteria and completely suspended. Then, 1% sodium dodecyl sulfate (SDS) and proteinase K were added. After lysozyme was added thereto and left for 30 minutes, 1% Hexadecyl trimethyl ammonium bromide (CTAB) solution was added thereto and left at 37 ^° C. An equal volume of chloroform was added thereto and left at 4 ^o C for one day, followed by centrifugation at 12,000 rpm and 4 ^o C for 30 minutes to remove the chloroform layer. Subsequently, extraction with chloroform was further performed twice. 200 vol% of ethanol was added thereto to precipitate the DNA containing lipopolysaccharide, and the precipitate was dissolved in TE (10 mM Tris, 1 mM EDTA) buffer to separate the genetic DNA.

B. Isolation of LPS from Bacterial DNA

Example 1 Insert with respect to the DNA samples isolated from ammonyumreul 10M acetic acid in a volume of 30% ethanol and one-third volume, then allowed to stand at 4 ^o C, 12,000 rpm, 4 in ^o C, 30 bungan centrifuged to precipitate and separate The supernatant was recovered first. The first collected supernatant was added to 50% by volume of ethanol to the initial sample and left at 4 ^° C., and then centrifuged at 12,000 rpm and 4 ^° C. for 30 minutes to separate the precipitates. Subsequently, the ethanol concentration was added to 100% by volume with respect to the first sample to the secondary supernatant, and left at 4 ^o C, and then centrifuged at 12,000 rpm and 4 ^o C for 30 minutes to separate the precipitate and the supernatant was recovered third. Subsequently, the ethanol concentration of the first sample was added to 200 vol% based on the initial sample and left at 4 ^° C., followed by centrifugation at 12,000 rpm and 4 ^° C. for 30 minutes to discard the supernatant to obtain a precipitate. The recovered precipitate was dissolved in TE (10 mM Tris, 1 mM EDTA) without lipopolysaccharide.

Each step is shown in FIG. 1 to more briefly and clearly show the process of A and B.

C. Quantification of DNA

The content of DNA contained in the precipitate produced in each treatment step of A and B was measured. Specifically, the precipitate produced in each of the above steps was dissolved in TE (10 mM Tris, 1 mM EDTA) without lipopolysaccharide. A portion of the solution in which the precipitate was dissolved was diluted and quantified by measuring absorbance at 260 nm. The method for calculating the amount of DNA from the absorbance values is as follows.

Amount of DNA (μg / ml) = OD _260nm x 50 (μg / ml) x Dilution factor

D. Quantification of Lipopolysaccharide

The content of lipopolysaccharide contained in the cell crude of A and B and the precipitate produced in each step was measured. Lipopolysaccharide was measured by Limulus Amebocyte Lysate kit (BioWhittaker). Specifically, the precipitate produced in each step was dissolved in TE (10 mM Tris, 1 mM EDTA) without lipopolysaccharide.

To measure the content of lipopolysaccharide, 96-well plates pre-warmed at 37 ^° C. by diluting the test solution and the lipopolysaccharide standard solution, respectively, in which the precipitates produced in the respective treatment steps A and B were dissolved. 50 μl each into plates). Subsequently, 50 μl of a chromogenic solution was added thereto and left at 37 ^° C. for 10 minutes, and then a substrate solution was added to develop color at 37 ^° C. After 6 minutes, the reaction was stopped with a stop solution (25% glacial acetic acid) and the absorbance was measured at 405 nm. The lipopolysaccharide content of the standard solution is 27 EU / ml.

The method for calculating the amount of lipopolysaccharide from the absorbance values is as follows.

Amount of LPS (EU / ml) = (ΔO.D. _405nm -y intercept) / slope

ΔO.D. _{405 nm:} absorbance value of the test solution-absorbance value of water without lipopolysaccharide

N: Number of standard solutions used

x (EU / ml): Lipopolysaccharide concentration in standard solution

y: mean ΔO.D. of standard solution. Value of _405nm

Σx (EU / ml): Sum of the concentrations of lipopolysaccharide in standard solution

Σy: average ΔO.D. of standard solution _Sum of _{405 nm} values

Sx: standard deviation of x =

Sy: standard deviation of y =

y-intercept: Σy / N-(Σx / N x slope)

Tilt: (Sy / Sx) ^r

r: ((NΣxy- (Σx) (Σy)) / (N (N-1) SxSy)

The amount of DNA and the amount of lipopolysaccharide measured by the same method as described above in C and D of Example 1 are shown in Table 1 below.

Measurement of endotoxin (EU / mg DNA) DNA recovery (mg) Cell crude > 10 ¹² N.D.1 Chloroform treatment Chloroform treatment N.D. 2 N.D. 2 * 200% ethanol precipitate 0.54 x 10 ³ 70.6 Ethanol precipitation 30% ethanol precipitate 71 0.47 50% ethanol precipitate 37.5 0.35 100% ethanol precipitate 4.7 4.52 200% ethanol precipitate 0 7.9

N.D. = not determined: Tested but not possible.

N.D.1: Not available due to high impurities (proteins, lipids, sugars, etc.) in cellular crude.

N.D.2: After chloroform treatment the state cannot be determined due to residual chloroform.

*: In order to measure the amount of DNA and lipopolysaccharide contained in the solution after chloroform treatment, 200 vol% ethanol was precipitated, and the precipitate was dissolved in TE.

Example 2

Lipopolysaccharide Removal Method Including Repeated Process

Bacterial Shredding and Gene Isolation

In order to isolate the bacterial DNA DNA, after incubating for 10 to 12 hours, the bacteria were recovered by centrifugation at 5,000 rpm and 4 ^o C for 20 minutes. The recovered bacteria (75 g) were washed with 1000 ml of TEN (10 mM Tris, 1 mM EDTA, 0.1M NaCl) buffer, and recovered by centrifugation at 5,000 rpm and 4 ^° C. for 30 minutes. TE (10 mM Tris, 1 mM EDTA) buffer was added to the recovered bacteria and completely suspended. Then, 1% sodium dodecyl sulfate (SDS) and proteinase K were added. After lysozyme was added thereto and left for 30 minutes, 1% hexadecyl trimethyl ammonium bromide (CTAB) solution was added thereto and left at 37 ^° C. An equal volume of chloroform was added thereto and left at 4 ^° C. for one day, followed by centrifugation at 12,000 rpm and 4 ^° C. for 30 minutes to remove the chloroform layer. Subsequently, extraction with chloroform was further performed twice. 200 vol% of ethanol was added thereto to precipitate the DNA containing lipopolysaccharide, and then dissolved in TE (10 mM Tris, 1 mM EDTA) buffer, followed by repeated treatment of lipopolysaccharide using ethanol B As in.

B. Isolation of LPS from Bacterial DNA

30 mL of ethanol and 1/3 volume of 10M ammonium acetate were added to the DNA sample isolated from A, and left at 4 ^o C. After centrifugation at 12,000 rpm and 4 ^o C for 30 minutes, the precipitate was separated from the precipitate. The supernatant was first recovered. The first collected supernatant was added to 50% by volume of ethanol to the initial sample and left at 4 ^° C., and then centrifuged at 12,000 rpm and 4 ^° C. for 30 minutes to separate the precipitates. Then, the ethanol concentration was added to 100% by volume with respect to the first sample to the secondary supernatant and left at 4 ^o C, and then centrifuged at 12,000 rpm and 4 ^o C for 30 minutes to separate the precipitate to recover the supernatant 3 times. Subsequently, the ethanol concentration of the first sample was added to 200 vol% based on the initial sample and left at 4 ^° C., followed by centrifugation at 12,000 rpm and 4 ^° C. for 30 minutes to discard the supernatant to obtain a precipitate. The precipitate was taken up in TE (10 mM Tris, 1 mM EDTA) without lipopolysaccharide.

C. Iterative Process of LPS Removal: (2), (3), (4) of FIG.

(2) process of Figure 3

The precipitate was the ethanol concentration of said is 100% by volume precipitated TE dissolved in (10 mM Tris, 1mM EDTA) was added and an ethanol concentration of 50% by volume of 4 ^o then allowed to stand at C, 12,000 rpm, 4 ^o C at 30 The supernatant was recovered again by centrifugation for a minute. Subsequently, the ethanol concentration was added to 100% by volume with respect to the first sample to the first supernatant and left at 4 ^o C, and then centrifuged at 12,000 rpm and 4 ^o C for 30 minutes to recover the supernatant. Subsequently, the ethanol concentration of the first sample was added to 200% by volume, and the mixture was left at 4 ^° C., and then centrifuged at 12,000 rpm and 4 ^° C. for 30 minutes to recover the supernatant to obtain a precipitate. The precipitate was dissolved in lipopolysaccharide-free TE (10 mM Tris, 1 mM EDTA).

(3) process of Figure 3

The 100 vol% precipitate was recovered, dissolved in TE (10mM Tris, 1mM EDTA), ethanol concentration was added to 50 vol%, and left at 4 ^o C, followed by centrifugation at 12,000 rpm and 4 ^o C for 30 minutes. The supernatant was recovered again. Subsequently, the ethanol concentration was added to 100% by volume with respect to the first sample to the first supernatant and left at 4 ^o C, and then centrifuged at 12,000 rpm and 4 ^o C for 30 minutes to recover the supernatant. Subsequently, the ethanol concentration of the first sample was added to 200% by volume, and the mixture was left at 4 ^° C., and then centrifuged at 12,000 rpm and 4 ^° C. for 30 minutes to recover the supernatant to obtain a precipitate. The precipitate was taken up in TE (10 mM Tris, 1 mM EDTA) without lipopolysaccharide.

Process of Fig. 3 (4)

100% by volume of ethanol containing DNA content and lipopolysaccharide was recovered and dissolved in TE (10mM Tris, 1mM EDTA), added to 75% by ethanol concentration, and left at 4 ^° C. , And centrifuged at 4 ^o C for 30 minutes to separate from the precipitate to recover the supernatant again. Following the primary supernatant supernatant and the ethanol concentration and centrifuged at 90 after added was allowed to stand at 4 ^o C to a volume%, 12,000 rpm, 4 ^o C for 30 minutes to separate the precipitates with respect to the first sample to the second was recovered. Subsequently, the ethanol concentration of the first sample was added to 200% by volume, and the mixture was left at 4 ^° C., and then centrifuged at 12,000 rpm and 4 ^° C. for 30 minutes to discard the supernatant to obtain a precipitate. The precipitate was dissolved in lipopolysaccharide-free TE (10 mM Tris, 1 mM EDTA).

The precipitate recovered in each step in C of Example 2 was dissolved in lipopolysaccharide-free TE (10 mM Tris, 1 mM EDTA) and the amount of DNA and the amount of lipopolysaccharide were measured and shown in FIG. 3.

D. Determination of Mass, Purity and LPS of DNA in Precipitates of Each Stage

The mass and purity of the DNA contained in the precipitate produced in each treatment step of B and C were tested for the presence of LPS. The method for measuring the mass of DNA and the presence or absence of LPS was performed in the same manner as in C and D of Example 1.

The purity of DNA was measured as the ratio of absorbance (260/280 nm) measured in light of 280 nm wavelength to absorbance measured in light of 260 nm wavelength. If the ratio is 1.8 to 2.0, it can be said that pure DNA.

The measurement result is shown in FIG.

As described above, according to the present invention, due to the commonality between the DNA or RNA and the lipopolysaccharide as a polysaccharide, it has been difficult to separate from the bacterial DNA or RNA, and can be easily and efficiently removed from the bacterial DNA or RNA. Lipopolysaccharide can be removed.

Claims (7)

Alcohol is added to the bacterial DNA or RNA sample to remove the precipitated LPS, and then the supernatant is taken, and then the alcohol is added to the supernatant to remove the precipitated LPS and take the supernatant. Repeating until%; And taking a DNA or RNA precipitate formed by adding more alcohol to the supernatant thus obtained so that the alcohol is 100-1000% by volume with respect to the sample. 17. A method for removing lipopolysaccharide from bacterial DNA or RNA. delete The method of claim 1 wherein the alcohol is preferably methanol, ethanol, or isopropanol. The method of claim 1, wherein the alcohol is directed to a bacterial DNA or RNA sample. A first step of adding supernatant after adding alcohol to the sample to 1 to 30% by volume, centrifuging at 0 ^° C to 30 ^° C for 1 second to 72 hours at 5,000 to 15,000 rpm; A second step of adding supernatant taken in the first step to 31 to 50% by volume, followed by centrifugation under the same centrifugation conditions as step 1 and taking the supernatant; Adding to the supernatant taken in the second step so as to be 51-100% by volume, followed by centrifugation under the same centrifugation conditions as in step 1, and then taking the supernatant; Lipopoly from bacterial DNA or RNA, comprising adding a supernatant taken in step 3 to 100 to 1000% by volume, followed by centrifugation under the same range of step 1 as centrifugation, and taking a precipitate. How to get rid of saccharides. The method according to claim 4, wherein the precipitate obtained in step 3 is diluted and subjected to the first to fourth steps again, wherein the same procedure is repeated with the precipitate obtained in the third step to repeat DNA or RNA. A method for removing lipopolysaccharide from bacterial DNA or RNA characterized by increasing the yield. The method according to claim 1, wherein before the lipopolysaccharide is removed with the alcohol, chloroform, ether or phenol is added and left at -70 ^° C to 30 ^° C for 1 second to 72 hours and at 5,000 to 15,000 rpm for 1 second. And removing lipopolysaccharide by removing the chloroform layer, ether layer or phenol layer by centrifugation at 0-30 ^° C. for ˜72 hours. delete