阅读说明：本技术 一种改性琼脂粉 (Modified agar powder ) 是由 朱艳冰 梁梅芳 倪辉 姜泽东 肖安风 李利君 杨远帆 杜希萍 于 2019-08-20 设计创作，主要内容包括：本发明涉及琼脂粉的改性,具体涉及一种改性琼脂粉,其通过以下方法制得：将琼脂粉用50mmol/L pH 9.0的Tris-HCl缓冲液溶解,以便获得琼脂粉溶液；将芳香基硫酸酯酶加入所述琼脂粉溶液中,再置于50℃下温浴6h,以便获得所述改性琼脂粉。本发明制得的改性琼脂粉的性质类似于商品琼脂糖,在核酸电泳试验时,对凝胶电泳的DNA片段具有较好的分离分辨率。(The invention relates to modification of agar powder, in particular to modified agar powder, which is prepared by the following method: dissolving agar powder with 50mmol/L Tris-HCl buffer solution with pH of 9.0 to obtain agar powder solution; adding aryl sulfatase into the agar powder solution, and then placing the agar powder solution in a warm bath at 50 ℃ for 6 hours to obtain the modified agar powder. The modified agar powder prepared by the invention has the property similar to commercial agarose, and has better separation resolution ratio on DNA fragments subjected to gel electrophoresis in a nucleic acid electrophoresis test.)

1. The modified agar powder is characterized by being prepared by the following method:

dissolving agar powder with 50mmol/L Tris-HCl buffer solution with pH of 9.0 to obtain agar powder solution;

Adding aryl sulfatase into the agar powder solution, and then placing the agar powder solution in a warm bath at 50 ℃ for 6 hours to obtain the modified agar powder.

2. The modified agar powder according to claim 1, wherein: after the completion of the warm bath, 1mL of the sample was collected and centrifuged to obtain a supernatant, and BaCl was added to the supernatant₂·2H₂Standing the O-Tween 80 mixed solution and concentrated hydrochloric acid at room temperature for 30min, measuring absorbance at 420nm, and calculating the released sulfate amount by substituting the absorbance into the standard curve of sulfate radical content.

3. The modified agar powder according to claim 1, wherein: the enzyme adding amount of the aryl sulfatase is 1.20U/mL.

4. The modified agar powder according to claim 2, wherein: the supernatant fluid and BaCl₂·2H₂The ratio of the O-Tween 80 mixed solution to the concentrated hydrochloric acid is 9:6: 2.

5. The modified agar powder according to claim 1, wherein: the aryl sulfatase is prepared by the following method: the engineering bacteria containing the aryl sulfatase gene are prepared by mixing the following components in a volume ratio of 1: 100 is transferred into 250mL LB liquid culture medium, cultured to OD at 37 ℃ under 180r/min₆₀₀adding LIPTG with final concentration of 0.1mmol/LIPTG, culturing at 18 deg.C and 180r/min for 20 hr, centrifuging, and collecting thallus; and (3) re-suspending the thalli in a binding buffer solution, placing the thalli on ice for ultrasonic disruption treatment, centrifuging, collecting supernatant, performing Ni-NTA affinity chromatography on the supernatant, washing the supernatant by using a washing buffer solution, eluting by using an elution buffer solution, and collecting eluent so as to obtain the aryl sulfatase.

Technical Field

The invention relates to the field of modification of agar powder, and particularly relates to modified agar powder.

background

The agarose is chain neutral polysaccharide mainly formed by combining D-galactose and 3, 6-dehydrated-L-galactose; because the content of charged groups such as sulfate, pyruvic acid and the like is low, the electrophoresis medium has small electroosmosis and no adsorption to protein and basic dye, can be used as an ideal electrophoresis medium and is generally used for nucleic acid electrophoresis tests.

at present, domestic agarose is mainly obtained by separating agarose from sulfur agar so as to achieve the aim of purifying the agarose; therefore, the quality of domestic agarose products is not high, so that commercial agarose for laboratories mostly depends on import from abroad.

Therefore, a substance which can replace commercial agarose is to be researched.

Disclosure of Invention

the present invention is directed to solving, at least to some extent, one of the technical problems in the related art. Therefore, an object of the present invention is to provide a modified agar powder. The modified agar powder prepared by the invention has the property similar to commercial agarose, and has better separation resolution ratio on DNA fragments subjected to gel electrophoresis in a nucleic acid electrophoresis test.

In view of the above, in one aspect of the present invention, the present invention provides a modified agar powder, which is prepared by the following method:

Dissolving agar powder with 50mmol/L Tris-HCl buffer solution with pH of 9.0 to obtain agar powder solution;

Adding aryl sulfatase into the agar powder solution, and then placing the agar powder solution in a warm bath at 50 ℃ for 6 hours to obtain the modified agar powder.

The invention adopts aryl sulfatase to carry out desulfurization treatment on the agar powder, changes the property of the agar powder, ensures that the property of the modified agar powder is close to that of commercial agarose, can be applied to nucleic acid electrophoresis, and has better separation resolution ratio on DNA fragments of gel electrophoresis during the nucleic acid electrophoresis test.

In addition, the modified agar powder provided by the above embodiment of the invention may also have the following additional technical features:

according to the embodiment of the invention, after the warm bath is finished, 1mL of sample is taken and centrifuged to obtain supernatant, and BaCl is added into the supernatant₂·2H₂Standing the O-Tween 80 mixed solution and concentrated hydrochloric acid at room temperature for 30min, measuring absorbance at 420nm, and calculating the released sulfate amount by substituting the absorbance into the standard curve of sulfuric acid.

according to the embodiment of the invention, the enzyme adding amount of the aryl sulfatase is 1.20U/mL.

according to an embodiment of the invention, the supernatant, BaCl₂·2H₂the ratio of the O-Tween 80 mixed solution to the concentrated hydrochloric acid is 9:6: 2.

According to an embodiment of the present invention, the arylsulfatase is prepared by: the engineering bacteria containing the aryl sulfatase gene are prepared by mixing the following components in a volume ratio of 1: 100 is transferred into 250mL LB liquid culture medium, cultured to OD at 37 ℃ under 180r/min₆₀₀Adding IPTG (0.1 mmol/L) at a final concentration of 0.8, culturing at 18 deg.C for 20 hr at 180r/min, centrifuging, and collecting thallus; and (3) re-suspending the thalli in a binding buffer solution, placing the thalli on ice for ultrasonic disruption treatment, centrifuging, collecting supernatant, performing Ni-NTA affinity chromatography on the supernatant, washing the supernatant by using a washing buffer solution, eluting by using an elution buffer solution, and collecting eluent so as to obtain the aryl sulfatase.

additional aspects and advantages of the invention will be set forth in part in the description which follows and, in part, will be obvious from the description, or may be learned by practice of the invention.

Drawings

FIG. 1 shows the dissolution temperatures of modified agar powder, commercial agar powder and commercial agarose in accordance with an embodiment of the present invention;

FIG. 2 is a graph showing the melting temperatures of modified agar powder, commercial agar powder, and commercial agarose in accordance with an embodiment of the present invention;

FIG. 3 is the solidification temperatures of modified agar powder, commercial agar powder, and commercial agarose of an embodiment of the invention;

FIG. 4 is a graph showing the gel strength of modified agar powder, commercial agar powder, and commercial agarose according to an embodiment of the present invention;

FIG. 5 is a graph showing the transparency of modified agar powder, commercial agar powder, and commercial agarose according to an embodiment of the invention;

FIG. 6 is a graph of the viscosities of modified agar powder, commercial agar powder, and commercial agarose of an embodiment of the invention;

FIG. 7 shows the whiteness of modified agar powder, commercial agar powder and commercial agarose in accordance with an embodiment of the present invention;

FIG. 8 is a gel electrophoresis image of modified agar powder, commercial agar powder, and commercial agarose according to an embodiment of the invention;

Wherein, P is less than 0.05, and represents that the modified agar powder and the commercial agar powder have significant difference; indicates that the modified agar powder and the commercial agar powder have very significant difference, and P is less than 0.01.

Detailed Description

The technical solution of the present invention is illustrated by specific examples below. It is to be understood that one or more method steps mentioned in the present invention do not exclude the presence of other method steps before or after the combination step or that other method steps may be inserted between the explicitly mentioned steps; it should also be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. Moreover, unless otherwise indicated, the numbering of the various method steps is merely a convenient tool for identifying the various method steps, and is not intended to limit the order in which the method steps are arranged or the scope of the invention in which the invention may be practiced, and changes or modifications in the relative relationship may be made without substantially changing the technical content.

In order to better understand the above technical solutions, exemplary embodiments of the present invention are described in more detail below. While exemplary embodiments of the invention have been shown, it should be understood that the invention may be embodied in various forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the invention to those skilled in the art.

The test materials adopted by the invention are all common commercial products and can be purchased in the market.

the present invention proposes a modified agar powder, which will be described in detail below.

according to an embodiment of the invention, the modified agar powder is prepared by the following method:

Dissolving agar powder with 50mmol/L Tris-HCl buffer solution (pH 9.0) to obtain agar powder solution; in this step, agar powder is dissolved to prepare an agar powder solution. Specifically, 4g of agar powder is weighed and placed in 100mL of buffer solution to obtain an agar powder solution with the concentration of 4%. The inventor finds that when the pH of the agar powder solution is 9, the agar powder solution is beneficial to aromatic sulfatase to carry out better desulfurization treatment on the agar powder so as to obtain modified agar powder with the property close to that of commercial agarose.

According to an embodiment of the present invention, the kind of the agar powder is not particularly limited, and may be selected by those skilled in the art according to actual needs, and according to an embodiment of the present invention, the agar powder may be selected from commercially available agar powder (manufacturer: Guangdong Huanji Microscience Co., Ltd.).

Adding aryl sulfatase into the agar powder solution, and then placing the agar powder solution in a warm bath at 50 ℃ for 6 hours to obtain the modified agar powder. In the step, aryl sulfatase is added into the agar powder solution, and the solution is subjected to warm bath for desulfurization treatment, so that the modified agar powder is obtained. The inventor finds that the property of the agar powder can be changed by adopting aryl sulfatase to carry out desulfurization treatment on the agar powder solution, so that the quality of the commercial agar powder is obviously improved, the property of the modified agar powder is close to that of commercial agarose, the modified agar powder can be applied to nucleic acid electrophoresis, and the agar powder has better separation resolution ratio on DNA fragments subjected to gel electrophoresis in a nucleic acid electrophoresis test.

According to an embodiment of the present invention, the enzyme adding amount of the arylsulfatase enzyme-treated agar powder solution is not particularly limited, and can be selected by those skilled in the art according to actual needs, and according to an embodiment of the present invention, the enzyme adding amount of the arylsulfatase enzyme is 1.20U/mL. The inventors have found that the addition of a small amount of arylsulfatase leads to a slow desulfurization rate of agar powder. Therefore, the enzyme adding amount can ensure the desulfurization rate of the agar powder and the quality of the modified agar powder.

According to another embodiment of the present invention, after the agar powder and the arylsulfatase are subjected to warm bath, 1mL of sample is taken, centrifuged to obtain a supernatant, and BaCl is added to the supernatant₂·2H₂Standing the O-Tween 80 mixed solution and concentrated hydrochloric acid at room temperature for 30min, measuring absorbance at 420nm, and calculating the released sulfate amount by substituting the absorbance into the standard curve of sulfuric acid. According to a specific example of the present invention, after 6 hours of warm bath, 1mL of the supernatant was sampled and centrifuged at 12000 Xg for 10min to obtain a supernatant, and 0.9mL of the supernatant was mixed with 0.2mL of concentrated HCl and 0.6mL of 13.33% BaCl₂·2H₂the O-2.67% Tween 80 mixed solution was mixed, and after standing at room temperature for 30min, the absorbance was measured at 420 nm. The amount of sulfate released was calculated using a stable standard curve in the range of 2-20 μ g/mL sulfuric acid. Inactivated enzyme treated agar powder was used as a reference. And (3) measuring the sulfate content of the agar powder treated by the enzyme, so as to calculate the desulfurization rate of the agar powder acted by aryl sulfatase, and further control the condition of the agar powder modification process.

According to another embodiment of the present invention, the specific type of the arylsulfatase is not particularly limited and may be selected by those skilled in the art according to actual needs, and according to one embodiment of the present invention, the arylsulfatase is prepared by: carrying out site-directed mutagenesis on an engineering bacterium containing an aryl sulfatase gene (the engineering bacterium is escherichia coli, and the aryl sulfatase gene (GenBank accession number: KJ509595) derived from Pseudoalteromonas carrageenovora is used for obtaining a mutant enzyme K253H/H260L group with higher heat stability, and expressing the mutant enzyme K253H/H260L group in the escherichia coli), wherein the volume ratio of the engineering bacterium to the engineering bacterium is 1: 100 was transferred to 250mL of LB liquid medium (containing 50. mu.g/mL kanamycin), and cultured at 37 ℃ and 180r/min to OD₆₀₀Adding IPTG (0.1 mmol/L) at final concentration of 0.8, culturing at 18 deg.C for 20 hr at 180r/min, centrifuging at 6000 Xg for 10min, and collecting thallus; the cells were resuspended in binding buffer (50mmol/L NaH)₂PO₄300mmol/L sodium chloride, 15mmol/L imidazole, pH 8.0), ultrasonication on ice, centrifugation at 18000 Xg for 20min at 4 deg.C, collecting supernatant, Ni-NTA affinity chromatography, washing with buffer (50mmol/L NaH)₂PO₄300mmol/L NaCl, 30mmol/L imidazole, pH 8.0) and then eluted with elution buffer (50mmol/L NaH)₂PO₄300mmol/L sodium chloride, 250mmol/L imidazole, pH 8.0), and collecting the eluate to obtain aryl sulfatase.

The invention will now be described with reference to specific examples, which are intended to be illustrative only and not to be limiting in any way: