阅读说明：本技术 一种提取油泥砂中宏蛋白质组的提取液及提取方法 (The extracting solution and extracting method of macro protein group in a kind of extraction oil sludge and sand ) 是由 王新新 韩增 吴亮 李凤娟 宿辉 杨勇 于 2019-08-01 设计创作，主要内容包括：本发明提供一种提取油泥砂中宏蛋白质组的提取液及提取方法。提取液由聚苯基甲基硅氧烷和pH8.0～8.2磷酸盐缓冲液组成；两者体积比1:2～2:1,聚苯基甲基硅氧烷密度1010-1120kg/m<Sup>3</Sup>。提取液震荡时宏观上形成乳浊液,微观上分散为聚苯基甲基硅氧烷和磷酸盐缓冲液微粒。聚苯基甲基硅氧烷微粒结合油泥砂,使石油进入聚苯基甲基硅氧烷微粒中,疏水性降低。磷酸盐缓冲液微粒结合疏水性降低的油泥砂,使其中的宏蛋白质组进入水相提取液。经离心、过滤、沉淀、洗涤和干燥,获得提纯的宏蛋白质组。本发明有助于对油泥砂中微生物宏基因组翻译产生的宏蛋白质组进行分析,从而获得油藏微生物代谢信息,为微生物采油提供数据基础。(The present invention provides a kind of extracting solution and extracting method for extracting macro protein group in oil sludge and sand.Extracting solution is made of polyphenyl methyl siloxane and the phosphate buffer of pH8.0~8.2；The two volume ratio 1:2~2:1, polyphenyl methyl siloxane density 1010-1120kg/m 3 .Extracting solution macroscopically forms emulsion when shaking, it is microcosmic on be separated into polyphenyl methyl siloxane and phosphate buffer particle.Polyphenyl methyl siloxane particle combination oil sludge and sand, enters petroleum in polyphenyl methyl siloxane particle, and hydrophobicity reduces.The oil sludge and sand that phosphate buffer particle combination hydrophobicity reduces, makes macro protein group therein enter aqueous extract.Through centrifugation, filtering, precipitating, washing and drying, the macro protein group of purification is obtained.The macro protein group that the present invention helps to translate the macro genome of microorganism in oil sludge and sand generation is analyzed, to obtain oil pool microorganisms metabolic information, provides data basis for microbe oil production.)

1. a kind of extracting solution for extracting macro protein group in oil sludge and sand, it is characterized in that by polyphenyl methyl siloxane and pH8.0~ 8.2 phosphate buffers composition；Polyphenyl methyl siloxane density is 1010-1120kg/m³, polyphenyl methyl siloxane and phosphorus Phthalate buffer volume ratio 1:2~2:1.

2. the extracting method of macro protein group in oil sludge and sand is carried out using the extracting solution of claim 1, it is characterized in that including as follows Step:

1): oil sludge and sand being added in the extracting solution of polyphenyl methyl siloxane and phosphate buffer composition；After ultrasonic vibration Shaking table concussion is uniformly mixed oil sludge and sand and extracting solution, centrifugation removal large granular impurity；

2): upper strata aqueous phase extracting solution is sucked out, uses filtering with microporous membrane；

3): acetone being added into filtrate, is centrifuged out and precipitates；

4): being precipitated with acetone washing；

5): the precipitating after washing is dry with vacuum drier, obtains the macro protein group of purification.

3. method according to claim 2, it is characterized in that it is 0~4 DEG C that polyphenyl methyl siloxane is added in step 1).

4. method according to claim 2, it is characterized in that the ultrasonic vibration time is 5~30min in step 1).

5. method according to claim 2, it is characterized in that the shaking table concussion time is 8~12h in step 1).

6. method according to claim 2, it is characterized in that the centrifugation rate of centrifugation removal large granular impurity is in step 1) 6000~10000r/min.

7. method according to claim 2, it is characterized in that the amount of acetone is the body of 1~4 times of phosphate buffer in step 3) Product；Temperature is 0~4 DEG C.

8. method according to claim 2, it is characterized in that the amount of acetone is 0.01~0.05 times of phosphate-buffered in step 4) The volume of liquid；Temperature is 0~4 DEG C.

9. method according to claim 2, it is characterized in that vacuum drier drying time 10 in the step 5)~ 60min；Drying temperature is -80~-10 DEG C.

10. method according to claim 2, it is characterized in that step 1), 3) environment temperature, 4) operated are 0~4 DEG C.

Technical field

The invention belongs to oil recovery technique field, it is related to the extracting solution of macro protein group and extraction side in a kind of extraction oil sludge and sand Method.

Background technique

The Microbial Enhanced Oil Recovery that oil recovery is improved using the metabolic activity of stratum depths oil pool microorganisms can be with The utilization rate of petroleum resources is greatly improved, therefore fully realizes the microorganism in oil reservoir to the field conduct of Microbial Enhanced Oil Recovery It is of great significance.Since more difficult, the source generated to oil recovery process is implemented in situ sampling analysis at layer depth over the ground It is sampled in the oil sludge and sand of stratum depths oil reservoir, further carrying out analysis to the microorganism wherein carried becomes preferable choosing It selects.Generally microorganism is analyzed using macro genome DNA analytical technology at present, however macro genome DNA analytical technology is only It is the analysis to microorganism hereditary information level, the information of its metabolism level can not be obtained, it is difficult to reflects that stratum depths oil reservoir is micro- The metabolic activity situation of biology.And macro Proteomic analysis technology is divided for the protein generated after genome translation Analysis, can obtain metabolism section information abundant, facilitate the metabolic activity for fully realizing oil pool microorganisms.

Macro protein group extraction is the basis of the macro protein group of analysis detection, substantially influences the accurate of macro Proteomic analysis Property.Existing extracting method is preferable to the Protein Extraction effect in hydrophilic material due to using water base extracting solution.Such as it sends out Bright patent 2015104424587,2016108014158,2015102104160,201910203088X, 2017100688388, 2009102430747 and 200910111462X each provides a kind of " extraction of the macro protein group of thallus in spontaneous fermentation beans sauce Method ", " extracting method of macro protein group outside extracellular microbial in a kind of spontaneous fermentation beans sauce ", " one kind is used for proteomics The extracting method of the Siraitia grosvenorii blade gross protein of analysis ", " a kind of efficient soil protein extracting method ", a kind of " soil The extracting method of protein ", " preparation method of soil member protein group " and " point of soil Protein Extraction and intracellular protein From method ".Above-mentioned patent is preferable to the Protein Extraction effect in the hydrophilic materials such as beans sauce, plant and soil, but due to Oil sludge and sand contains a large amount of petroleum, causes hydrophobicity higher, therefore existing method can not be in the hydrophobic materials such as oil sludge and sand Macro protein group extracts, and hinders subsequent macro Proteomic analysis detection.

Summary of the invention

The purpose of the present invention is overcome the deficiencies in the prior art, for the macro egg in the oil sludge and sand of stratum depths oil reservoir White matter group extracts problem, provides a kind of extracting solution and extracting method for extracting macro protein group in oil sludge and sand.

Technical scheme is as follows:

The extracting solution of macro protein group, is by polyphenyl methyl siloxane and the phosphorus of pH8.0~8.2 in a kind of extraction oil sludge and sand Phthalate buffer composition；Polyphenyl methyl siloxane density is 1010-1120kg/m³, polyphenyl methyl siloxane and phosphate Buffer volume ratio 1:2~2:1.

Included the following steps using the extracting method that extracting solution of the invention carries out macro protein group in oil sludge and sand.

1): oil sludge and sand being added in the extracting solution of polyphenyl methyl siloxane and phosphate buffer composition, shaken through ultrasound Swinging rear shaking table concussion is uniformly mixed oil sludge and sand and extracting solution, centrifugation removal large granular impurity；

2): upper strata aqueous phase extracting solution is sucked out, uses filtering with microporous membrane；

3): acetone being added into filtrate, is centrifuged out and precipitates；

4): being precipitated with acetone washing；

5): the precipitating after washing is dry with vacuum drier, obtains the macro protein group of purification.

It is 0~4 DEG C that polyphenyl methyl siloxane preferable temperature is added in the step 1).

The ultrasonic vibration time is preferably 5~30min in the step 1).

The shaking table concussion time is preferably 8~12h in the step 1).

The centrifugation rate of centrifugation removal large granular impurity is preferably 6000~10000r/min in the step 1).

The amount of acetone is preferably the volume of 1~4 times of phosphate buffer in the step 3)；Preferable temperature is 0~4 ℃。

The rate of departure of centrifuge separation precipitating is preferably 8000~16000r/min in the step 3).

The amount of acetone is preferably the volume of 0.01~0.05 times of phosphate buffer in the step 4)；Preferable temperature is 0~4 DEG C.

Vacuum drier drying time preferably 10~60min in the step 5)；Preferably -80~-10 DEG C of drying temperature.

The step 1), 3), 4) environment temperature of operation is preferably 0~4 DEG C.

Macro protein group content is measured using Coomassie Brilliant Blue, macro albumen is detected using SDS-PAGE and LC-MS instrument Kinds of protein in matter group.

Uniform emulsion is macroscopically formed when polyphenyl methyl siloxane and phosphate buffer earthquake, on microcosmic It is separated into polyphenyl methyl siloxane particle and phosphate buffer particle.Polyphenyl methyl siloxane particle and hydrophobicity are higher Oil sludge and sand combine, petroleum is transferred in polyphenyl methyl siloxane particle, to reduce the hydrophobicity of oil sludge and sand.Phosphate Buffer particle is in conjunction with the oil sludge and sand after reducing hydrophobicity, to extract macro protein group therein.Since phosphate is slow The density of fliud flushing is lower than polyphenyl methyl siloxane, and emulsion is layered after high speed centrifugation, the phosphate containing macro protein group Buffer floats on polyphenyl methyl siloxane upper layer, detects through purification for macro Proteomic analysis after suction.

Advantages of the present invention: (1) extracting solution is made of polyphenyl methyl siloxane and phosphate buffer, and when concussion disperses For polyphenyl methyl siloxane particle and phosphate buffer particle, reduce oil sludge and sand it is hydrophobic on the basis of extract macro egg White matter group, therefore this patent is suitable for oil sludge and sand, it is with strong points.(2) density of phosphate buffer is lower than polyphenyl methyl silicon The density of oxygen alkane, the phosphate buffer after high speed centrifugation containing macro protein group float on polyphenyl methyl siloxane upper layer, just In subsequent operation.(3) polyphenyl methyl siloxane toxicity is low, influences on ecological environment and personnel health smaller.This patent helps The macro protein group generated after the macro genome translation of microorganism in oil sludge and sand is analyzed, to obtain metabolism layer abundant Face information provides data convenient for fully realizing the microbiota metabolic activity in oil reservoir for the field conduct of Microbial Enhanced Oil Recovery Basis.

Specific embodiment

Below in conjunction with specific example, this patent is described in detail.