阅读说明：本技术 一种基于免疫印迹法鉴别皮革的方法 (method for identifying leather based on immunoblotting method ) 是由 杨海亮 郑浩然 尚亚廷 翟雨洁 王秉 彭志勤 于 2019-09-08 设计创作，主要内容包括：本发明涉及皮革鉴定技术领域,公开了一种基于免疫印迹法鉴别皮革的方法,本发明采用碱法提取皮革中的蛋白,然后通过鹿蛋白标准序列筛选,得到了特征肽段,接着通过免疫反应得到了蛋白序列抗体,并采用western blot技术对其进行分析,探究其样品皮革是否为鹿皮。这种方法环保无污染。具有所需测样量较少,并且不受被测样表面损坏或降解等不利因素的影响、能准确高效的解决动物皮革中的仿冒问题等优点。(the invention relates to the technical field of leather identification, and discloses a method for identifying leather based on an immunoblotting method. The method is environment-friendly and pollution-free. The method has the advantages of less sample measurement amount, no influence of adverse factors such as surface damage or degradation of the measured sample, accurate and efficient solution of counterfeit problem in animal leather, and the like.)

1. a method for identifying leather based on an immunoblotting method is characterized by comprising the following steps:

1) taking a leather sample to be detected, grinding the leather sample into powder, adding distilled water according to a bath ratio of 1: 50-70g/mL, and soaking for 20-30 h;

2) Adjusting the pH value to be alkaline by using alkaline earth metal hydrolysate, heating to boil, continuously stirring slowly in the heating process, and reacting for 1-2 h in the state;

3) filtering while hot, adjusting pH of the filtrate to neutral, filtering, concentrating under reduced pressure to viscous state, oven drying, and pulverizing to obtain soluble protein powder;

4) washing the gel making frame with distilled water, drying at 55-65 deg.C for 10-20min, taking out, building gel making frame, checking whether water leakage occurs, and performing subsequent gel making operation if water leakage does not occur to obtain two glass plates with multiple lanes sandwiched between middle bags;

5) dissolving soluble protein powder in CB to obtain protein mixed solution with the concentration of 8-12mg/mL, putting 2.5-3.5ul of reduced 5XSDS loading buffer solution into a test tube, then adding the protein mixed solution, heating in a water bath at 95-100 ℃ for 8-12min, and centrifuging;

6) Taking the supernatant for electrophoresis, adding the supernatant into a lane for electrophoresis, stripping glue between glass plates after the electrophoresis is finished, cleaning the glass plates by using distilled water, and observing an electrophoresis result by using a chemiluminescence imager after the cleaning;

7) After observation, cutting a PVDF membrane, soaking the PVDF membrane in methanol for 0.5-1.5min for excitation, then soaking the PVDF membrane and sponge filter paper in a transfer buffer solution to build a sandwich structure, then performing transfer printing, wrapping the periphery of the reaction device with an ice bag in the transfer printing process, wherein the transfer printing current is 190-210mA, and the transfer printing process lasts for 20-40 min;

8) Pouring 18-22mL of sealing liquid on a PVDF membrane, sealing a shaking table for 0.5-1.5h, pouring off, adding 8-12mL of primary antibody diluted by TBST, incubating the primary antibody for 0.5-1.5h by the shaking table, then cleaning for multiple times by TBST, using 18-22mL of each time, shaking the table for 8-12min, cleaning for multiple times, adding 8-12mL of diluted secondary antibody, incubating for 0.5-1.5h, cleaning for multiple times by TBST after incubation is finished, finally uniformly dripping the color developing liquid on the PVDF membrane, covering the PVDF membrane with tinfoil paper for 3-7min in dark, observing an immune result by a chemiluminescence imager, and if a shadow band exists, determining the PVDF membrane as genuine leather, and if the PVDF membrane does not exist, determining the imitation leather.

2. the method of claim 1, wherein in step 3), the pH is adjusted to a value of 7.1 to 7.3.

3. the method according to claim 1, wherein in step 4), the glue making operation is specifically: injecting the separation gel between the two glass plates by using a liquid-transferring gun, and then injecting isopropanol into the glass plates to enable the liquid level of the isopropanol to be flush; then placing the glass plate at 35-40 ℃ for 15-20 min; after the separation gel is solidified, pouring off the isopropanol, then adding the concentrated gel, immediately inserting a comb, placing the glass plate in the device at 35-40 ℃ for 10-20min, and placing the glass plate after gel preparation in an electrophoresis tank for leak detection; when the glass plate is placed, the short glass plate faces inwards, and the long glass plate faces outwards; the comb was then removed to form the lane.

4. the method of claim 1, wherein in step 6), the electrophoresis operation is performed by: regulating the voltage to 75-85V, increasing the voltage to 110-130V after electrophoresis for 8-12min, and stopping electrophoresis when the bromophenol blue reaches the vicinity of the bottom.

5. the method of claim 1, wherein in step 8), the antigen of the primary antibody is a deer protein signature sequence; the characteristic sequence is used as hapten, and is combined with carrier protein to immunize animals to obtain protein sequence antibody.

Technical Field

The invention relates to the technical field of leather identification, in particular to a method for identifying leather based on an immunoblotting method.

Background

At present, leather products are more and more popular, the market potential of the leather and the products thereof is very large, the global total demand of the leather is about 1.0 hundred million square meters, which is equivalent to the yield of 3 hundred million cowhide (standard leather), and the yield of Chinese leather is reduced to about 7000 million standard leather, which accounts for about 23.33 percent of the global yield of the leather. Leather products sold in the market are mainly classified into animal leather (genuine leather) and artificial leather (fake leather), wherein the animal leather is mainly the raw leather peeled from cattle, sheep, pigs, deer or other animals. In the aspect of distinguishing artificial leather from animal leather, since artificial leather is generally a plastic product, one can generally distinguish artificial leather from animal leather by a simple identification method, but many bad vendors in the market replace valuable deer leather with cow leather and sheep leather, which is very difficult for consumers to distinguish. Many of the complaints about leather products on the 12315 website are about materials "in false. In addition, the infrared spectroscopy used for identifying the artificial leather and the animal leather has the advantages of no sample damage, simple operation and the like, but is not suitable for identifying the animal leather.

disclosure of Invention

in order to solve the technical problems, the invention provides a method for identifying leather based on an immunoblotting method, which comprises the steps of extracting protein in leather by an alkaline method, screening a deer protein standard sequence to obtain a characteristic peptide segment, obtaining a protein sequence antibody by immunoreaction, analyzing the protein sequence antibody by a western blot technology, and researching whether the sample leather is deer leather. The method is environment-friendly and pollution-free. The method has the advantages of less sample measurement amount, no influence of adverse factors such as surface damage or degradation of the measured sample, accurate and efficient solution of counterfeit problem in animal leather, and the like.

the specific technical scheme of the invention is as follows: a method for identifying leather based on an immunoblotting method comprises the following steps:

1) taking a leather sample to be detected, grinding the leather sample into powder, adding distilled water according to a bath ratio of 1: 50-70g/mL, and soaking for 20-30 h;

2) adjusting the pH value to be alkaline by using alkaline earth metal hydrolysate, heating to boil, continuously stirring slowly in the heating process, and reacting for 1-2 h in the state;

3) filtering while hot, adjusting pH of the filtrate to neutral, filtering, concentrating under reduced pressure to viscous state, oven drying, and pulverizing to obtain soluble protein powder;

4) Washing the gel making frame with distilled water, drying at 55-65 deg.C for 10-20min, taking out, building gel making frame, checking whether water leakage occurs, and performing subsequent gel making operation if water leakage does not occur to obtain two glass plates with multiple lanes sandwiched between middle bags;

5) Dissolving soluble protein powder in CB to obtain protein mixed solution with the concentration of 8-12mg/mL, putting 2.5-3.5ul of reduced 5XSDS loading buffer solution into a test tube, then adding the protein mixed solution, heating in a water bath at 95-100 ℃ for 8-12min, and centrifuging;

6) taking the supernatant for electrophoresis, adding the supernatant into a lane for electrophoresis, stripping glue between glass plates after the electrophoresis is finished, cleaning the glass plates by using distilled water, and observing an electrophoresis result by using a chemiluminescence imager after the cleaning;

7) After observation, cutting a PVDF membrane, soaking the PVDF membrane in methanol for 0.5-1.5min for excitation, then soaking the PVDF membrane and sponge filter paper in a transfer buffer solution to build a sandwich structure, then performing transfer printing, wrapping the periphery of the reaction device with an ice bag in the transfer printing process, wherein the transfer printing current is 190-210mA, and the transfer printing process lasts for 20-40 min;

8) pouring 18-22mL of sealing liquid on a PVDF membrane, sealing a shaking table for 0.5-1.5h, pouring off, adding 8-12mL of primary antibody diluted by TBST, incubating the primary antibody for 0.5-1.5h by the shaking table, then cleaning for multiple times by TBST, using 18-22mL of each time, shaking the table for 8-12min, cleaning for multiple times, adding 8-12mL of diluted secondary antibody, incubating for 0.5-1.5h, cleaning for multiple times by TBST after incubation is finished, finally uniformly dripping the color developing liquid on the PVDF membrane, covering the PVDF membrane with tinfoil paper for 3-7min in dark, observing an immune result by a chemiluminescence imager, and if a shadow band exists, determining the PVDF membrane as genuine leather, and if the PVDF membrane does not exist, determining the imitation leather.

preferably, in step 3), the pH is adjusted to a value of 7.1 to 7.3.

preferably, in the step 4), the glue making operation specifically comprises: injecting the separation gel between the two glass plates by using a liquid-transferring gun, and then injecting isopropanol into the glass plates to enable the liquid level of the isopropanol to be flush; then placing the glass plate at 35-40 ℃ for 15-20 min; after the separation gel is solidified, pouring off the isopropanol, then adding the concentrated gel, immediately inserting a comb, placing the glass plate in the device at 35-40 ℃ for 10-20min, and placing the glass plate after gel preparation in an electrophoresis tank for leak detection; when the glass plate is placed, the short glass plate faces inwards, and the long glass plate faces outwards; the comb was then removed to form the lane.

Preferably, in step 6), the electrophoresis operation process is as follows: regulating the voltage to 75-85V, increasing the voltage to 110-130V after electrophoresis for 8-12min, and stopping electrophoresis when the bromophenol blue reaches the vicinity of the bottom.

Preferably, in step 8), the antigen of the primary antibody is a deer protein characteristic sequence; the characteristic sequence is used as hapten, and is combined with carrier protein to immunize animals to obtain protein sequence antibody. Compared with the prior art, the invention has the beneficial effects that:

(1) The invention can accurately and efficiently solve the problem of imitation leather (such as deerskin) in animal leather.

(2) The immunoblotting technology adopted in the invention has high feasibility, can be popularized to identify different animal and plant leather types, and is expected to become a scientific method for identifying leather.

(3) the invention needs less sample measurement amount and is not influenced by the adverse factors such as the surface damage or degradation of the sample to be measured.

Detailed Description

The present invention will be further described with reference to the following examples.