A method for identifying the species of goat skin based on immune antibody technology

Abstract

The application discloses a goat skin species identification method based on an immune antibody technology, and the specific protein of the goat skin is identified through proteomics; a polyclonal antibody is prepared according to the specific protein sequence; the specific antibody is combined with the natural antigen in the cortex by using the immune antibody technology; and finally, whether the to-be-detected skin is the goat skin is identified through result analysis. The polyclonal goat skin species identification antibody, i.e., the goat specific antibody, is produced by multiple B lymphocyte clones of New Zealand rabbits, can specifically recognize the natural antigen in the goat skin, has good sensitivity and specificity, and can be combined with the determination method provided by the application to quickly and accurately identify whether the cortex is the goat skin. The method has the characteristics of high accuracy, low equipment requirement, strong specificity and short detection period compared with other methods, and can be used for quickly identifying whether the skin is the goat skin.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of goat skin detection, and relates to a goat skin species identification method based on immune antibody technology. BACKGROUND

[0002] Leather is a product made from animal skin after mechanical and chemical processing, commonly known as "real leather" on the market. Generally, "real leather" refers to the difference from various artificial similar leather, which is only made from animal rawhide. There are various types of "real leather" on the market, with different qualities and huge price differences. The processing technology of "real leather" is complex, and it usually needs to go through more than ten processes to be made. According to the raw materials, it can be divided into sheep leather, cow leather, horse leather, pig leather, snake leather, crocodile leather, etc. Among them, sheep, cow and pig are the three major rawhide for leather making. Cow leather has high gloss, soft and delicate skin, wear resistance and tensile resistance, and therefore high-end cow leather is more expensive, while cheap cow leather is easy to crack and break. Pig leather has good air and water vapor permeability, high strength and wear resistance, and is suitable for making underwear and children's products, but the pig skin has deep hair roots, resulting in rough grain, poor softness and appearance, and lower smoothness than cow leather. Sheep leather is light, thin and soft, with good air permeability, smooth surface, softness, fullness, good air permeability and elasticity, and the skin surface retains the natural and original state of the sheep skin, which is an ideal material for fashion. Sheep leather is mainly divided into goat leather and sheep leather: sheep leather has fine and smooth grain, better softness than goat leather, and is mainly used for making clothes and gloves, and also for making shoes and soft packages. Goat leather has fine and beautiful grain, good softness, and its products are comfortable and beautiful to wear, and are mainly used for making high-end shoes, leather clothes, gloves and soft packages. The raw materials, processing technology and characteristics of the above leathers are different, resulting in large market price differences. The characteristics of sheep, cow and pig skin are covered, and the identification of leather species becomes a difficult problem after cutting and segmentation. In addition, there are many goods on the market that use artificial leather, horse leather and donkey leather to imitate cow leather and sheep leather, and how to use effective methods to identify the skin has become a problem that people pay close attention to.

[0003] At present, the method for identifying animal skin species mostly depends on sensory identification, such as visual identification method, which is based on the pattern, pores, animal fibers and side section of the skin to distinguish; olfactory identification method, which is based on the special smell of the skin (natural skin smell or chemically synthesized smell) to distinguish; touch identification method, which is based on the bending wrinkle, fold thickness, grain performance, etc. to distinguish; and combustion identification method, which is based on the smell of the skin when burning to distinguish. The above methods are simple and fast, and are most widely used, but the requirements for the identifier are high, and the identifier needs to have relevant knowledge and familiarity of animal skin, and there is no accurate standard and data support for the identification results, and the reliability needs to be improved.

[0004] To improve the accuracy and scientificity of cortex identification, many technology-related identification methods have been proposed and applied. For example, the drop test using the different water absorption of the skin and the tension and elasticity test using the difference in elasticity. More accurate methods include leather microscope identification method: by comparing the cross-section image of the material under the microscope or electron microscope with the cross-section mirror image of the known species leather sample, observing the morphological characteristics of the skin plate fibers and artificial materials and textile materials, and identifying the material of the test sample; infrared spectroscopy: using the difference in the type and composition ratio of amino acids in the skin, using continuous wavelength infrared light to irradiate the surface of the skin sample, causing molecular vibration and energy level transition, and obtaining different spectra in the infrared absorption spectrum; DNA identification method: extracting the DNA of animal origin in the skin, designing primers for specific gene sequences of species, amplifying the mitochondrial endogenous gene by PCR, obtaining the gene sequence of the target species, and determining the animal origin component in the skin according to the amplification product by fluorescence quantitative PCR method or sequencing comparison molecular biology technology. These methods are more accurate and scientific than sensory identification, but the addition of chemicals during skin processing can have unpredictable effects on the above methods, such as pattern damage, infrared interference, DNA degradation, etc., making it difficult to operate. Therefore, it is necessary to propose more detailed identification methods.

[0005] Summary of the Invention Bos taurus (SEQ ID NO. 1), Ovis aries (SEQ ID NO. 2), Capra hircus (SEQ ID NO. 3), Sus scrofa (SEQ ID NO. 4)

[0006] The purpose of the present application is to solve the technical problems existing in the prior art, and provide a goat skin species identification method based on immune antibody technology, which is realized by the following steps:

[0007] (1) Preparation of goat specific antibody; the specific protein used for antibody preparation is: NHS actin remodeling regulatory protein, and the amino acid sequence of the NHS actin remodeling regulatory protein is shown in SEQ NO. 3: CISLSDLGKSGSGSN;

[0008] (2) Protein extraction and sample preparation of domestic pig skin, domestic cow skin, goat skin and sheep skin samples;

[0009] (3) The sample prepared from the extracted protein is subjected to polyacrylamide gel electrophoresis, and the goat specific antibody is incubated to detect the band of the sample, so as to determine the species of domestic pig skin, domestic cow skin, goat skin and sheep skin.

[0010] The goat specific antibody of step (1) is prepared by the following method:

[0011] According to the protein group results of bovine skin, sheep skin and pig skin, the protein sequences of each detected protein were compared with the protein sequences of Sus scrofa domesticus, Bos taurus and Ovis aries, and candidate proteins with specific protein sequences were screened, i.e., the protein sequence comparison results were significantly different among the three,

[0012] Protein name Gene name Uniprot Accessions Antibody name NHS actin remodelling regulatory protein NHS A0A452EPD8 Goat specific antibody

[0013] The specific sequence suitable for antibody preparation screened by protein sequence comparison is: NHS actin remodeling regulatory protein: CISLSDLGKSGSGSN (goat specific sequence).

[0014] The specific steps for preparing "goat specific antibody" are as follows:

[0015] (1) Synthesize the polypeptide sequence "CISLSDLGKSGSGSN" using a polypeptide synthesizer, and couple the polypeptide with keyhole limpet hemocyanin (KLH) to obtain a polypeptide antigen;

[0016] (2) Dilute the polypeptide antigen with physiological saline to 250 μg / 500 μl·rabbit, and mix with an equal volume of complete Freund's adjuvant (CFA) to inject healthy New Zealand rabbits for the first immunization;

[0017] (3) Two weeks later, dilute the polypeptide antigen with physiological saline to 125 μg / 500 μl·rabbit, and mix with an equal volume of incomplete Freund's adjuvant (IFA) to inject New Zealand rabbits for the second immunization;

[0018] (4) Two weeks later, dilute the polypeptide antigen with physiological saline to 250 μg / 500 μl·rabbit, and mix with an equal volume of adjuvant IFA to inject New Zealand rabbits every two weeks for the third to sixth immunizations;

[0019] (5) Collect the blood of the immunized rabbits, and allow the blood clot to fully contract and the antiserum to fully separate, then collect the antiserum and purify to obtain the antibody.

[0020] The sample protein extraction and sample preparation method of step (2) are as follows:

[0021] (1) Collect the skin sample to be tested;

[0022] (2) In a 1.5 ml centrifuge tube, weigh 100 mg of the collected sample to be tested, cut it into small pieces, add 400 μl of RIPA lysis buffer and 4 μl of protease inhibitor PMSF, add 2-3 zirconium oxide grinding beads, and then use a grinder to grind for 5 min, and then take it out and place it on ice for 30 min;

[0023] (3) Centrifuge the sample at 12000 rpm, 4°C for 5 min, and transfer the supernatant to a new 1.5 ml centrifuge tube and store on ice;

[0024] (4) Detect the protein concentration of the sample using the BCA kit, and add 20 μl of 0, 0.1, 0.5, 1, and 2 mg / ml protein standard solution and sample supernatant to each well of a 96-well plate, then add 200 μl of prepared working solution to each well, and place the 96-well plate in a 37°C incubator for 8-10 min;

[0025] (5) Using a fluorescence spectrophotometer, absorbance is 562 nm, and the standard curve is calculated, the above 96-well plate is placed in the fluorescence spectrophotometer to detect the absorbance, and the corresponding protein concentration of each sample can be obtained on the standard curve according to the measured absorbance value of the sample;

[0026] (6) Correct the concentration of each sample according to the formula: sample amount (μl) = 100 * minimum concentration / sample concentration, PBS amount (μl) = 100-sample amount, and calculate the required sample stock solution content and PBS dilution amount for each sample concentration correction, and dilute the sample to obtain a standardized sample protein sample of 100 μl;

[0027] (7) Add 100 μl of 2x SDS containing cationic denaturing detergent to each sample in a 1:1 ratio, and boil in boiling water for 5 min, then cool on ice, and store at -30°C.

[0028] The method of step (3) using goat-specific antibody for polyacrylamide gel electrophoresis is as follows:

[0029] (1) Extract the protein from the leather to be tested, and prepare a polyacrylamide gel electrophoresis (PAGE) sample;

[0030] (2) Prepare an 8% polyacrylamide gel, add protein marker and the above sample, and start electrophoresis, with a protein marker and sample loading amount of 5 μl and 10 μl, respectively;

[0031] (3) The above electrophoresis conditions are set to a constant current mode with a voltage of 200 V, a current of gel block number x 20 mA, and a time of 60-75 min;

[0032] (4) After electrophoresis, use a membrane transfer instrument to transfer the strip from the electrophoresis gel to a PVDF membrane with a pore size of 0.45 μm, and block with skim milk blocking solution for 3-5 h;

[0033] (5) Discard the blocking solution, and add the corresponding antibody diluent for primary antibody incubation;

[0034] (6)Discard the first antibody diluent, and after washing with TBST, add a horseradish enzyme-labeled rabbit secondary antibody diluent to incubate the secondary antibody;

[0035] (7)Discard the secondary antibody, and after washing with TBST, view the result bands through ECL development.

[0036] The above goat skin species identification method based on the immune antibody technology uses "goat specific antibody", and the standard for judging whether the skin is a certain goat skin is:

[0037] The goat specific antibody: according to the above steps, polyacrylamide gel electrophoresis is carried out, no band is generated in the 100-180kDa interval of the pig skin sample, the cow skin sample and the sheep sample, and one band is generated in the goat sample.

[0038] The above polyacrylamide gel electrophoresis detection result determines whether the test skin is a certain goat skin, and the determination standard is:

[0039] The same sample is subjected to polyacrylamide gel electrophoresis with the goat specific antibody, and no band is generated in the 100-180kDa interval after incubation with the goat specific antibody, and it is determined that the leather is not goat skin.

[0040] The same sample is subjected to polyacrylamide gel electrophoresis with the goat specific antibody, and one band is generated in the 100-180kDa interval after incubation with the goat specific antibody, and it is determined that the leather is goat skin.

[0041] The same sample is subjected to polyacrylamide gel electrophoresis with the goat specific antibody, and multiple bands or trailing bands are generated in the 100-180kDa interval after incubation with the goat specific antibody, and the sample should be prepared again for polyacrylamide gel electrophoresis, and the result after re-electrophoresis is determined again according to the above conditions.

[0042] Another object of the present application is to provide the application of the method in goat skin species identification.

[0043] The present application provides a specific antibody and a specific detection method for identifying goat skin based on immune antibody technology, which has good sensitivity and specificity, and can quickly and accurately identify whether the cortex is goat skin by combining the determination method provided by the present application. The method provided by the present application identifies the specific protein of goat skin through proteomics, prepares polyclonal antibodies according to the specific protein sequence, uses immune antibody technology to make the specific antibody combine with the natural antigen in the cortex, and finally identifies whether the test skin is goat skin through result analysis. The method has the characteristics of high accuracy, low equipment requirement, strong specificity and short detection period compared with other methods, and can be used for quickly identifying whether the skin is goat skin. BRIEF DESCRIPTION OF DRAWINGS

[0044] Figure 1is the sequence alignment of the bovine (SEQ ID NO. 1), sheep (SEQ ID NO. 2), goat (SEQ ID NO. 3), and pig (SEQ ID NO. 4) NHS actin remodeling regulatory protein sequences in Example 1 of the present application. The highlighted part is the goat-specific sequence.

[0045] Figure 2 is a graph showing the detection of the titer of the antiserum after the third immunization in the preparation of the goat-specific antibody in Example 1 of the present application.

[0046] Figure 3 is a polyacrylamide gel electrophoresis result graph for verifying the accuracy of the goat-specific antibody "goat-specific antibody" in Example 1 of the present application; wherein 1 is pigskin; 2 is cowhide; 3 is goat skin; and 4 is sheepskin.

[0047] Figure 4 is a polyacrylamide gel electrophoresis result graph for verifying the accuracy of the goat-specific antibody after different concentrations of acid-base pollution in Example 2 of the present application; wherein 1 is pigskin; 2 is cowhide; 3 is goat skin; and 4 is sheepskin. DETAILED DESCRIPTION

[0048] The present application will be further described in detail below in conjunction with the accompanying drawings and examples.

[0049] Example 1: Verification of the accuracy of the goat-specific antibody

[0050] 1. Preparation of goat-specific antibody

[0051] According to the analysis results of the pig, cow, and sheep skin proteomes, the Uniprot database (www.uniprot.org) was used to find the protein sequences of the pig (Sus scrofa domesticus), cow (Bos taurus), sheep (Ovis aries), and goat (Capra hircus) for each detected protein, and protein sequence alignment was performed to screen out candidate proteins with significant sequence differences. Among them, the protein NHS actin remodeling regulatory protein (NHS) with obvious differences was designed as a specific sequence suitable for antibody preparation: NHS actin remodeling regulatory protein: CISLSDLGKSGSGSN (goat-specific sequence).

[0052] A polypeptide synthesizer was used to synthesize the "CISLSDLGKSGSGSN" hapten, and a cysteine was added at the N-terminus of the hapten to couple the polypeptide with the keyhole limpet hemocyanin (KLH) to obtain a complete antigen.

[0053] The polypeptide antigen is diluted with physiological saline to 250 μg / 500 μl·rabbit, mixed with an equal volume of complete Freund's adjuvant (CFA), and emulsified with a solution of streptomycin and penicillin. A healthy New Zealand rabbit is selected for the first immunization. Before immunization, a rabbit ear blood sample is taken for comparison.

[0054] After two weeks, the polypeptide antigen is diluted with physiological saline to 125 μg / 500 μl·rabbit, mixed with an equal volume of incomplete Freund's adjuvant (IFA), and injected into a New Zealand rabbit for the second immunization.

[0055] After two weeks, the polypeptide antigen is diluted with physiological saline to 250 μg / 500 μl·rabbit, mixed with an equal volume of adjuvant IFA, and injected into a New Zealand rabbit every two weeks for the third to sixth immunizations. Ten days after the third immunization, the rabbit is intravenously bled to test the titer of the antiserum.

[0056] After six immunizations, the rabbit is killed, and a large amount of blood is collected at room temperature to coagulate. The coagulated blood clot is placed at 37°C for 30 min, then transferred to 4°C overnight to allow the blood clot to fully shrink and the antiserum to fully separate out. The antiserum is then collected at 4°C, centrifuged at 3000g for 10 min to obtain the supernatant, which is stored at -70°C for later use.

[0057] The antiserum is purified by immunoaffinity chromatography. The synthesized hapten (i.e., the polypeptide "CISLSDLGKSGSGSN") is cross-linked to the chromatography filler Sulfo-link-gel to block the N-terminal cysteine of the polypeptide. The column is pretreated with PBS (20 ml, 50 mM, pH 7.4) at a flow rate of 60 ml / h. The 10 ml antiserum is diluted one-fold with PBS (50 mM, pH 7.4), and the diluted sample is loaded and repeated once. The column is washed with PBS at a flow rate of 60 ml / h. The glycine-HCL (pH 3.0, 0.1 M) is used to elute the antibodies to obtain the purified antibodies, which are stored at 4°C.

[0058] 2. Sample protein extraction and sample preparation

[0059] (1) Pig skin, cow skin, goat skin, and sheep skin samples are collected from three different places.

[0060] (2) In a 1.5 ml centrifuge tube, 100 mg of pig skin, cow skin, goat skin, and sheep skin collected from different places (sample site ①, sample site ②, and sample site ③) are weighed, cut into small pieces, and added with 400 μl of RIPA lysis buffer and 4 μl of protease inhibitor PMSF. After adding 2-3 zirconium oxide grinding beads, the mixture is ground for 5 min using a grinder, and then placed on ice for 30 min.

[0061] (3) Centrifuge the sample at 12000 rpm, 4℃ for 5 min, and take the supernatant to a new 1.5 ml centrifuge tube and store on ice;

[0062] (4) Detect the protein concentration of the sample using the BCA kit, take 20 μl of 0, 0.1, 0.5, 1, 2 mg / ml protein standard solution and sample supernatant into a 96-well plate, then add 200 μl of prepared working solution to each well, and place the 96-well plate in a 37℃ incubator for 8-10 min after covering the lid;

[0063] (5) Use a fluorescence spectrophotometer with an absorbance of 562 nm, add a standard curve to calculate, and place the above 96-well plate into the fluorescence spectrophotometer to detect the absorbance, and according to the measured absorbance of the sample, the corresponding protein concentration of each sample can be obtained on the standard curve;

[0064] (6) Correct the concentration of each sample according to the formula: sample amount (μl) = 100 * minimum concentration / sample concentration, PBS amount (μl) = 100-sample amount, and calculate the required sample stock solution content and PBS dilution amount for each sample concentration correction, and dilute the sample to obtain 100 μl of standardized sample protein with uniform concentration;

[0065] (7) Add 100 μl of 2x SDS containing cationic denaturing detergent to each sample in a 1:1 ratio, and boil in boiling water for 5 min, then cool on ice and store at -30℃.

[0066] 3. Polyacrylamide gel electrophoresis

[0067] (1) Prepare 8% polyacrylamide gel electrophoresis gel

[0068] The 8% gel formula is shown in Table 1:

[0069]

[0070] ① Assemble the gel preparation device, clamp the glass plate on the gel preparation frame, and tightly attach the bottom to the sponge rubber strip;

[0071] ② Prepare 8% separation gel according to the above separation gel formula, mix thoroughly, and carefully pour the separation gel solution into the assembled glass plate to two-thirds of the way with a Pasteur pipette;

[0072] ③ Add ddH2O to the surface of the separation gel to seal the separation gel and make the surface flat with a Pasteur pipette, and let it stand at room temperature for about 30-60 min, and solidify in a 37℃ incubator for about 20 min;

[0073] (4) Use the above formula to prepare concentrated glue, mix well, first tilt to discard the upper layer of the separation glue ddH2O, use clean filter paper to absorb the remaining ddH2O on the edge of the glass plate, then use a Pasteur pipette to pour the concentrated glue solution into the upper layer of the separation glue, insert the 15-hole sample comb, make sure there are no air bubbles in the gel or around the comb teeth, stand at room temperature for about 20-30 min, and solidify in a 37°C incubator for about 5-10 min;

[0074] (5) The solidified glue is placed in a sealed bag with a small amount of pure water and stored at 4°C.

[0075] (2) Polyacrylamide gel electrophoresis specific operation method

[0076] (1) Assemble the electrophoresis tank: place the gel plate vertically against the power supply stand in the electrophoresis tank, with the concave side of the gel plate facing the power supply stand. Usually two gel plates share one power supply stand. Fix the gel plate and power supply stand in the power supply tank, add 1x SDS electrophoresis buffer. Gently pull out the comb in the gel plate;

[0077] (2) After centrifugation at 12000 rpm, 4°C for 5 min, place the prepared sample on ice, use a 10 μl pipette to suck up the protein marker and sample liquid respectively, slowly add them to the concave part of the gel plate (sample point entry), the protein marker loading amount is 5 μl, the sample loading amount is 10 μl, pay attention not to scatter the sample;

[0078] (3) Electrophoresis: connect the electrophoresis tank and the electrophoresis instrument with two wires, pay attention to the plug and socket of red and black electrodes. Turn on the power supply and set it to constant current mode, voltage to 200v, current according to the number of gel blocks (current mA = gel block number * 20), electrophoresis time is 50-60 min, generally stop electrophoresis when bromophenol blue reaches the bottom of the gel;

[0079] (4) Transfer: After the completion of the electrophoresis, the two glass plates of the gel were gently pried apart with a thin plate, and the gel was poured onto one of the glass plates. According to the size of the band of interest, the gel was cut and transferred to the transfer buffer. A PVDF membrane with a pore size of 0.45 pm was cut to the same size as the gel block, soaked in methanol for 10 s, and placed on the PVDF membrane. The semi-dry transfer sandwich was soaked in the transfer buffer and squeezed to semi-dryness. The semi-dry transfer sandwich was placed on the semi-dry transfer instrument, and the transfer device was placed from bottom to top in the order of semi-dry transfer sandwich gasket, PVDF membrane, gel block, semi-dry transfer sandwich gasket. Air bubbles must be excluded, especially between the gel and the membrane. The transfer instrument cover was closed, and the transfer current was set to constant current, 0.16 A, 2 W, 25 V, and the time was about 20-45 min (the time required was different for different protein sizes, and the degree of transfer could be judged according to the residual marker). After the transfer was completed, the membrane was removed and placed in an incubation box, 10 ml of 5% skim milk blocking solution (5 g of skim milk powder + 100 ml of TBST) was added, and the blocking was performed at 4°C for 3-5 h or at room temperature for 1 h with shaking;

[0080] (5) Incubation of primary antibody: The goat-specific antibody was diluted in 10 ml of 5% skim milk blocking solution at a ratio of 1:1000 to obtain the primary antibody. After the blocking solution was completely absorbed, the diluted primary antibody was immediately added, and the incubation was performed at room temperature for 1 h with slow shaking on a shaker or overnight at 4°C;

[0081] (6) Incubation of secondary antibody: The rabbit anti-horseradish peroxidase-labeled secondary antibody was diluted in 10 ml of 5% skim milk blocking solution at a ratio of 1:4000 to obtain the secondary antibody. After the primary antibody was recovered, 10 ml of TBST was added to the incubation box, and the PVDF membrane was washed on a shaker for 5 min, repeated 3 times, and then the secondary antibody was immediately added. The incubation was performed at room temperature for 1 h with slow shaking on a shaker;

[0082] (7) Chemiluminescence: After the incubation was completed, the secondary antibody was removed, 10 ml of TBST was added to the incubation box, and the PVDF membrane was washed on a shaker for 5 min, repeated 3-5 times. The A and B solutions of the ECL kit were mixed in a light-proof centrifuge tube at a ratio of 1:1, and the PVDF membrane was placed on the operating table after the ECL instrument was cooled to -30°C. 200 pl of the luminescent solution was taken with a pipette to cover the PVDF membrane, and the image was exposed and collected on the ECL luminescence instrument.

[0083] 4. Result determination:

[0084] The same sample was subjected to polyacrylamide gel electrophoresis with goat-specific antibody, and there was no band in the 100-180 kDa interval after the goat-specific antibody incubation, indicating that the leather was not goat leather.

[0085] The same sample is subjected to polyacrylamide gel electrophoresis with goat-specific antibody, and a band is generated in the 100-180 kDa interval after goat-specific antibody incubation, and the leather is determined to be goat leather;

[0086] The same sample is subjected to polyacrylamide gel electrophoresis with goat-specific antibody, and a band is generated in the 100-180 kDa interval after goat-specific antibody incubation, and the leather is determined to be goat leather;

[0087] 5. Detection results:

[0088] Referring to Figure 1 The pig leather, cow leather, goat leather and sheep leather samples are detected by using the goat-specific antibody provided by the application, the protein of the sample to be detected is extracted and sampled, and then subjected to polyacrylamide gel electrophoresis; only the goat leather sample generates a band in the 100-180 kDa interval after goat-specific antibody incubation, and no band is generated for the pig leather, cow leather and sheep leather samples, which is consistent with the actual situation. The embodiment shows that the goat-specific detection antibody provided by the application can well identify goat leather.

[0089] Example 2: Accuracy verification of goat leather-specific antibody under acid-base pollution

[0090] 1. Preparation of goat-specific antibody

[0091] The polypeptide synthesizer is used to synthesize a hapten "CISLSDLGKSGSGSN", and a cysteine is added at the N-terminus of the hapten to couple the polypeptide with keyhole limpet hemocyanin (KLH) to obtain a complete antigen;

[0092] The polypeptide antigen is diluted with physiological saline to 250 μg / 500 μl·rabbit, mixed with an equal volume of complete Freund's adjuvant (CFA), and then emulsified by adding a streptomycin and penicillin solution, and a healthy New Zealand rabbit is selected for the first immunization. Rabbit ear blood sample is taken before immunization for comparison;

[0093] Two weeks later, the polypeptide antigen is diluted with physiological saline to 125 μg / 500 μl·rabbit, mixed with an equal volume of incomplete Freund's adjuvant (IFA), and injected into the New Zealand rabbit for the second immunization;

[0094] Two weeks later, the polypeptide antigen is diluted with physiological saline to 250 μg / 500 μl·rabbit, mixed with an equal volume of adjuvant IFA, and injected into the New Zealand rabbit every two weeks for the third to sixth immunizations. The antiserum titer is detected by intravenous blood sampling on the tenth day after the third immunization;

[0095] After the sixth immunization, the rabbits were killed and the whole blood was collected. The blood was allowed to clot at room temperature, and the clot was incubated at 37°C for 30 min and then transferred to 4°C overnight to allow the clot to shrink and the antiserum to be completely separated. The antiserum was collected and centrifuged at 3000g for 10 min at 4°C to obtain the supernatant, which was stored at -70°C for later use.

[0096] The antiserum was purified by immunoaffinity chromatography. The synthesized hapten (i.e., the polypeptide "CISLSDLGKSGSGSN") was cross-linked to the chromatography filler Sulfo-link-gel to block the N-terminal cysteine of the polypeptide. The column was pretreated with PBS (20 ml, 50 mM, pH 7.4) at a flow rate of 60 ml / h. The antiserum was diluted 1:1 with PBS (50 mM, pH 7.4), and the diluted sample was loaded onto the column and repeated once. The column was washed with PBS at a flow rate of 60 ml / h. The antibody was eluted with glycine-HCL (pH 3.0, 0.1 M) to obtain the purified antibody, which was stored at 4°C.

[0097] 2. Acid-base contamination treatment of the sample

[0098] (1) Contamination with NaOH solutions of different concentrations:

[0099] NaOH solutions of 0.1%, 1%, and 5% were prepared, and the fresh skins of pigs, cattle, goats, and sheep were soaked in the solutions for 6 h.

[0100] (2) Contamination with HCl solutions of different concentrations:

[0101] HCl solutions of 0.1%, 1%, and 5% were prepared, and the fresh skins of pigs, cattle, goats, and sheep were soaked in the solutions for 6 h.

[0102] 3. Protein extraction and sample preparation

[0103] (1) 100 mg of the treated pig skin, cattle skin, sheep skin, and goat skin were weighed into 1.5 ml centrifuge tubes, respectively, and cut into small pieces. RIPA lysis buffer (400 μl) and protease inhibitor PMSF (4 μl) were added, and 2-3 zirconium oxide grinding beads were added. The mixture was ground using a grinder for 5 min, and then placed on ice for 30 min.

[0104] (2) The sample was centrifuged at 12000 rpm and 4°C for 5 min, and the supernatant was transferred to a new 1.5 ml centrifuge tube and stored on ice.

[0105] (3) Using BCA kit to detect sample protein concentration, 0, 0.1, 0.5, 1, 2 mg / ml of protein standard solution and sample supernatant were each taken 20 μl into 96-well plate, and then 200 μl of prepared working solution was added to each well. After covering the lid, the 96-well plate was placed in a 37°C incubator for 8-10 min;

[0106] (4) Using a fluorescence spectrophotometer, the absorbance was 562 nm, and the standard curve was added. The above 96-well plate was placed in a fluorescence spectrophotometer to detect the absorbance. According to the measured absorbance of the sample, the corresponding protein concentration of each sample could be obtained on the standard curve;

[0107] (5) Correct the concentration of each sample according to the formula: sample amount (μl) = 100 * minimum concentration / sample concentration, PBS amount (μl) = 100-sample amount. The sample original liquid content and PBS dilution required for concentration correction of each sample were calculated respectively, and the standardized sample protein sample 100 μl after uniform concentration was obtained by diluting the sample;

[0108] (6) According to the ratio of 1:1, 100 μl of 2x SDS containing cationic denaturing detergent was added to each sample, and boiled in boiling water for 5 min. After cooling on ice, it was stored at -30°C.

[0109] 4. Polyacrylamide gel electrophoresis

[0110] (1) Preparation of 8% polyacrylamide gel electrophoresis gel

[0111] 8% gel formula is as follows Table 2:

[0112]

[0113] The specific operation is as follows:

[0114] ① Assemble the gel preparation device, clamp the glass plate on the gel preparation frame, and tightly paste the bottom with the sponge rubber strip;

[0115] ② According to the above separation gel formula, prepare 8% separation gel, mix thoroughly, and then carefully pour the separation gel solution into the assembled glass plate to two-thirds with a Pasteur pipette;

[0116] ③ Add ddH2O liquid to the surface of the separation gel with a Pasteur pipette to seal the separation gel and make the surface flat. After standing at room temperature for 30-60 min, solidify in a 37°C incubator for about 20 min;

[0117] (4) Use the above formula to prepare concentrated glue, mix well, first tilt to discard the upper layer of the separation glue ddH2O, use clean filter paper to absorb the remaining ddH2O on the edge of the glass plate, then use a Pasteur pipette to pour the concentrated glue solution into the upper layer of the separation glue, insert the 15-hole sample comb, make sure there are no air bubbles in the gel or around the comb teeth, stand at room temperature for about 20-30 min, and solidify in a 37°C incubator for about 5-10 min;

[0118] (5) The solidified glue is placed in a sealed bag with a small amount of pure water and stored at 4°C.

[0119] (2) Polyacrylamide gel electrophoresis specific operation method

[0120] (1) Assemble the electrophoresis tank: place the gel plate vertically against the power supply stand in the electrophoresis tank, with the concave side of the gel plate facing the power supply stand. Usually two gel plates share one power supply stand. Fix the gel plate and power supply stand in the power supply tank, add 1x SDS electrophoresis buffer. Gently pull out the comb in the gel plate.

[0121] (2) After centrifugation at 12000 rpm and 4°C for 5 min, place the prepared sample on ice, use a 10 μl pipette to extract the protein marker and sample liquid, slowly add them to the concave part of the gel plate (sample point), the protein marker loading amount is 5 μl, the sample loading amount is 10 μl, pay attention not to scatter the sample;

[0122] (3) Electrophoresis: connect the electrophoresis tank and the electrophoresis instrument with two wires, pay attention to the plug and socket of the red and black electrodes. Turn on the power supply and set it to constant current mode, voltage to 200v, current according to the number of gel blocks (current mA = gel block number * 20), electrophoresis time is 50-60 min, generally stop electrophoresis when the bromophenol blue reaches the bottom of the gel;

[0123] (4) Transfer: After the completion of the electrophoresis, the two glass plates of the gel were gently pried apart with a thin plate, and the gel was poured onto one of the glass plates. According to the size of the band of interest, the corresponding region of the gel was cut off and transferred to the transfer buffer. A PVDF membrane with a pore size of 0.45 μm was cut to the same size as the gel block, soaked in methanol for 10 s, and placed on the PVDF membrane. The semi-dry transfer sandwich was fully soaked in the transfer buffer and then squeezed to semi-dryness. The semi-dry transfer sandwich was placed on the semi-dry transfer instrument, and the transfer device was placed from bottom to top in the order of semi-dry transfer sandwich gasket, PVDF membrane, gel block, semi-dry transfer sandwich gasket. Air bubbles must be excluded during placement, especially between the gel and the membrane. The transfer instrument cover was closed, and the transfer current was set to constant current, 0.16 A, 2 W, 25 V, and the time required was about 20-45 min (the time required varies depending on the size of the protein, and the degree of transfer can be determined according to the residual marker). After the transfer was completed, the membrane was removed and placed in an incubation box, 10 ml of 5% skim milk blocking solution (5 g of skim milk powder + 100 ml of TBST) was added, and the blocking was performed at 4°C for 3-5 h or at room temperature for 1 h with shaking;

[0124] (5) Incubation of primary antibody: The goat-specific antibody was diluted in 10 ml of 5% skim milk blocking solution at a ratio of 1:1000 to obtain the primary antibody. After the blocking solution was completely absorbed, the diluted primary antibody was immediately added, and the incubation was performed at room temperature for 1 h with slow shaking on a shaker or overnight at 4°C;

[0125] (6) Incubation of secondary antibody: The rabbit anti-horseradish peroxidase-labeled secondary antibody was diluted in 10 ml of 5% skim milk blocking solution at a ratio of 1:4000 to obtain the secondary antibody. After the primary antibody was recovered, 10 ml of TBST was added to the incubation box, and the PVDF membrane was washed on a shaker for 5 min, repeated 3 times, and then the secondary antibody was immediately added. The incubation was performed at room temperature for 1 h with slow shaking on a shaker;

[0126] (7) Chemiluminescence: After the incubation was completed, the secondary antibody was removed, 10 ml of TBST was added to the incubation box, and the PVDF membrane was washed on a shaker for 5 min, repeated 3-5 times. The A and B solutions of the ECL kit were mixed in a light-proof centrifuge tube at a ratio of 1:1, and the PVDF membrane was placed on the operating table after the ECL instrument was cooled to -30°C. 200 μl of the luminescent solution was taken with a pipette to cover the PVDF membrane, and the image was exposed and collected on the ECL luminescence instrument.

[0127] 5. Result determination:

[0128] The same sample was subjected to polyacrylamide gel electrophoresis with goat-specific antibody, and there was no band in the 100-180 kDa region after incubation with goat-specific antibody, indicating that the leather was not goat leather.

[0129] The same sample is subjected to polyacrylamide gel electrophoresis with goat-specific antibody, and a band is generated in the 100-180 kDa interval after goat-specific antibody incubation, and the leather is determined to be goat leather;

[0130] The same sample is subjected to polyacrylamide gel electrophoresis with goat-specific antibody, and a band is generated in the 100-180 kDa interval after goat-specific antibody incubation, and the leather is determined to be goat leather;

[0131] 6. Detection result:

[0132] The goat leather-specific antibody provided in the application is used to detect pig leather, cow leather, goat leather and sheep leather samples contaminated by different concentrations of acid and alkali, and the proteins of the samples to be detected are extracted to prepare samples, and then subjected to polyacrylamide gel electrophoresis; only the goat leather sample after treatment generates a band in the 100-180 kDa interval after goat-specific antibody incubation, and the pig leather, cow leather and sheep leather samples after treatment do not generate bands, which is consistent with the actual situation. Example 2 shows that the goat leather-specific detection antibody provided in the application can still identify goat leather well under acid and alkali contamination.

Claims

1. Use of a specific antibody for goat skin species identification in the preparation of a goat skin species identification reagent, characterized in that: The antibody is prepared by polypeptide immunization, the polypeptide is derived from goat NHS actin remodeling regulatory protein, and the amino acid sequence of the polypeptide is CISLSDLGKSGSGSN.

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