Sericin antigen, sericin antibody and application

Abstract

The present invention relates to the technical field of sericin detection, and provides a sericin antigen, sericin antibody and application thereof. The sericin antigen includes sericin X1, sericin X3, sericin X10, sericin X12 and sericin X9; the sericin antibody is obtained by mixing the sericin antigen with an adjuvant in equal volumes, injecting it subcutaneously into a rabbit, immunizing it, taking blood and separating serum. The sericin antibody in the present invention can accurately detect five types of sericins, and has good specificity and high detection sensitivity, and can detect trace sericin components at the level of 0.01ng.

Description

Technical Field

[0001] The present invention relates to the technical field of sericin detection, and in particular to sericin antigens, sericin antibodies and applications. Background Art

[0002] Silk production and utilization has a long history in China. In addition to being used as a textile, silk has also been developed and utilized as high-tech materials such as cosmetics, food additives, and biomedicine. The main components of silk include sericin and fibroin. The outer part of silk fiber is sericin, which accounts for about 20% of the total silk protein; the inner fiber is fibroin. Although sericin and fibroin belong to silk fiber protein, they are both natural protein materials and are rich in 18 kinds of amino acids, but their properties are different due to different protein compositions. Among them, the main component of sericin fiber is sericin, and it contains a variety of sericin molecules. Sericin is easily soluble in water and easy to form colloids, while fibroin requires saline and alkali solutions to dissolve, and its gelling ability after dissolution is slightly poor. Sericin is considered to be an ideal natural moisturizing factor with antioxidant, sunscreen and whitening properties. It has been used in the cosmetics field as an additive to enhance the efficacy of cosmetics.

[0003] Since there are many methods for extracting silk protein from silk cocoons, different manufacturers use different saline-alkali solutions or enzymatic reactions for extraction, which damage silk protein to varying degrees, so that the silk protein raw material products produced are not all macromolecular silk protein, but substances hydrolyzed into amino acid forms. At present, the sericin added to cosmetics mainly consists of two raw materials: sericin peptides or hydrolyzed amino acids. Since sericin hydrolyzed into amino acids does not have the characteristics of high-molecular sericin peptides, adding hydrolyzed amino acid sericin ingredients cannot effectively improve the performance of cosmetics. Therefore, being able to detect and distinguish whether sericin peptides are added to cosmetics is of great significance for product quality testing.

[0004] Protein polyclonal antibody technology is widely used. The antigen required for antibody preparation can be a macromolecular protein or a peptide, so the antibody can only detect proteins or protein peptides, and cannot detect amino acid products. On the other hand, the antibody obtained with a single peptide as an antigen can only recognize the peptide or the protein molecule containing the peptide. For protein samples with complex protein molecular composition such as sericin, the coverage of the antibody obtained with a single peptide or a single protein molecule as an antigen is small, resulting in inaccurate test results. Summary of the invention

[0005] In order to solve the above technical problems, the present invention provides sericin antigens, sericin antibodies and applications, which can accurately detect five different sericins at the same time.

[0006] The first aspect of the present invention provides a sericin antigen, wherein the sericin antigen is a sericin liquid;

[0007] The sericin liquid includes sericin X1, sericin X3, sericin X10, sericin X12 and sericin X9;

[0008] The amino acid sequence of the sericin X1 is shown in SEQ ID NO.1, the amino acid sequence of the sericin X3 is shown in SEQ ID NO.2, the amino acid sequence of the sericin X10 is shown in SEQ ID NO.3, the amino acid sequence of the sericin X12 is shown in SEQ ID NO.4, and the amino acid sequence of the sericin X9 is shown in SEQ ID NO.5;

[0009] The specific process of obtaining the sericin solution is as follows:

[0010] Soak silkworm cocoons with a mass volume ratio of 10 g: 200 mL in boiling water for 5 minutes, stirring continuously with a glass rod to remove the remaining undissolved silk cocoon fibers, which are the silk fibroin components. The sericin outside the silk is dissolved in water, reducing the damage to the sericin components, and obtaining a sericin stock solution;

[0011] The sericin stock solution was dissolved and diluted by adding 40 mL of phosphate solution for every 10 mL, and the solution was shaken for 2 hours to promote dissolution. The solution was dialyzed for desalination and diluted to 0.5 mg / mL with deionized water to obtain the sericin solution.

[0012] The sericin antigen in the present invention has 5 different sericins, and compared with the prior art which uses a single protein molecule as an antigen, it has a wider coverage.

[0013] The second aspect of the present invention provides a sericin antibody, which is obtained by mixing the sericin antigen and an adjuvant in equal volumes, injecting the mixture subcutaneously into a rabbit for immunization, collecting blood, and separating serum.

[0014] The sericin antibody obtained by using the sericin antigen of the present invention has good specificity, can effectively detect the presence of 5 different sericin components, and has high detection sensitivity, and can detect trace sericin components at the level of 0.01 ng.

[0015] In another preferred embodiment, the number of immunizations is 4 to 5 times, and the interval between two adjacent immunizations is 3 weeks; and the adjuvant is Freund's adjuvant.

[0016] The third aspect of the present invention provides the use of the sericin antibody in detecting sericin peptides.

[0017] A fourth aspect of the present invention provides a method for detecting sericin, comprising the following steps:

[0018] The sample to be tested is dropped onto the PVDF membrane, and a blocking solution is added for blocking. The sericin antibody is added for incubation, and the membrane is rinsed with a buffer solution. A goat anti-rabbit antibody is added for further incubation, and the membrane is rinsed with a buffer solution. The PVDF membrane is transferred to a glass plate, and after the luminescent liquid is added, if luminescence occurs, it indicates that the sample to be tested contains sericin, and if no luminescence occurs, it indicates that the sample to be tested does not contain sericin.

[0019] In another preferred embodiment, the volume ratio of the sericin antibody to the blocking solution is 1:5000; the volume ratio of the goat anti-rabbit antibody to the blocking solution is 1:5000.

[0020] In another preferred embodiment, the blocking solution is 10 wt% skim milk solution; and the buffer solution is a phosphate buffer solution containing 0.1 wt% Tween-20.

[0021] In another preferred embodiment, the incubation time is 1 h to 1.2 h.

[0022] In another preferred embodiment, the luminescent liquid is an ECL ultrasensitive luminescent liquid.

[0023] Compared with the prior art, the present invention has the following beneficial effects:

[0024] The sericin antigens in the present invention include five types of sericin. Compared with the prior art which uses a single protein as an antigen, the sericin antigens in the present invention have a wider coverage.

[0025] The sericin antibody obtained by using the sericin antigen of the present invention can detect 5 types of sericin, and has good specificity during the detection process, avoiding the interference of silk fibers and improving the detection accuracy. When the sericin antibody of the present invention is used to detect sericin, the detection sensitivity is high, and the sensitivity can detect trace sericin components at the level of 0.01ng.

[0026] The sericin antibody of the present invention can detect a variety of sericins, and thus can be applied to detect protein samples with complex molecular structures, and effectively detect the presence of sericin components. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 The polyacrylamide gel electrophoresis and Coomassie brilliant blue staining diagram in Example 2 of the present invention.

[0028] Figure 2 This is a diagram showing the specificity detection results of sericin antibodies in Example 3 of the present invention.

[0029] Figure 3This is a diagram showing the detection results of sericin peptides in a sample by the sericin antibody in Example 4 of the present invention.

[0030] Figure 4 This is a graph showing the detection results of sericin peptides in different samples using the sericin antibody in Example 5 of the present invention. DETAILED DESCRIPTION

[0031] The technical solutions in the present invention will be described clearly and completely below in conjunction with the specific embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0032] The methods described in the embodiments of the present invention are conventional methods unless otherwise specified. The materials, reagents, etc. used can be obtained from commercial sources unless otherwise specified.

[0033] The New Zealand experimental rabbits in the following examples were purchased from Jiangsu Academy of Agricultural Sciences.

[0034] Example 1: A sericin antibody.

[0035] The preparation process of sericin antibody is as follows:

[0036] S1. Soak silkworm cocoons with a mass volume ratio of 10 g: 200 mL in boiling water for 5 minutes. During this period, stir continuously with a glass rod to remove the remaining undissolved silk cocoon silk, which is the silk fibroin fiber component. The sericin protein outside the silk has been dissolved in water, reducing the damage to the sericin protein component, and obtaining the sericin protein stock solution.

[0037] S2. The sericin stock solution was dissolved and diluted by adding 40 mL of 1 M phosphate solution for every 10 mL of the sericin stock solution, and placed on a shaker at room temperature for 2 hours to promote dissolution. After desalting by dialysis, the sericin stock solution was diluted to 0.5 mg / mL with deionized water. The sericin solution, i.e., sericin antigen, can be further extracted from the sericin by the phosphate solution, and the sericin structure is further protected, thereby improving the extraction effect of the sericin.

[0038] S3. Take 500 μg of sericin antigen and mix it with Freund's adjuvant in a volume ratio of 1:1, and inject it subcutaneously into New Zealand experimental rabbits weighing 2.5 kg. The injection volume is 2 mL / time for immunization. The interval between two adjacent immunizations is 3 weeks. After a total of 5 immunizations, whole blood is collected from the rabbit heart, and the serum is separated to obtain sericin antibodies.

[0039] Example 2: Determination of sericin in sericin antigen.

[0040] Take 20 μL of the sericin solution in Example 1 and perform polyacrylamide gel electrophoresis and Coomassie brilliant blue staining. Figure 1 As shown, from Figure 1 It can be seen that sericin of different molecular weights in the sericin solution is diffusely distributed, with large molecular weight concentrated in the 55KDa-130KDa region, and the hydrolyzed small molecular weight sericin is more at 17KDa, so an obvious band can be seen at 17KDa. In order to further analyze the sericin solution in the sericin solution, the 17KDa and larger sericin strips were collected and then sequenced for proteins. The results are shown in Tables 1 to 4, and the main sericin components were determined.

[0041] Table 1 Protein sequencing results

[0042]

[0043] Table 2 Protein sequencing results

[0044]

[0045] Table 3 Protein sequencing results

[0046]

[0047] Table 4 Protein sequencing results

[0048]

[0049] From the results in Tables 1 to 4, it can be seen that the sum of the intensities of the five sericin peptides 1 to 5 in Table 1 are all above 400, and the credibility is all above 1600. The sum of the intensities of the other 65 detected protein peptides is all below 40, and the credibility is all below 65, accounting for less than 20% in total.

[0050] From the above results, it can be seen that among the 70 types of sericin proteins detected, only 5 types of sericin proteins have high peptide intensity and reliability, and account for a large proportion. Therefore, these five types of sericin proteins are considered to be the main components of the sericin liquid, and these 5 types of sericin proteins are named as sericin X1, sericin X3, sericin X10, sericin X12, and sericin X9 respectively.

[0051] Combined with the results in Tables 1 to 4, the proportions of the above five types of sericin proteins and the proportions of the remaining 65 types of sericin proteins were statistically analyzed to further illustrate that the five types of sericin proteins are the main components of the sericin liquid. The specific statistical results are shown in Table 5.

[0052] Table 5 Proportion of different sericin proteins

[0053]

[0054] As can be seen from Table 5, the main sericins in the sericin liquid are sericin X1, sericin X3, sericin X10, sericin X12, and sericin X9, which account for 81.98%. These five proteins were sequenced separately, and the results are as follows.

[0055] The sericin X1 is shown as SEQ ID NO.1, the sericin X3 is shown as SEQ ID NO.2, the sericin X10 is shown as SEQ ID NO.3, the sericin X12 is shown as SEQ ID NO.4, and the sericin X9 is shown as SEQ ID NO.5.

[0056]

[0057]

[0058]

[0059]

[0060]

[0061] Example 3: Specific detection of sericin by sericin antibodies.

[0062] The silk fiber component in Example 1 was washed with Na2CO3 solution for 30 minutes, fully washed with distilled water, and dried naturally to obtain silk fiber; the silk fiber was heated and dissolved with CaCl2 solution at a mass volume ratio of 1:25, the dissolution temperature was 98°C, and the time was 10 minutes; the silk fibroin solution obtained after dissolution was dialyzed and air-dried and concentrated, and diluted with deionized water to 0.5 mg / mL to obtain a silk fibroin solution. The obtained silk fibroin solution was used to detect the specificity of sericin antibody, and the specificity means that the obtained silk fibroin solution will not be interfered by the silk fibroin in the silk fibroin solution during the detection process.

[0063] The sericin solution obtained in Example 1 was diluted to 1 μg / mL, and further gradient diluted to obtain 0.1 μg / mL, 0.01 μg / mL, and 0.001 μg / mL sericin solutions for use. Cut 3 pieces of PVDF membrane with the same area, soak the membrane with methanol for 5 seconds, drop 1μL of each dilution on the PVDF membrane in turn, dry for 30 minutes, and then use 10% skim milk as blocking solution for 1 hour; discard the blocking solution, add 10mL of new blocking solution, add 2μL of sericin antibody, and incubate for 2 hours; discard the antibody blocking solution, add phosphate buffer containing 0.1% Tween-20 to rinse for 5 minutes, repeat 3 times; discard the phosphate buffer, add 10mL of new blocking solution, add 2μL of goat anti-rabbit antibody, and incubate for 1 hour; discard the antibody blocking solution, add PBST to rinse for 5 minutes, repeat 3 times; discard PBST, transfer the membrane to a glass plate, drop ECL supersensitive luminescent solution to cover the entire membrane, and finally place it in a chemiluminescence instrument to observe the luminescence. The results are as follows Figure 2 As shown, the obtained antibody can clearly detect the sericin sample, and its sensitivity can detect trace sericin components at the 0.01 ng level.

[0064] Example 4: Detection of sericin peptides in silk protein samples using sericin antibodies.

[0065] Four brands of silk protein raw materials purchased from the market were tested. The purchased products included silk fibroin and sericin products. The detection of silk fibroin was used to verify the specificity of the antibody for sericin detection. The comparative samples were the sericin solution obtained in Example 1, marked as WS, and the silk fibroin solution, marked as WF. The test samples were all diluted to 1 μg / mL, and 1 μL of each was dropped on the PVDF membrane. The subsequent steps were the same as described in Example 3. The results are shown in FIG. Figure 3 shown.

[0066] from Figure 3 It can be seen that the sericin antibody can clearly detect WS, but cannot detect WF, indicating that the prepared antibody has a strong specificity for sericin and can be used to specifically detect sericin components.

[0067] Among the four silk products tested, the silk peptide product of brand 1 is recorded as Y1, the silk peptide product of brand 2 is recorded as Y2, the silk powder product of brand 2 in 2018 is recorded as Y3, and the silk powder product of brand 2 in 2021 is recorded as Y4. The results show that no sericin was detected in the silk peptide products of Y1 and Y2, indicating that these two products do not contain sericin; sericin can be detected in Y3, but not in Y4, which may be that the manufacturer has optimized the production process, and the 2021 product does not contain mixed sericin. The detection of these silk protein raw material products shows that the sericin antibody in Example 1 of the present invention can well distinguish between silk and sericin components, and can be used for the detection of sericin components in raw material products.

[0068] The test results of different brands and products are as follows:

[0069] The sericin product of brand 1 is denoted as Y5, the sericin peptide product of brand 3 is denoted as Y6, the sericin product of brand 3 is denoted as Y7, and the sericin peptide liquid of brand 4 is denoted as Y8.

[0070] No sericin was detected in products Y6 and Y7, indicating that these products do not contain sericin or are only hydrolyzed amino acid sericin products; while trace sericin was detected in Y5, indicating that the sericin or sericin peptide components in this product are relatively small, which may be related to the strong degree of damage to sericin in the production process; in contrast, sericin components in Y8 were detected, and the intensity was comparable to that of the tested sericin liquid, indicating that the purity and content of sericin peptides in this product were good. The detection of these sericin raw materials shows that the obtained antibodies can well detect the sericin components in the raw materials and can be used for the detection of sericin components in the raw materials.

[0071] Example 5: Detection of different forms of cosmetics using sericin antibodies.

[0072] Eight brands of cosmetics purchased from the market were tested in different forms. The products that did not contain silk protein were marked as T1, T2, T3, and T4, respectively, to verify whether the detection method was interfered by other ingredients in cosmetics. The comparative sample was the sericin solution in Example 1, marked as WS. 1 μL of the test sample solution and 1 μL of the sericin solution were taken, and each was diluted 10 times in a gradient, and 1 μL of each dilution was dropped on the appropriate area of ​​the PVDF membrane in turn. The subsequent detection was as described in Example 3. The results are shown in FIG. Figure 4 shown.

[0073] from Figure 4 It can be seen that no sericin component was detected in T1, T2, T3, and T4, which indicates that this detection method is not interfered by other ingredients in cosmetics and can be used to detect whether cosmetics contain sericin components. Brand 9 is a silk protein moisturizing mask, denoted as T5. No sericin component was detected in the test, indicating that the product does not contain sericin, but the possibility of added fibroin and unhydrolyzed amino acid type silk protein cannot be ruled out. The two silk masks of Brand 4 are denoted as T6-1 and T6-2, both of which can detect sericin component signals. Among them, the signal of the moisturizing repair mask T6-1 is stronger, indicating that both masks contain sericin components, and the sericin component in the moisturizing repair mask T6-1 is higher. The silk protein anti-dandruff shampoo of Brand 10 is denoted as T7, and no sericin component was detected. Among the three products of brand 11, T8-1, T8-2 and T8-3, only two were detected with sericin ingredients. No sericin ingredients were detected in the Silk Peptide Soothing Spray T8-1, while sericin ingredients were detected in both Silk Peptide Soothing Cream T8-2 and Silk Peptide Repair Essence T8-3. The sericin content of Silk Peptide Repair Essence T8-3 was higher.

[0074] The test results of these cosmetics show that this test method can detect whether the cosmetic products contain sericin ingredients and can be used as a test method for the content of sericin ingredients.

[0075] The above are only preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A sericin antibody, characterized in that: The sericin antibody is obtained by mixing equal volumes of sericin antigen and adjuvant, injecting subcutaneously into rabbits for immunization, taking blood, and separating serum; The sericin antigen is sericin liquid; The sericin liquid includes sericin X1, sericin X3, sericin X10, sericin X12 and sericin X9; The amino acid sequence of the sericin X1 is shown in SEQ ID NO.1, the amino acid sequence of the sericin X3 is shown in SEQ ID NO.2, the amino acid sequence of the sericin X10 is shown in SEQ ID NO.3, the amino acid sequence of the sericin X12 is shown in SEQ ID NO.4, and the amino acid sequence of the sericin X9 is shown in SEQ ID NO.5; The specific process of obtaining the sericin solution is as follows: Soak silkworm cocoons with a mass volume ratio of 10 g: 200 mL in boiling water for 5 minutes to remove the remaining undissolved silk cocoon fibers, which are the silk fibroin components. The sericin protein outside the silk has been dissolved in water, reducing the damage to the sericin protein components, and obtaining the sericin protein stock solution; The sericin stock solution was dissolved and diluted by adding 40 mL of phosphate solution for every 10 mL, and the solution was shaken for 2 hours to promote dissolution. The solution was dialyzed for desalination and diluted to 0.5 mg / mL with deionized water to obtain the sericin solution.

2. The sericin antibody according to claim 1, characterized in that: The number of immunizations is 4 to 5 times, and the interval between two adjacent immunizations is 3 weeks; the adjuvant is Freund's adjuvant.

3. Use of the sericin antibody according to claim 2 in detecting sericin.

4. A method for detecting sericin, characterized in that: The following steps are involved: The sample to be tested is dripped onto the PVDF membrane, a blocking solution is added for blocking, the sericin antibody according to claim 1 is added for incubation, and the membrane is rinsed with a buffer solution; goat anti-rabbit antibody is added for further incubation, and the membrane is rinsed with a buffer solution; The PVDF membrane is transferred to a glass plate, and after the luminescent liquid is added, if luminescence occurs, it means that the sample to be tested contains sericin, and if no luminescence occurs, it means that the sample to be tested does not contain sericin.

5. The method for detecting sericin according to claim 4, characterized in that: The volume ratio of the sericin antibody to the blocking solution is 1:5000; The volume ratio of the goat anti-rabbit antibody to the blocking solution is 1:5000.

6. The method for detecting sericin according to claim 4, characterized in that: The blocking liquid is skim milk liquid with a mass percentage of 10wt%; The buffer solution is a phosphate buffer solution containing 0.1 wt % Tween-20.

7. The method for detecting sericin according to claim 4, characterized in that: The incubation time is 1 h to 1.2 h.

8. The method for detecting sericin according to claim 4, characterized in that: The luminescent liquid is an ECL ultra-sensitive luminescent liquid.

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