CN111732657B - Polypeptide antibody for directly detecting sericin protein and preparation method and application thereof - Google Patents
Polypeptide antibody for directly detecting sericin protein and preparation method and application thereof Download PDFInfo
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Abstract
The invention belongs to the technical field of biology, and relates to a polypeptide antibody for directly detecting silk sericin, and preparation and application thereof, wherein the sericin polypeptide antibody comprises one or more of polypeptide I, polypeptide II, polypeptide III, polypeptide IV, polypeptide V, polypeptide VI and polypeptide VII, the amino acid sequence of polypeptide I is shown in SEQ ID NO.1, the amino acid sequence of polypeptide II is shown in SEQ ID NO.2, the amino acid sequence of polypeptide III is shown in SEQ ID NO.3, the amino acid sequence of polypeptide IV is shown in SEQ ID NO.4, the amino acid sequence of polypeptide V is shown in SEQ ID NO.5, the amino acid sequence of polypeptide VI is shown in SEQ ID NO.6, and the amino acid sequence of polypeptide VII is shown in SEQ ID NO. 7. The detection method can be used for detecting various silk materials, is rapid, direct, accurate and sensitive, and provides a realized channel for qualitative and quantitative detection of sericin.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a polypeptide antibody for directly detecting sericin in silk, and a preparation method and application thereof.
Background
Silk is a natural high molecular protein fiber, and is mainly divided into two categories, namely, domestic silk and tussah silk. China is the country which utilizes tussah and puts in stock tussah at the earliest, and silk is continuous long fiber which is formed by coagulation of secreted silk liquid when mature silkworms are cocooned, is natural fiber, and is one of the earliest animal fibers utilized by human beings. At present, silk fibroin, sericin and other substances extracted from silk are widely applied to the fields of medicine, chemical industry, food and the like.
The silk is mainly composed of two parts including sericin and fibroin, and the sericin wraps and permeates the two fibroin to form the silk. Although both sericin and fibroin are proteins, the structure and amino acid composition of both are different. The silk fibroin is the main component of silk, and accounts for about 70-73% of the mass, and the sericin accounts for about 23-25%. Sericin is mainly composed of at least 8 proteins with different molecular weights produced by selective splicing of three genes of sericin 1, sericin 2 and sericin 3. Wherein, the sericin 2 is mainly present on the net frame which is spitted out by silkworms at the first stage, and the content of the sericin 2 in silks spitted out at the later stage is very small; the sericin 3 is mainly positioned on the outermost layer of the silk sericin and has good hydrophilicity; sericin 1 is the main component of silk sericin, is located in the inner layer of sericin, and is tightly attached to fibroin. The overall molecular conformation of sericin is mainly in a random coil shape, the molecular space structure is loose, and the sericin contains a disordered beta structure but does not have an alpha helical structure. The sericin chain has a plurality of amino acids with longer side chains, such as arginine lysine, glutamic acid, methionine, tryptophan, tyrosine and the like, and a plurality of polar hydrophilic groups (such as-OH, -COOH, -NH2, > NH and the like) on the surface of polypeptide chain, and the structural characteristics endow the sericin with excellent humidity conditioning and moisture preserving effects and well protect the internal fibroin component.
In the production process of silk, residual sericin is required to be removed from raw silk so as to obtain the boiled silk for silk production. In the process of removing residual sericin from raw silk, it is often necessary to perform a treatment using a relevant chemical agent for refining. However, the technical problems that the raw silks in different batches have different residual sericin content, the refining reagent components required to be prepared are different, and the internal silk fibroin is damaged and a large amount of sericin remains due to refining transition and refining deficiency, so that the quality of the cooked silks is influenced exist. In actual production, the production rate needs to be improved, raw silks of different batches or different sources are often treated by the same refining reagent, and thus the rough treatment mode often causes uneven quality of refined boiled silks and influences the quality of silk products in later period. In turn, silk is wasted in pursuit of quality, resulting in less profit for the manufacturer.
In the process of silk fabric circulation, quality inspection is also needed to detect whether the fabric is a real silk fabric or not and meet the quality requirement. The sericin content of different varieties of silk or different silk fabrics is different, the sericin content of silk of different processing degrees and silk of different varieties also has difference, the silk sold on the market is also true and false, the quality of silk fabrics is also good and uneven, the silk material is discriminated and identified if the silk material is good or bad, and the silk material discrimination method is also a difficult problem.
At present, the qualitative and quantitative detection of sericin is carried out by some protein detection means, such as BCA method quantitative detection, Kjeldahl method detection and the like, but the requirements of specific detection and ultra-sensitive detection are difficult to meet. For example, the method of Kjeldahl method for detecting the sericin protein concentration in the sericin wastewater recovery experiment, the result is easily influenced by other nitrogen-containing organic matters in the wastewater. The concentration of sericin is calculated by an evaporation moisture method, which has a limited capability of detecting a low-concentration sericin solution and a detection result is easily affected by other impurities in water, although the method is simple to operate.
The sericin can be qualitatively detected by WB or ELISA detection by using a sericin antibody. ELISA (enzyme linked immunosorbent assay) has the characteristics of high detection sensitivity and strong specificity, and is widely applied to various biochemical experiments. To complete the ELISA experiments, appropriate ELISA antibodies are required. At present, the ELISA antibody is rich in types, but the detection of all proteins is difficult to meet. The sericin protein component is complex and difficult to separate; large molecular weight (>100Kd) and cannot be obtained by prokaryotic expression. Therefore, there is no antibody available on the market for direct detection of sericin.
Disclosure of Invention
In order to solve the technical problems, the invention provides a polypeptide antibody for directly detecting sericin, a preparation method and an application thereof, wherein the polypeptide antibody of sericin is obtained by first purification, is used for quickly, directly, accurately and sensitively detecting sericin, and provides a channel for quickly, accurately and sensitively detecting sericin.
The polypeptide antibody for directly detecting the sericin protein is characterized in that:
the sericin polypeptide antigen comprises one or more than two of polypeptide I, polypeptide II, polypeptide III, polypeptide IV, polypeptide V, polypeptide VI and polypeptide VII, wherein the amino acid sequence of polypeptide I is shown in SEQ ID NO.1, the amino acid sequence of polypeptide II is shown in SEQ ID NO.2, the amino acid sequence of polypeptide III is shown in SEQ ID NO.3, the amino acid sequence of polypeptide IV is shown in SEQ ID NO.4, the amino acid sequence of polypeptide V is shown in SEQ ID NO.5, the amino acid sequence of polypeptide VI is shown in SEQ ID NO.6, and the amino acid sequence of polypeptide VII is shown in SEQ ID NO. 7.
The polypeptide antibody can be used for detecting any object to be detected so as to determine whether sericin exists in the object to be detected. The sericin class can also be tested, i.e., one or several of the 7 polypeptide antibodies, to distinguish which protein sericin is.
In an optimized scheme, the sericin polypeptide antibody comprises a polypeptide I and a polypeptide II, or a polypeptide III, a polypeptide IV and a polypeptide V, or a polypeptide VI and a polypeptide VII.
In the detection of the sericin class, the polypeptide I and the polypeptide II can detect sericin 1, the polypeptide III, the polypeptide IV and the polypeptide V can detect sericin 2, and the polypeptide VI and the polypeptide VII can detect sericin 3.
In a further optimization scheme, the sericin polypeptide antibody comprises a polypeptide I, a polypeptide II, a polypeptide III, a polypeptide IV, a polypeptide V, a polypeptide VI and a polypeptide VII. Seven polypeptides are put together to detect objects, and the appearance of any signal indicates that the objects contain sericin, so that the detection objects have wide range and high accuracy.
The letters in the amino acid sequence are the single letter symbols of the corresponding amino acids, the right end is C-terminal sequencing, and the left end is N-terminal sequencing.
The active site loops (a) to (g) (including the two terminal amino acid residues shown) include the amino acid residues in the region between amino acid residues 1201-1217, also the amino acid residues in the region between amino acid residues 1201-1217, and also the amino acid residues in the region between 741-757, 902-916, 214-230, 1710-1727, 205-223 and 35-51.
Ser-1B:
741-757: GSSSNTDSSTKNAGSRT polypeptide-A
21-37:GHHPGNRDTVEVKNRKY
1201-1217: FKNIFDIPYHLRKNIGV polypeptide two
>Ser-2-2:
902-916: TEKAKPNDRSPSDRD polypeptide III
214-230: SAERTKSKRGERKEVEG polypeptide IV
1710-1727: NRVVEKSTDGDNEESYRS polypeptide five
>Ser-3-2:
482-499:DEDSDDSSGATKGNSSKS
205-223: EESSNGGSGSGRTGSAGGT polypeptide six
35-51: GGGRGRGSGVRRLDSG polypeptide hepta
Although polypeptide sites can be designed to produce antibodies by knowing the protein sequence, antibodies are not available at all polypeptide sites and the choice of antigenic polypeptide site is important.
The invention discloses a preparation method of a polypeptide antibody for directly detecting sericin, which is characterized by comprising the following steps of: the cocoon layer of the sample is subjected to polypeptide synthesis, enzyme-resistant labeling, polypeptide and KLH coupling to serve as immune antigen, PBS is used for diluting the immune antigen respectively, the antigen is added into Freund's incomplete adjuvant and is subjected to emulsification injection, blood centrifugation of small animals and antibody purification.
The PBS stock solution (10X) was prepared:
wherein 2.85g of Na2HPO4 & 12H2O can be 1.13g of Na2HPO4 & 2H2O, and the raw materials are dissolved in 100ml of deionized water, and the pH value is adjusted to 7.2.
The antibody purification step is followed by an immunoassay step in which secondary enzymatic labeling is performed when added to a secondary antibody.
The first enzyme identification is applicable to the case of large sample quantity; if more accurate and less sample amount is needed, enzyme labeling is carried out again during sensitive detection, and a secondary antibody is added.
The enzyme in the enzyme label is horseradish peroxidase.
The invention discloses an application of a sericin polypeptide antibody, and relates to an application of the sericin polypeptide antibody in preparation of a kit for detecting sericin.
In the preparation method, the polypeptide of the sericin is artificially synthesized, then the synthesized polypeptide is used as a complete antigen and injected to a living animal for animal immunization and purification to obtain the polyclonal antibody of the polypeptide fragments of the sericin 1, the sericin 2 and the sericin 3, and meanwhile, the antibody is marked by HRP (horse radish peroxidase), so that the sericin polypeptide antibody can quantitatively determine the sericin content in cocoon fibers.
The polypeptide antibody of the sericin is obtained by the first purification of the invention, and the polypeptide antibody of the sericin can be screened and identified for various silk materials, the detection is rapid, direct, accurate and sensitive, thereby providing an implementation channel for qualitative and quantitative detection of the sericin in actual production and quality inspection, filling the blank in industrial application research and ensuring the purchase of high-quality silk.
Drawings
The invention will be described in further detail with reference to the following drawings and detailed description:
FIG. 1 is a table of abbreviations for the twenty amino acids of the present invention;
FIG. 2 is a graph showing the results of immunofluorescence assay of the polypeptide antibodies of the present invention;
FIG. 3 is a graph showing the results of dot blot hybridization of the polypeptide antibody of the present invention
FIG. 4 is a Western blot detection chart of different sericin antibodies on contents of different regions of silkworm MSG
(wherein, A: 872 strain silk gland; B: SDS-PAGE of MSG content; C-E: Western blot identification of different sericin antibodies)
FIG. 5 is a diagram showing the removal of sericin in the processing of cocoon filaments according to the present invention
Detailed Description
The invention will be further illustrated with reference to specific embodiments:
example 1
The sericin polypeptide antibody comprises a polypeptide I, a polypeptide II, a polypeptide III, a polypeptide IV, a polypeptide V, a polypeptide VI and a polypeptide VII. Seven polypeptides are put together to detect objects, and the appearance of any signal indicates that the objects contain sericin, so that the detection objects have wide range and high accuracy.
The sample containing sericin with the total content of more than 1ug/mg can be detected.
The amino acid sequence of peptide one is: GSSSNTDSSTKNAGSRT, the amino acid sequence of the polypeptide II is: FKNIFDIPYHLRKNIGV, the amino acid sequence of polypeptide III is: TEKAKPNDRSPSDRD, the amino acid sequence of polypeptide IV is: SAERTKSKRGERKEVEG, the amino acid sequence of polypeptide five is: NRVVEKSTDGDNEESYRS, the amino acid sequence of polypeptide six is: EESSNGGSGSGRTGSAGGT, the amino acid sequence of polypeptide seven is: GGGRGRGSGVRRLDSG are provided. The letters in the above amino acid sequences are the single letter symbols of the corresponding amino acids, as shown in FIG. 1 for details.
The letters in the amino acid sequence are the single letter symbols of the corresponding amino acids, the right end is C-terminal sequencing, and the left end is N-terminal sequencing.
Example 2
The sericin polypeptide antibody comprises a polypeptide I and a polypeptide II, and sericin 1 is detected. The sample with sericin 1 content of more than 1ug/mg can be detected. The amino acid sequence of the polypeptide I is as follows: GSSSNTDSSTKNAGSRT, the amino acid sequence of the polypeptide II is: FKNIFDIPYHLRKNIGV are provided.
Example 3
The sericin polypeptide antibody comprises polypeptide three, polypeptide four and polypeptide five, and is used for detecting sericin 2. The sample with the sericin 2 content of more than 1ug/mg can be detected.
The amino acid sequence of the polypeptide III is as follows: TEKAKPNDRSPSDRD, the amino acid sequence of polypeptide IV is: SAERTKSKRGERKEVEG, the amino acid sequence of polypeptide five is: NRVVEKSTDGDNEESYRS are provided.
Example 4
The sericin polypeptide antibody comprises a polypeptide six and a polypeptide seven, and sericin 3 is detected. The sample containing sericin 3 of more than 1ug/mg can be detected.
The amino acid sequence of the polypeptide six is as follows: EESSNGGSGSGRTGSAGGT, the amino acid sequence of polypeptide seven is: GGGRGRGSGVRRLDSG are provided.
Example 5
The application of the sericin polypeptide antibody is the application of the sericin polypeptide antibody in the preparation of a kit for detecting sericin.
Example 6
The active site loops (a) to (g) (including the amino acid residues at both termini shown) to include the amino acid residues in the region between amino acid residues 1201 and 1217. Also included are amino acid residues in the region between amino acid residues 1201-1217 and also amino acid residues in the region between 741-757, 902-916, 214-230, 1710-1727, 205-223 and 35-51.
Ser-1B:
741-757: GSSSNTDSSTKNAGSRT polypeptide-A
21-37:GHHPGNRDTVEVKNRKY
1201-1217: FKNIFDIPYHLRKNIGV polypeptide two
>Ser-2-2:
902-916: TEKAKPNDRSPSDRD polypeptide III
214-230: SAERTKSKRGERKEVEG polypeptide IV
1710-1727: NRVVEKSTDGDNEESYRS polypeptide five
>Ser-3-2:
482-499:DEDSDDSSGATKGNSSKS
205-223: EESSNGGSGSGRTGSAGGT polypeptide six
35-51: GGGRGRGSGVRRLDSG polypeptide hepta
Example 7
The specific preparation steps of the sericin polypeptide antibody are as follows:
an immunization process:
(1) synthesizing a polypeptide: degumming cocoon shells in boiling water, removing silk fibroin to obtain sericin, freeze-drying the sericin to obtain sericin, hydrolyzing the sericin by a chain enzyme method, and extracting to obtain sericin polypeptide; (high temperature and high pressure method) safety of biological materials, cytotoxicity in vitro; as a replacement for serum; in the process, enzyme labeling is carried out, wherein the enzyme is horseradish peroxidase. The method of enzyme labeling is a routine procedure.
(2) Coupling polypeptide and KLH to serve as an immune antigen, and coupling polypeptide and BSA to serve as a detection antigen, wherein a KLH coupling agent is Sulfo-SMCC, and a BSA coupling agent is glutaraldehyde;
(3) diluting the immune antigen to 1mg/ml with PBS respectively, subpackaging and freezing at-20 ℃ in a refrigerator for later use;
the preparation method of the PBS comprises the following steps:
preparation of PBS stock solution (10 ×):
wherein 2.85g of Na2HPO4 & 12H2O can also be 1.13g of Na2HPO4 & 2H2O, and the raw materials are dissolved in 100ml of deionized water, and the pH value is adjusted to 7.2;
(1X) PBS preparation method: adding 50ml of storage solution into 450ml of deionized water, and storing at room temperature or 4 ℃ for later use;
(4) on day 1, 1ml of each antigen is taken and added with 1ml of Freund's complete adjuvant, the mixture is emulsified until one drop of emulsified antigen liquid is dropped into physiological saline without dispersion, the emulsification meets the requirement, then the mixture is injected subcutaneously at the back of the neck at multiple points, the number of the injected points is not less than 8, and each antigen immunizes 2 New Zealand white rabbits;
(5) on day 15, 1ml of each antigen is taken and added with 1ml of Freund incomplete adjuvant, emulsification is carried out, then subcutaneous multi-point injection is carried out on the neck and the back, the number of injection points is not less than 8, and each antigen immunizes 2 New Zealand white rabbits;
(6) on the 29 th day, 1ml of each antigen is taken and added with 1ml of Freund incomplete adjuvant, emulsification is carried out, then subcutaneous multi-point injection is carried out on the neck and the back, the number of injection points is not less than 8, and each antigen immunizes 2 New Zealand white rabbits;
(7) on day 43, 1ml of each antigen is taken and added with 1ml of Freund incomplete adjuvant, emulsification is carried out, then subcutaneous multi-point injection is carried out on the neck and the back, the number of injection points is not less than 8, and each antigen immunizes 2 New Zealand white rabbits;
(8) day 53, carotid bleeding and rabbits were sacrificed;
(9) standing the rabbit blood at 4 ℃ overnight, centrifuging at the temperature of 4 ℃ at the speed of 10000rpm for 30 minutes, and collecting supernatant;
antibody purification
(1) Attaching the polypeptide to activated Sulfolink Resin to prepare an antigen affinity column, 1ml Sulfolink Resin coupled to 1mg polypeptide;
(2) the affinity column was equilibrated with 10 column volumes of PBS, and the solution was drained off; filtering rabbit serum with 0.45um filter membrane;
(3) then passing the rabbit serum through an antigen affinity column, draining the solution, and collecting the flow-through;
(4) then balancing by using PBS (phosphate buffer solution) with 10 times of column volume, and completely draining the solution;
(5) adding 5ml of antibody eluent, collecting the eluent in different tubes, wherein each tube contains 1ml of the eluent, and the preparation method of the antibody eluent comprises the following steps: weighing 7.5g of glycine, dissolving in 100ml of deionized water, adjusting the pH value to 2.7 by using concentrated hydrochloric acid, and storing at 4 ℃ for later use;
(6) detecting absorbance at 280nm with the collected eluate, mixing the components with absorbance greater than 1.0, and dialyzing with PBS;
(7) antibody identification after dialysis: and detecting the protein concentration by using an ultraviolet absorption method, and determining the antibody titer by Elisa to finally obtain the polyclonal antibody of 6 polypeptide fragments of sericin 1, sericin 2 and sericin 3.
Example 7
Antibody immunoassay by Elisa method:
(1) coating: coating the antigen with an antigen coating solution, diluting the coated antigen to 1ug/ml with CBS, adding into an ELISA plate with each well at 100ul, and standing overnight at 4 deg.C; the preparation method of the antigen coating solution (CBS, 1 x) comprises the following steps: weighing Na2CO31.59g, NaHCO32.93g and 950ml of deionized water, adjusting the pH value to 9.6, adding deionized water to a constant volume of 1000ml, and storing at 4 ℃;
(2) and (3) sealing: discarding the coating solution, adding 200ul of sealing solution into each hole, and standing at 37 ℃ for 1.5 hours, wherein the preparation method of the sealing solution comprises the following steps: weighing 5g of skimmed milk powder, dissolving in 100ml of PBS, mixing, and storing at 4 ℃ for use, wherein the storage time is less than or equal to 3 days;
(3) adding a sample: removing the confining liquid, adding serum or antibody samples, adding an enzyme label plate into each hole at 100ul, and standing at 37 ℃ for 1 hour;
(4) washing: washing with tap water for 10 times, and drying;
(5) adding a secondary antibody: diluting the enzyme-labeled goat anti-rabbit with a sealing solution to a working concentration, adding an enzyme-labeled plate into each hole at 100ul, and standing at 37 ℃ for 30 minutes;
(6) washing: washing with tap water for 10 times, and drying;
(7) addition of TMB chromogenic substrate: adding an enzyme label plate into each hole at 100ul, and standing for 15 minutes at 37 ℃; the preparation method of the TMB chromogenic substrate comprises the following steps:
TMB stock solution: weighing TMB powder, preparing into 1.5mg/ml stock solution with DMSO, and subpackaging at-20 deg.C for storage;
(ii) NaAc buffer: preparing 200mM NaAc solution, and adjusting the pH value to 5.3 by HAc;
(iii) H2O2 solution: preparing 0.03% of H2O2 solution;
storing the solution according to TMB when in use: NaAc buffer solution: preparing a color developing solution by using the H2O2 solution in a ratio of 1:4:5, and using the color developing solution as it is;
(8) adding a stop solution: 2M H2SO 450 ul is added into each hole, and the preparation method of the stop solution comprises the following steps: slowly adding 100ml of concentrated sulfuric acid into 900ml of water, and storing at room temperature for later use;
(9) reading: and (4) reading by using an enzyme labeling instrument OD450nm, and obtaining an OD value to obtain the quantity of the antibody.
Example 8
The other contents are the same, and a secondary antibody is added: diluting the enzyme-labeled goat anti-rabbit with a sealing solution to a working concentration, adding an enzyme-labeled plate into each hole at 100ul, and standing at 37 ℃ for 30 minutes; enzyme labeling is carried out in an enzyme label plate, wherein the enzyme is horseradish peroxidase.
The experimental results for examples 7 or 8 are as follows:
(1) number 374-1 (polypeptide one: GSSSNTDSSTKNAGSRT)
Finally obtaining 2.3ml of 374-1-R1 antibody, 0.57 mg/ml; 374-1-R2 antibody 1.8ml, 1.02mg/ml, the detailed test results are shown in the following table:
(2) number 374-2 (polypeptide two: FKNIFDIPYHLRKNIGV)
Finally obtaining 1.7ml of 374-2-R1 antibody, 1.85 mg/ml; the 374-2-R2 antibody is 3.1ml and 1.11mg/ml, and the detailed detection results are shown in the following table:
(3) no. 374-3 (polypeptide III: TEKAKPNDRSPSDRD)
Finally obtaining 2.5ml of 374-3-R1 antibody, 0.89 mg/ml; 374-3-R2 antibody 1.1ml, 0.73mg/ml, the detailed test results are shown in the following table:
(4) number 374-4 (polypeptide four: SAERTKSKRGERKEVEG)
3.3ml of 374-4-R1 antibody is finally obtained, 1.3 mg/ml; 374-4-R2 antibody 3.3ml, 0.87mg/ml, the detailed test results are shown in the following table:
(5) number 374-5 (polypeptide five: NRVVEKSTDGDNEESYRS)
Finally obtaining 1.7ml of 374-5-R1 antibody, 2.5 mg/ml; 374-5-R2 antibody 2.3ml, 1.76mg/ml, the detailed test results are shown in the following table:
(6) number 374-6 (polypeptide six: EESSNGGSGSGRTGSAGGT)
Finally obtaining 1.7ml of 374-6-R1 antibody, 1.7 mg/ml; 374-6-R2 antibody 3.1ml, 0.54mg/ml, the detailed test results are shown in the following table:
(7) number 374-7 (polypeptide seven: GGGRGRGSGVRRLDSG)
Finally obtaining 1.7ml of 374-7-R1 antibody, 2.06 mg/ml; 374-7-R2 antibody 2.9ml, 1.02mg/ml, the detailed test results are shown in the following table:
the titer measurement shows that the titer of the prepared antibody reaches 1:80000, and the antibody can be completely used for subsequent immunodetection. The immunofluorescence detection result of the polypeptide antibody is shown in figure 2, and the checkpoint hybridization result of the polypeptide antibody is shown in figure 3. The result shows that the prepared antibody can be used for in-situ detection and immunodetection of sericin except that the antibody with the number of 374-4 cannot be used for immunohistochemical experiments.
One of the antibodies for preparing sericin 1, sericin 2 and sericin 3 is selected for the immunodetection of the contents of the tissue sample, and the result shows that sericin at each part of the silk gland can be specifically detected, and the result is shown in figure 4.
The detection of cocoons and silk products in different processing processes shows that the removal condition of sericin in the cocoon silk processing process can be detected by using the antibody group. The reeling cocoons do not contain sericin 2 protein, which is consistent with the reported result that sericin 2 protein exists only in cocoon net, and the cocoon net and cocoon silk are removed from the reeling cocoons. In the reeling process, sericin 3 protein is removed firstly, sericin 1 protein can be effectively removed only in a special refining process, and silk is basically free of sericin, and the result is shown in figure 5.
While the foregoing shows and describes the fundamental principles and principal features of the invention, together with the advantages thereof, the foregoing embodiments and description are illustrative only of the principles of the invention, and various changes and modifications can be made therein without departing from the spirit and scope of the invention, which will fall within the scope of the invention as claimed. The scope of the invention is defined by the appended claims and equivalents thereof.
Sequence listing
SEQUENCE LISTING
<110> university of southwest
<120> polypeptide antibody for directly detecting sericin protein, preparation method and application thereof
<160> 4
<170> PatentIn version 3.3
<210> 1
<211> 17 bp
<212> DNA
<213> Artificial Synthesis
<220>
<223> polypeptide-
<400> 1
gsssntdsstknagsrt 17
<210> 2
<211> 17 bp
<212> DNA
<213> Artificial Synthesis
<220>
<223> polypeptide two
<400> 2
fknifdipyhlrknigv 17
<210> 3
<211> 15 bp
<212> DNA
<213> Artificial Synthesis
<220>
<223> polypeptide III
<400> 3
tekakpndrspsdrd 15
<210> 4
<211> 17 bp
<212> DNA
<213> Artificial Synthesis
<220>
<223> polypeptide IV
<400> 4
saertkskrgerkeveg 17
<210> 5
<211> 21 bp
<212> DNA
<213> Artificial Synthesis
<220>
<223> polypeptide five
<400> 5
nrvvekstdgdneesyrs 18
<210> 6
<211> 19 bp
<212> DNA
<213> Artificial Synthesis
<220>
<223> polypeptide hexa
eessnggsgsgrtgsaggt 19
<400> 6
<210> 7
<211> 16 bp
<212> DNA
<213> Artificial Synthesis
<220>
<223> polypeptide hepta
<400> 7
gggrgrgsgvrrldsg 16
Claims (9)
1. A polypeptide antibody for directly detecting sericin is characterized in that: the antigen comprises a polypeptide I, wherein the amino acid sequence of the polypeptide I is shown in SEQ ID NO. 1.
2. The polypeptide antibody for direct detection of sericin according to claim 1, wherein: the sericin polypeptide antigen further comprises at least one of a polypeptide II, a polypeptide III, a polypeptide IV, a polypeptide V, a polypeptide VI and a polypeptide VII, wherein the amino acid sequence of the polypeptide II is shown in SEQ ID NO.2, the amino acid sequence of the polypeptide III is shown in SEQ ID NO.3, the amino acid sequence of the polypeptide IV is shown in SEQ ID NO.4, the amino acid sequence of the polypeptide V is shown in SEQ ID NO.5, the amino acid sequence of the polypeptide VI is shown in SEQ ID NO.6, and the amino acid sequence of the polypeptide VII is shown in SEQ ID NO. 7.
3. The polypeptide antibody for direct detection of sericin according to claim 1, wherein: the sericin polypeptide antigen comprises a polypeptide I and a polypeptide II.
4. The polypeptide antibody for direct detection of sericin according to claim 1, wherein: the sericin polypeptide antigen comprises a polypeptide I, a polypeptide II, a polypeptide III, a polypeptide IV, a polypeptide V, a polypeptide VI and a polypeptide VII.
5. The method for preparing the polypeptide antibody for directly detecting the sericin protein according to claim 1, wherein the polypeptide antibody comprises: coupling polypeptide I with KLH to serve as an immune antigen, diluting the immune antigen with PBS respectively, adding Freund's incomplete adjuvant into the antigen, emulsifying and injecting, centrifuging the blood of the small animal and purifying the antibody.
6. The method for preparing the polypeptide antibody for directly detecting the sericin protein according to claim 5, wherein the polypeptide antibody comprises: the PBS stock solution (10X) was prepared:
NaCl 8.5g，
KCl 0.2g，
Na2HPO4•12H2O 2.85g，
KH2PO4 0.27g；
2.85g of Na2HPO4•12H2O may also be 1.13g of Na2HPO4•2H2And O, dissolving the raw materials in 100ml of deionized water, and adjusting the pH value to 7.2.
7. The method for preparing the polypeptide antibody for directly detecting the sericin protein according to claim 5, wherein the polypeptide antibody comprises: the antibody purification step is followed by an immunoassay step in which enzyme labeling is performed again when a secondary antibody is added.
8. The method for preparing the polypeptide antibody for directly detecting the sericin protein according to claim 7, wherein the polypeptide antibody comprises: the enzyme in the enzyme label is horseradish peroxidase.
9. Use of a polypeptide antibody for the direct detection of sericin according to claim 1, wherein: the application is to prepare a kit for detecting sericin 1.
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| CN202111158475.XA CN113861290B (en) | 2020-06-19 | 2020-06-19 | Polypeptide antibody for directly detecting silk sericin, preparation method and application |
| CN202111148723.2A CN113735972B (en) | 2020-06-19 | 2020-06-19 | Polypeptide antibody for directly detecting silk sericin, preparation method and application |
| CN202111148026.7A CN113773383B (en) | 2020-06-19 | 2020-06-19 | Polypeptide antibody for directly detecting silk sericin, preparation method and application |
| CN202111149126.1A CN113773384B (en) | 2020-06-19 | 2020-06-19 | Polypeptide antibody for directly detecting silk sericin, preparation method and application |
| CN202010563917.8A CN111732657B (en) | 2020-06-19 | 2020-06-19 | Polypeptide antibody for directly detecting sericin protein and preparation method and application thereof |
| CN202111148343.9A CN113754765B (en) | 2020-06-19 | 2020-06-19 | Polypeptide antibody for directly detecting silk sericin, preparation method and application |
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| CN202111148343.9A Division CN113754765B (en) | 2020-06-19 | 2020-06-19 | Polypeptide antibody for directly detecting silk sericin, preparation method and application |
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| CN113735972B (en) | 2023-08-15 |
| CN113754765A (en) | 2021-12-07 |
| CN113754765B (en) | 2023-10-03 |
| CN113773384A (en) | 2021-12-10 |
| CN113861290A (en) | 2021-12-31 |
| CN113735972A (en) | 2021-12-03 |
| CN113773384B (en) | 2023-07-04 |
| CN113773383B (en) | 2024-02-06 |
| CN113861290B (en) | 2023-06-16 |
| CN111732657A (en) | 2020-10-02 |
| CN113773383A (en) | 2021-12-10 |
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