CN113773384B - Polypeptide antibody for directly detecting silk sericin, preparation method and application - Google Patents
Polypeptide antibody for directly detecting silk sericin, preparation method and application Download PDFInfo
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- CN113773384B CN113773384B CN202111149126.1A CN202111149126A CN113773384B CN 113773384 B CN113773384 B CN 113773384B CN 202111149126 A CN202111149126 A CN 202111149126A CN 113773384 B CN113773384 B CN 113773384B
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/531—Production of immunochemical test materials
- G01N33/532—Production of labelled immunochemicals
- G01N33/535—Production of labelled immunochemicals with enzyme label or co-enzymes, co-factors, enzyme inhibitors or enzyme substrates
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
- G01N2333/43504—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from invertebrates
- G01N2333/43552—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from invertebrates from insects
- G01N2333/43578—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from invertebrates from insects from silkworm
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
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 than two of a first polypeptide, a second polypeptide, a third polypeptide, a fourth polypeptide, a fifth polypeptide, a sixth polypeptide and a seventh polypeptide, the amino acid sequence of the first polypeptide is shown as SEQ ID NO.1, the amino acid sequence of the second polypeptide is shown as SEQ ID NO.2, the amino acid sequence of the third polypeptide is shown as SEQ ID NO.3, the amino acid sequence of the fourth polypeptide is shown as SEQ ID NO.4, the amino acid sequence of the fifth polypeptide is shown as SEQ ID NO.5, the amino acid sequence of the sixth polypeptide is shown as SEQ ID NO.6, and the amino acid sequence of the seventh polypeptide is shown as SEQ ID NO. 7. Can detect various silk materials, is quick, direct, accurate and sensitive, and provides a realized channel for qualitative and quantitative detection of sericin.
Description
The application is a divisional application, the application date of the original application is 2020-06-19, the application number is 2020105639178, and the invention creates a polypeptide antibody for directly detecting silk sericin, and a preparation method and application thereof.
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a polypeptide antibody for directly detecting silk sericin, a preparation method and application thereof.
Background
Silk is a natural high molecular protein fiber and is mainly divided into two major classes of domestic silk and tussah silk. China is the country which utilizes tussah and puts tussah in the earliest, silk is continuous long fiber which is formed by solidifying secretion liquid when mature silkworms are cocooned, and is a natural fiber and one of the earliest animal fibers used by human beings. At present, substances such as silk fibroin and sericin extracted from silk are widely applied to the fields of medicines, chemical engineering, foods and the like.
The silk is mainly composed of two parts, including sericin and silk fibroin, and the silk is formed by wrapping and penetrating two silk fibroin by the sericin. Sericin and silk fibroin are proteins, but the structures and amino acid components of the sericin and the silk fibroin are different. Silk fibroin is a main component in silk, and accounts for about 70% -73% of the mass, and sericin accounts for about 23% -25%. Sericin mainly consists of at least 8 proteins with different molecular weights generated by selective splicing of three genes of sericin 1, sericin 2 and sericin 3 through mRNA. The sericin 2 mainly exists on a net rack which is firstly discharged by silkworms, and the content of the sericin 2 in the silk which is discharged in the later period is 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, and is positioned in the inner layer of sericin and clings to silk fibroin. The overall molecular conformation of sericin is mainly random coil, the molecular space structure is loose, disordered beta structure is contained, but alpha helix structure is not contained. The sericin 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 the polypeptide chain, and the structural characteristics endow the sericin with excellent humidity regulating and preserving effects, and also well protect the internal silk fibroin components.
In the silk processing production process, the raw silk is required to be subjected to residual sericin removal treatment so as to obtain cooked silk which can be used for silk production. In the removal of residual sericin from raw silk, it is often necessary to use a treatment with a relevant chemical agent for refining. However, the raw silk in different batches has different content of sericin residues, the components of the refining reagent to be prepared are different, and the internal sericin is damaged and a large amount of sericin residues are caused by insufficient refining transition and insufficient refining, so that the technical problem of influencing the quality of cooked silk is solved. In actual production, the production rate needs to be improved, raw silk of different batches or different sources is often treated by using the same refining reagent, so that the rough treatment mode often leads to uneven quality of refined cooked silk and influences the quality of later silk products. In turn, silk is wasted for quality pursuit, and the profit obtained by manufacturers is reduced.
In the silk fabric circulation process, quality inspection is also required to detect whether the fabric is silk fabric or not and meet the quality requirement. The sericin content of different varieties of silks or different silk fabrics is different, the sericin content of the silks with different processing degrees and the sericin content of the silks with different varieties of silks are different, the silks sold on the market are true and false, the quality of the silk fabrics is good and uneven, and the discrimination and identification of silk materials are good and bad, so that the method is a difficult problem.
Qualitative and quantitative detection of sericin is currently performed by detection means of some proteins, such as BCA assay, kjeldahl assay, etc., but is difficult to meet the requirements of specific detection and ultrasensitive detection. For example, the Kjeldahl method is used for detecting the concentration of sericin in a sericin wastewater recovery experiment, and the result is easily influenced by other nitrogen-containing organic matters in the wastewater. There are also methods for calculating the concentration of sericin by evaporation of water, which, although simple to operate, have limited detection capability for low concentration sericin solutions and the detection result is susceptible to other impurities in water.
Sericin can be qualitatively detected by WB or ELISA detection using sericin antibodies. 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, a suitable ELISA antibody is required. At present, ELISA antibodies are rich in variety, but detection of all proteins is difficult to meet. The sericin has complex components and is difficult to separate; the molecular weight is large (> 100 Kd) and cannot be obtained by means of prokaryotic expression. Thus, 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 silk sericin, a preparation method and application thereof, and the polypeptide antibody of the sericin is obtained by first purification, is used for rapidly, directly, accurately and sensitively detecting the sericin, and provides a realization channel for rapidly, accurately and sensitively detecting the sericin.
The polypeptide antibody for directly detecting silk sericin in the invention for solving the technical problems is characterized in that:
the sericin polypeptide antigen comprises one or more than two of a first polypeptide, a second polypeptide, a third polypeptide, a fourth polypeptide, a fifth polypeptide, a sixth polypeptide and a seventh polypeptide, wherein the amino acid sequence of the first polypeptide is shown as SEQ ID NO.1, the amino acid sequence of the second polypeptide is shown as SEQ ID NO.2, the amino acid sequence of the third polypeptide is shown as SEQ ID NO.3, the amino acid sequence of the fourth polypeptide is shown as SEQ ID NO.4, the amino acid sequence of the fifth polypeptide is shown as SEQ ID NO.5, the amino acid sequence of the sixth polypeptide is shown as SEQ ID NO.6, and the amino acid sequence of the seventh polypeptide is shown as SEQ ID NO. 7.
The polypeptide antibody can detect any one of the objects to be detected, and can determine whether sericin exists in the detected object. Sericin class detection, i.e., testing with one or more of the 7 polypeptide antibodies, can also be performed to distinguish which protein sericin is.
In an optimized scheme, the sericin polypeptide antigen comprises a first polypeptide and a second polypeptide, or a third polypeptide, a fourth polypeptide and a fifth polypeptide, or a sixth polypeptide and a seventh polypeptide.
In the detection of sericin category, the first polypeptide and the second polypeptide can detect sericin 1, the third polypeptide, the fourth polypeptide and the fifth polypeptide can detect sericin 2, and the sixth polypeptide and the seventh polypeptide can detect sericin 3.
In a further preferred embodiment, the sericin polypeptide antigen comprises a first polypeptide, a second polypeptide, a third polypeptide, a fourth polypeptide, a fifth polypeptide, a sixth polypeptide and a seventh polypeptide. Seven polypeptides are put together to detect an object, any signal shows that sericin is contained in the object, the detection object has wide range and high accuracy of results.
The letters in the amino acid sequences are single letter symbols of corresponding amino acids, the right end is C-end sequencing, and the left end is N-end sequencing.
The active site loops (a) through (g) (including the two terminal amino acid residues shown) are intended to include amino acid residues in the region between amino acid residues 1201-1217, as well as amino acid residues in the regions between 741-757, 902-916, 214-230, 1710-1727, 205-223, and 35-51.
Ser-1B:
741-757 GSSSNTDSSS TKAGSRT polypeptide one
21-37:GHHPGNRDTVEVKNRKY
1201-1217 FKNIFDIPYIHLRKNIGV polypeptide two
>Ser-2-2:
902-916 TEKAKPNDRSPSDRD polypeptide three
214-230 SAERKSKRGERKEVEG polypeptide four
1710-1727 NRVVEKESTDGDNEESYRS polypeptide five
>Ser-3-2:
482-499:DEDSDDSSGATKGNSSKS
205-223 EESSNGGSGSGRTGSAGGT polypeptide six
35-51 GGGRGRGSGVRRRLDSG polypeptide seven
While it is possible to design polypeptide sites to produce antibodies by knowing the protein sequence, not all polypeptide sites can be used to obtain antibodies, and the choice of antigenic polypeptide sites is important.
The invention discloses a preparation method of a polypeptide antibody for directly detecting silk sericin, which is characterized by comprising the following steps of: the cocoon shells of the samples are subjected to steps of polypeptide synthesis, enzyme resistance labeling, polypeptide coupling and KLH as immune antigens, PBS (poly-vinyl acetate) respectively diluting the immune antigens, adding Freund's incomplete adjuvant into the antigens, emulsion injection, small animal blood centrifugation and antibody purification.
The PBS stock solution (10×) formulation method:
wherein 2.85g of Na2HPO4.12H2O can be 1.13g of Na2HPO4.2H2O, and the above 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 enzyme labeling is performed when a secondary antibody is added.
The first enzyme identification is applicable to the case of large sample size; if more accurate and small sample size is needed, enzyme labeling is performed again during sensitive detection, and secondary antibodies are added.
The enzyme in the enzyme label is horseradish peroxidase.
The invention discloses application of a silk sericin polypeptide antibody, which is application of the silk sericin polypeptide antibody in preparation of a kit for detecting sericin.
In the preparation method, the polypeptide of sericin is synthesized artificially, then the synthesized polypeptide is injected into living animals as complete antigen, animal immunity is carried out, polyclonal antibodies of polypeptide fragments of sericin 1, sericin 2 and sericin 3 are obtained after purification, and simultaneously, the antibodies are marked by HRP (horseradish peroxidase), so that the sericin polypeptide antibody can quantitatively determine the sericin content in cocoon filaments.
The polypeptide antibody of sericin is obtained by first purification, can be used for screening and identifying various silk materials, is rapid, direct, accurate and sensitive in detection, provides a realization channel for qualitative and quantitative detection of sericin in actual production and quality inspection, fills up the blank of industrial application research and ensures that high-quality silk is purchased.
Drawings
The invention will be described in further detail with reference to the accompanying drawings and detailed description:
FIG. 1 is an alphabet of twenty amino acids according to the present invention;
FIG. 2 is a graph showing immunofluorescence detection results of a polypeptide antibody of the present invention;
FIG. 3 is a graph showing the hybridization results of detection spots of the polypeptide antibody of the present invention
FIG. 4 is a Western blot detection chart of different sericin antibodies of the invention on contents of different regions of silkworm MSG
( Wherein A:872 strain silk gland; b: SDS-PAGE of MSG contents; C-E: western blot identification of different sericin antibodies )
FIG. 5 is a drawing showing the removal of sericin during cocoon silk processing in the present invention
Detailed Description
The invention is further described in connection with the following embodiments:
example 1
The silk sericin polypeptide antigen comprises a first polypeptide, a second polypeptide, a third polypeptide, a fourth polypeptide, a fifth polypeptide, a sixth polypeptide and a seventh polypeptide. Seven polypeptides are put together to detect an object, any signal shows that sericin is contained in the object, the detection object has wide range and high accuracy of results.
The sample with the total sericin content of more than 1ug/mg can be detected.
The amino acid sequence of peptide one is: GSSSNTDSSTKNAGSRT, the amino acid sequence of polypeptide two is: FKNIFDIPYHLRKNIGV, the amino acid sequence of polypeptide three is: TEKAKPNDRSPSDRD, the amino acid sequence of polypeptide four 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. The letters in the amino acid sequences are single-letter symbols of corresponding amino acids, and the details are shown in figure 1.
The letters in the amino acid sequences are single letter symbols of corresponding amino acids, the right end is C-end sequencing, and the left end is N-end sequencing.
Example 2
Sericin polypeptide antigens include a first polypeptide and a second polypeptide, and sericin 1 is detected. The detection can be carried out on the sample with the sericin 1 content of more than 1 ug/mg. The amino acid sequence of polypeptide one is: GSSSNTDSSTKNAGSRT, the amino acid sequence of polypeptide two is: FKNIFDIPYHLRKNIGV.
Example 3
Sericin polypeptide antigens comprise a third polypeptide, a fourth polypeptide and a fifth polypeptide, and sericin 2 is detected. The detection can be carried out on the sample with the sericin 2 content of more than 1 ug/mg.
The amino acid sequence of polypeptide three is: TEKAKPNDRSPSDRD, the amino acid sequence of polypeptide four is: SAERTKSKRGERKEVEG, the amino acid sequence of polypeptide five is: NRVVEKSTDGDNEESYRS.
Example 4
Sericin polypeptide antigens include polypeptide six and polypeptide seven, and sericin 3 is detected. The sample containing more than 1ug/mg of sericin 3 can be detected.
The amino acid sequence of polypeptide six is: EESSNGGSGSGRTGSAGGT, the amino acid sequence of polypeptide seven is: GGGRGRGSGVRRLDSG.
Example 5
The application of the silk sericin polypeptide antibody is the application of the silk sericin polypeptide antibody in preparing a kit for detecting sericin.
Example 6
Active site loops (a) through (g) (including the two terminal amino acid residues shown) to include amino acid residues in the region between amino acid residues 1201-1217. Amino acid residues in the region between amino acid residues 1201-1217 are also included, as are amino acid residues in the regions between 741-757, 902-916, 214-230, 1710-1727, 205-223 and 35-51.
Ser-1B:
741-757 GSSSNTDSSS TKAGSRT polypeptide one
21-37:GHHPGNRDTVEVKNRKY
1201-1217 FKNIFDIPYIHLRKNIGV polypeptide two
>Ser-2-2:
902-916 TEKAKPNDRSPSDRD polypeptide three
214-230 SAERKSKRGERKEVEG polypeptide four
1710-1727 NRVVEKESTDGDNEESYRS polypeptide five
>Ser-3-2:
482-499:DEDSDDSSGATKGNSSKS
205-223 EESSNGGSGSGRTGSAGGT polypeptide six
35-51 GGGRGRGSGVRRRLDSG polypeptide seven
Example 7
The specific preparation steps of the sericin polypeptide antibody of the invention are as follows:
immunization procedure:
(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 adopting a chain enzyme method, and extracting to obtain sericin polypeptide; safety of biological material (high temperature high pressure method), cytotoxicity in vitro; as a surrogate for serum; the enzyme labeling is performed in the process, wherein the enzyme is horseradish peroxidase. The method of enzyme labelling is conventional.
(2) Coupling the polypeptide with KLH as an immune antigen, and coupling the polypeptide with BSA as a detection antigen, wherein the KLH coupling agent is Sulfo-SMCC, and the BSA coupling agent is glutaraldehyde;
(3) Diluting the immune antigen to 1mg/ml with PBS, and packaging and freezing in a refrigerator at-20deg.C;
the preparation method of the PBS comprises the following steps:
(1) PBS stock solution (10×) formulation method:
wherein 2.85g of Na2HPO4.12H2O can be 1.13g of Na2HPO4.2H2O, and the raw materials are dissolved in 100ml of deionized water to adjust the pH value to 7.2;
(2) PBS was prepared using the liquid preparation method (1×): adding 450ml of deionized water into 50ml of the storage solution, and keeping at room temperature or 4 ℃ for standby;
(4) On day 1, 1ml of Freund's complete adjuvant is added into 1ml of each antigen, and the mixture is emulsified until one drop of emulsified antigen liquid is dripped into normal saline and not dispersed, which indicates that the emulsification has reached the requirement, then the mixture is subcutaneously injected at multiple points at the back of the neck, the number of injection points is not less than 8, and each antigen is used for immunizing 2 New Zealand white rabbits;
(5) On day 15, 1ml of Freund's incomplete adjuvant is added into 1ml of each antigen, emulsification is carried out, then subcutaneous multipoint injection is carried out on the back of the neck, the injection point is not less than 8 points, and each antigen is used for immunizing 2 New Zealand white rabbits;
(6) On day 29, 1ml of Freund's incomplete adjuvant is added into 1ml of each antigen, emulsification is carried out, then subcutaneous multipoint injection is carried out on the back of the neck, the injection point is not less than 8 points, and each antigen is used for immunizing 2 New Zealand white rabbits;
(7) On day 43, 1ml of Freund's incomplete adjuvant is added into 1ml of each antigen, emulsification is carried out, then subcutaneous multipoint injection is carried out on the back of the neck, the injection point is not less than 8 points, and each antigen is used for immunizing 2 New Zealand white rabbits;
(8) On day 53, carotid artery blood was removed and rabbits were sacrificed;
(9) The rabbit blood was left overnight at 4℃and centrifuged at 10000rpm for 30 minutes at 4℃and the supernatant was collected;
antibody purification
(1) Attaching the polypeptide to an activated Sulfolink Resin to prepare an antigen affinity column, 1ml Sulfolink Resin coupled to 1mg of 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 rabbit serum through an antigen affinity column, draining off the solution, and collecting the flow-through;
(4) Balancing with 10 times of column volume PBS, and completely draining the solution;
(5) Adding 5ml of antibody eluent, collecting the eluent by a branch pipe, wherein each pipe comprises 1ml of antibody eluent, and the preparation method comprises the following steps: weighing 7.5g glycine, dissolving in 100ml deionized water, regulating pH to 2.7,4 ℃ with concentrated hydrochloric acid, and preserving for later use;
(6) Detecting absorbance at 280nm with the collected eluent, combining components with absorbance greater than 1.0, and dialyzing against PBS;
(7) Identification of antibodies after dialysis: detecting protein concentration by ultraviolet absorption method, determining antibody titer by Elisa, and finally obtaining polyclonal antibodies of 6 polypeptide fragments of sericin 1, sericin 2 and sericin 3.
Example 7
Elisa method antibody immunoassay:
(1) Coating: coating antigen with antigen coating liquid, diluting the coated antigen to 1ug/ml with CBS, adding into an ELISA plate, and standing overnight at 4 ℃ per well; the preparation method of the antigen coating liquid (CBS, 1X) comprises the following steps: weighing Na2CO3 1.59g,NaHCO3 2.93g, 950ml of deionized water, adjusting the pH value to 9.6, adding deionized water to 1000ml of constant volume, and preserving at 4 ℃;
(2) Closing: the coating solution is discarded, 200ul of sealing solution is added into each hole, and the mixture is placed for 1.5 hours at 37 ℃, wherein the sealing solution is prepared by the following steps: weighing 5g of skimmed milk powder, dissolving in 100ml of PBS, uniformly mixing, and preserving at 4 ℃ for standby, wherein the storage time is less than or equal to 3 days;
(3) Adding a sample: discarding the sealing solution, adding serum or antibody sample, adding an ELISA plate into 100ul of each hole, 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 100ul of each hole, and standing at 37 ℃ for 30 minutes;
(6) Washing: washing with tap water for 10 times, and drying;
(7) Adding TMB chromogenic substrate: adding an ELISA plate into 100ul of each hole, and standing at 37 ℃ for 15 minutes; the preparation method of the TMB chromogenic substrate comprises the following steps:
(1) TMB stock solution: weighing TMB powder, preparing 1.5mg/ml stock solution with DMSO, and sub-packaging at-20deg.C;
(2) NaAc buffer: preparing a 200mM NaAc solution, and regulating the pH value to 5.3 by using HAc;
(3) H2O2 solution: preparing 0.03% H2O2 solution;
(4) the use is carried out according to TMB stock solution: naAc buffer: preparing a color development solution according to the ratio of H2O2 solution=1:4:5, and preparing the color development solution for use at present;
(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 preserving at room temperature for later use;
(9) Reading: and (3) reading by using an enzyme-labeled instrument OD450nm, and obtaining an OD value to obtain the number of the antibodies.
Example 8
Other content is the same, add the secondary antibody: diluting the enzyme-labeled goat anti-rabbit with a sealing solution to a working concentration, adding an enzyme-labeled plate into 100ul of each hole, and standing at 37 ℃ for 30 minutes; the enzyme labeling is performed in an elisa 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 and 0.57mg/ml;374-1-R2 antibody 1.8ml,1.02mg/ml, 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 and 1.85mg/ml;374-2-R2 antibody 3.1ml,1.11mg/ml, detailed detection results are shown in the following Table:
(3) Number 374-3 (polypeptide three TEKAKPNDRSPSDRD)
Finally obtaining 374-3-R1 antibody 2.5ml and 0.89mg/ml;374-3-R2 antibody 1.1ml,0.73mg/ml, detailed detection results are shown in the following Table:
(4) Number 374-4 (polypeptide four: SAERTKSKRGERKEVEG)
Finally obtaining 3.3ml of 374-4-R1 antibody and 1.3mg/ml;374-4-R2 antibody 3.3ml,0.87mg/ml, 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 and 2.5mg/ml;374-5-R2 antibody 2.3ml,1.76mg/ml, 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 and 1.7mg/ml;374-6-R2 antibody 3.1ml,0.54mg/ml, detailed detection results are shown in the following Table:
(7) Number 374-7 (polypeptide seven: GGGRGRGSGVRRLDSG)
Finally obtaining 1.7ml of 374-7-R1 antibody and 2.06mg/ml;374-7-R2 antibody 2.9ml,1.02mg/ml, detailed test results are shown in the following Table:
the potency measurement shows that the prepared antibody potency reaches 1:80000, and the method can be completely used for subsequent immune detection. The immunofluorescence detection result of the polypeptide antibody is shown in figure 2, and the detection point hybridization result of the polypeptide antibody is shown in figure 3. The results show that the prepared antibody can not be used for immunohistochemical experiments, and other antibodies can be used for in-situ detection and immunodetection of sericin except the antibody with the number of 374-4.
The result of immunodetection of tissue sample contents by selecting one of antibodies for preparing sericin 1, sericin 2 and sericin 3 shows that sericin at each part of silk gland can be specifically detected, and the result is shown in figure 4.
The cocoons and silk products in different processing processes are detected, and the situation that sericin is removed in the cocoon silk processing process can be detected by utilizing the antibody group. In silk reeling cocoons, sericin 2 protein is not contained, which is consistent with the reported result that sericin 2 protein exists only in cocoon nets, and the cocoon nets and the cocoon silks are removed from the silk reeling cocoons. In the silk reeling process, the sericin 3 protein is firstly removed, the sericin 1 protein can be effectively removed in a special refining process, and silk basically does not contain sericin, so that the result is shown in figure 5.
While the basic principles and main features of the present invention and advantages thereof have been shown and described, the foregoing embodiments and description are merely illustrative of the principles of the present invention, and various changes and modifications can be made therein without departing from the spirit and scope of the invention, which is defined in the appended claims. 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 silk sericin, preparation method and application thereof
<160> 4
<170> PatentIn version 3.3
<210> 1
<211> 17 AA
<212> POLYPEPTIDE
<213> Synthesis
<220>
<223> polypeptide one
<400> 1
gsssntdsstknagsrt 17
<210> 2
<211> 17 AA
<212> POLYPEPTIDE
<213> Synthesis
<220>
<223> polypeptide two
<400> 2
fknifdipyhlrknigv 17
<210> 3
<211> 15 AA
<212> POLYPEPTIDE
<213> Synthesis
<220>
<223> polypeptide three
<400> 3
tekakpndrspsdrd 15
<210> 4
<211> 17 AA
<212> POLYPEPTIDE
<213> Synthesis
<220>
<223> polypeptide four
<400> 4
saertkskrgerkeveg 17
<210> 5
<211> 21 AA
<212> POLYPEPTIDE
<213> Synthesis
<220>
<223> polypeptide five
<400> 5
nrvvekstdgdneesyrs 18
<210> 6
<211> 19 AA
<212> POLYPEPTIDE
<213> Synthesis
<220>
<223> polypeptide six
eessnggsgsgrtgsaggt 19
<400> 6
<210> 7
<211> 16 AA
<212> POLYPEPTIDE
<213> Synthesis
<220>
<223> polypeptide seven
<400> 7
gggrgrgsgvrrldsg 16
Claims (7)
1. A polyclonal antibody for direct detection of silk sericin 3, characterized in that: the antigen comprises a polypeptide six, wherein the amino acid sequence of the polypeptide six is shown as SEQ ID NO. 6.
2. A polyclonal antibody for direct detection of silk sericin 3 according to claim 1, characterized in that: the antigen also comprises at least one of a first polypeptide, a second polypeptide, a third polypeptide, a fourth polypeptide, a fifth polypeptide and a seventh polypeptide, wherein the amino acid sequence of the first polypeptide is shown as SEQ ID NO.1, the amino acid sequence of the second polypeptide is shown as SEQ ID NO.2, the amino acid sequence of the third polypeptide is shown as SEQ ID NO.3, the amino acid sequence of the fourth polypeptide is shown as SEQ ID NO.4, the amino acid sequence of the fifth polypeptide is shown as SEQ ID NO.5, and the amino acid sequence of the seventh polypeptide is shown as SEQ ID NO. 7.
3. The method for preparing the polyclonal antibody for directly detecting the silk sericin 3 according to claim 1, wherein the method comprises the following steps: coupling the polypeptide six with KLH as an immune antigen, respectively diluting the immune antigen by PBS, adding the antigen into Freund's incomplete adjuvant, emulsifying and injecting, centrifuging the blood of the small animal and purifying the antibody.
4. A method for preparing polyclonal antibodies for direct detection of silk sericin 3 according to claim 3, characterized in that: stock solution of PBS (10×) formulation method:
wherein 2.85g of Na 2 HPO 4 ·12H 2 O or Na of 1.13g 2 HPO 4 ·2H 2 O, the above raw materials are dissolved in 100ml deionized water, and the pH value is adjusted to 7.2.
5. A method for preparing polyclonal antibodies for direct detection of silk sericin 3 according to claim 3, characterized in that: the antibody purification step is followed by an immunoassay step in which enzyme labeling is performed again when a secondary antibody is added.
6. The method for preparing polyclonal antibody for directly detecting silk sericin 3 according to claim 5, wherein: the enzyme in the enzyme label is horseradish peroxidase.
7. Use of a polyclonal antibody for direct detection of silk sericin 3 according to claim 1, characterized in that: the application of the polyclonal antibody of the silk sericin 3 in preparing a kit for detecting the silk sericin 3.
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