CN114805566A

CN114805566A - Mulberry silk fibroin heavy chain protein polyclonal antibody and preparation method and application thereof

Info

Publication number: CN114805566A
Application number: CN202210526317.3A
Authority: CN
Inventors: 刘春�; 魏欣; 李豪; 徐永平; 崔红娟; 程廷才
Original assignee: Southwest University
Current assignee: Southwest University
Priority date: 2022-05-16
Filing date: 2022-05-16
Publication date: 2022-07-29
Anticipated expiration: 2042-05-16
Also published as: CN114805566B

Abstract

The invention relates to the technical field of biology, in particular to a mulberry silk fibroin heavy chain protein polyclonal antibody and a preparation method and application thereof, wherein an antigen of the polyclonal antibody comprises polypeptide Fib-h-1, polypeptide Fib-h-2, polypeptide Fib-h-3 or polypeptide Fib-h-4, the amino acid sequence of the Fib-h-1 is shown as SEQ ID NO.1, the amino acid sequence of the Fib-h-2 is shown as SEQ ID NO.2, the amino acid sequence of the Fib-h-3 is shown as SEQ ID NO.3, and the amino acid sequence of the Fib-h-4 is shown as SEQ ID NO. N. In view of the wide application of the mulberry silk fibroin product and the detection method of the mulberry silk product at present, the invention utilizes the principle that the mulberry silk fibroin fiber is protein and utilizes the principle of antigen-antibody affinity combination to prepare the polyclonal antibody of the main component fibroin heavy chain of the mulberry silk fibroin.

Description

Mulberry silk fibroin heavy chain protein polyclonal antibody and preparation method and application thereof

Technical Field

The invention relates to the technical field of biology, in particular to a mulberry silk fibroin heavy chain protein polyclonal antibody and a preparation method and application thereof.

Background

Mulberry silk is a natural high molecular protein fiber and mainly divided into two major categories, namely, silkworm silk and wild silkworm silk. China is the country which uses wild silkworms to produce clothes and domesticate wild silkworms to become silkworms at the earliest, and has nearly 5000 years of cultural history of silkworm cultivation. The mulberry silk is a continuous long fiber formed by coagulating secreted silk liquid when the mature silkworms are cocooned, is a natural fiber, is one of the earliest animal fibers utilized by human beings, and is mainly used for manufacturing fiber clothing materials into clothes and home textile fabrics; at present, silk fibroin, sericin and other substances extracted from mulberry silk are widely applied to the fields of medicines, chemical engineering, foods, materials and the like due to the anti-inflammatory, antibacterial and antioxidant activities and good biocompatibility.

The mulberry silk is composed of two parts, wherein the part composed of collagen at the periphery is called sericin (sericin); the portion of the inner core made of crystalline protein is called fibroin (fibriin); the sericin wraps the two silk fibroin fibers to form the silk. Sericin and fibroin both belong to macromolecular proteins, but the structures and amino acid components of the sericin and the fibroin are different. Silk fibroin is the main component of silk, and accounts for about 70% of the mass, sericin accounts for about 25% of the mass, and other media account for about 5% of the mass. The silk Fibroin is mainly composed of Fibroin heavy chain (Fib-h), Fibroin light chain (Fib-l) and P25 protein with molecular weight of 25KDa according to molar ratio of 6:6: 1. The Fib-h protein contains 5632 amino acid residues and has a molecular weight of about 400KDa, and consists of 12 crystalline regions rich in GSGSGSA repetitive sequences, 11 amorphous regions and two ends of NC. Fib-l is composed of a domain-free protein which encodes 256 amino acid residues and has a molecular weight of 25KDa, wherein the C-terminal S bond is combined with a fibroin heavy chain, a fibroin secretory corpuscle formed by a 6-molecule heavy chain and a 6-molecule light chain and 1-molecule P25 protein is secreted into a silk gland cavity, then the silk gland cavity is changed from a gel state to a sol state, and the silk gland and sericin are mixed and then secreted from a mouth organ in a fiber form through the action of an anterior silk gland.

Because of low immunogenicity of mulberry silk fibroin to human body, silk fibroin is prepared into various forms of biomaterials by dissolution, such as silk sponge, silk fibroin scaffolds, nano silk fibroin particles and the like for drug sustained release carriers, nerve and blood vessel regeneration scaffolds and the like. With the wide application of silk fibroin, a molecular technology for qualitatively detecting components of silk fibroin is urgently needed. Meanwhile, the silk products sold on the market at present lack the corresponding molecular detection means, so that consumers are often deceived by silk counterfeit products to lose trust of the silk products. At present, the method for detecting the mulberry silk is mainly a physical method, or a pasteurization solution is used for dissolving the mulberry silk or a fire is used for detecting whether the mulberry silk has the smell of protein combustion or not for judging. But both of these methods may be misdetected. Similar results can be obtained by using pasteurization solution or combustion method for protein fiber. Therefore, the development of a molecular diagnostic method capable of detecting the silk fibroin of the mulberry silk is imperative.

Disclosure of Invention

The invention aims to provide a mulberry silk fibroin heavy chain protein polyclonal antibody, a preparation method and application thereof, and solves the technical problem that silk products lack corresponding molecular detection means in the prior art.

The invention discloses a mulberry silk fibroin heavy chain protein polyclonal antibody, the antigen of which comprises polypeptide Fib-h-1, polypeptide Fib-h-2, polypeptide Fib-h-3 or polypeptide Fib-h-4, the amino acid sequence of Fib-h-1 is shown in SEQ ID NO.1, the amino acid sequence of Fib-h-2 is shown in SEQ ID NO.2, the amino acid sequence of Fib-h-3 is shown in SEQ ID NO.3, and the amino acid sequence of Fib-h-4 is shown in SEQ ID NO. N.

Furthermore, the antigen comprises at least two of the polypeptide Fib-h-1, the polypeptide Fib-h-2, the polypeptide Fib-h-3 or the polypeptide Fib-h-4.

Furthermore, the antigen comprises at least three of polypeptide Fib-h-1, polypeptide Fib-h-2, polypeptide Fib-h-3 or polypeptide Fib-h-4.

Furthermore, the antigen comprises polypeptide Fib-h-1, polypeptide Fib-h-2, polypeptide Fib-h-3 and polypeptide Fib-h-4.

The polypeptide sequences are respectively shown as follows:

SEQ ID NO.1：GGYSRSDGYEY

SEQ ID NO.2：AWSSESDFGT

SEQ ID NO.3：SASSRSYDYSRRNV

SEQ ID NO.4：QITTKKMQRKNK

a preparation method of a mulberry silk fibroin heavy chain protein polyclonal antibody comprises the steps of immunizing animals with polypeptide-KLH by polypeptide antigen and KLH carrier protein, collecting blood after detection to prepare antiserum, and purifying.

Further, the animal immunization is subcutaneous immunization, and the immunization is carried out once in 2-3 weeks and 4 times.

Further, the purification is that the polypeptide antigen and an agarose medium are coupled to prepare an antigen affinity purification chromatographic column, the obtained antiserum and PBS are mixed in equal amount and then are slowly loaded, and after the antigen antibody is combined, glycine elution buffer solution is used for elution, so that the purified antibody required is obtained.

An application of a mulberry silk fibroin heavy chain protein polyclonal antibody is used for detecting whether silk fibroin is contained.

An application of a mulberry silk fibroin heavy chain protein polyclonal antibody is used for preparing a kit for detecting mulberry silk.

An application of a mulberry silk fibroin heavy chain protein polyclonal antibody is used for detecting whether a facial mask solution contains fibroin protein molecules.

Compared with the prior art, the invention has the beneficial effects that:

1. in view of the wide application of the mulberry silk fibroin product and the detection method of the mulberry silk product at present, the invention utilizes the principle that the mulberry silk fibroin fiber is protein and utilizes the principle of combining the affinity of antigen and antibody to prepare the polyclonal antibody of the fibroin heavy chain which is the main component of the mulberry silk fibroin.

2. The invention analyzes immune polypeptide sites of mulberry silk fibroin, artificially synthesizes polypeptide of fibroin heavy chain, then mixes the polypeptide with adjuvant as complete antigen to be injected to living animals for animal immunization, and obtains polyclonal antibody of 4 polypeptide segments after purification. Through WB experiments, the mulberry silk fibroin can be qualitatively judged; the antibody can be used for carrying out an immunity experiment of a bombyx mori silk gland tissue slice, and researching the secretion and crystallization process of silk protein; meanwhile, biological products containing mulberry silk fibers, such as facial masks, cosmetics and medical dressings, can be identified, and whether the fibers contain the mulberry silk or not can be identified.

3. The antibody can be used for detecting the mask which uses mulberry silk fiber as base cloth on the market at present; also can be used for the molecular detection of whether the facial mask solution contains silk fibroin.

Drawings

In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings needed to be used in the embodiments will be briefly described below, it should be understood that the following drawings only show some embodiments of the present invention, and therefore should not be considered as limiting the scope, and for those skilled in the art, other related drawings can be obtained according to the drawings without inventive efforts.

FIG. 1 is a specific detection diagram of a mulberry silk fibroin heavy chain protein polypeptide antibody and a corresponding antigen according to the present invention;

FIG. 2 is an in situ immunofluorescence detection map of mulberry silk fibroin heavy chain protein polypeptide antibody and antigen in silk gland cells according to the present invention;

FIG. 3 is an immunoblot detection map of total protein extracted from mulberry silk fibroin heavy chain polypeptide antibody and silk gland cells of the present invention;

FIG. 4 is a diagram of detection of cocoons of different species and different protein fibers by using antibodies, wherein A is a diagram of various silkworm cocoon sheets and protein fiber morphologies; b, using rabbit Ig-G primary antibody as WB negative control result; WB results for Ab-Fib-h antibody; d: the test staining results of the loading protein of each sample;

FIG. 5 is a result graph of detection of silkworm fibroin contained in a base cloth of a silk mask by using an antibody;

FIG. 6 is a graph showing the results of detecting the presence or absence of silk fibroin in the facial mask solution using antibodies.

Detailed Description

In order to make the objects, technical solutions and advantages of the embodiments of the present invention more apparent, the technical solutions of the embodiments of the present invention will be described clearly and completely with reference to the accompanying drawings of the embodiments of the present invention, and it is obvious that the described embodiments are some, but not all embodiments of the present invention.

Example 1

A method for preparing mulberry silk fibroin heavy chain protein polyclonal antibody comprises the following steps of animal immunization

The polypeptides Fib-h-1, Fib-h-2, Fib-h-3 and Fib-h-4 are respectively coupled with KLH carrier protein, and 2 New Zealand white rabbits (2-2.5KG) are respectively immunized by using the polypeptides-KLH, and are immunized for 500 ug/time, once in 2-3 weeks and 4 times. Blood sampling detection, namely determining the titer of antiserum against protein by an indirect ELISA method, finally sampling blood to prepare antiserum when the titer is more than 1:50,000, and preparing for purification;

antibody purification

Respectively coupling polypeptides Fib-h-1, Fib-h-2, Fib-h-3 and Fib-h-4 with an agarose medium to prepare an antigen affinity purification chromatographic column, mixing the obtained antiserum with PBS in equal amount, slowly loading the mixture, eluting with a glycine elution buffer solution after the antigen and antibody are combined to obtain the required purified antibody, immediately dialyzing in the PBS for 4 degrees overnight, and measuring the purity, concentration and titer every other day;

antibody identification

The titer of the purified antibody is Ab-Fib-h-1>1:512,000 by ELISA detection; Ab-Fib-h-2>1:128,000; Ab-Fib-h-3 is more than 1:512,000, Ab-Fib-h-4 is more than 1:512,000, and the concentration of the obtained antibody is measured by using a BCA protein concentration measuring kit, wherein the concentration of the Ab-Fib-h-1 is 5.4 mg/ml; Ab-Fib-h-2 is 2.1 mg/ml; Ab-Fib-h-3 is 3.8 mg/ml; Ab-Fib-h-4 is 1.45mg/ml, and the result is shown in figure 1 by point hybridization of antigen polypeptide and antibody, which shows that each antibody only recognizes autoantigen and has good antigen recognition.

Example 2

Recognition of antigen to silk fibroin heavy chain protein of mulberry silk gland tissue

The antibody obtained in the step of example 1 is used for immunofluorescence experiments on silk glands of 5-year-old silkworm DZ varieties, and as shown in the attached figure 2, the results show that the antibodies can obviously detect silk fibroin in silk gland cells and unassembled protein in partial gland cavities. Meanwhile, total protein of 5-year-old posterior silk gland of DZ variety is extracted, SDS-PAGE and immunoblotting experiments are carried out by using 3-8% gradient gel, and the result shows that the antibody can specifically recognize the fibroin heavy chain, as shown in figure 3. The research results show that the prepared fibroin heavy chain protein polypeptide antibody can well and specifically recognize fibroin heavy chain protein.

Example 3

Using the antibody of example 1, it was examined whether the silk cloth material contained mulberry silk.

Taking 5mg each of silkworm cocoon, silk and various cloth, shearing in a centrifuge tube, adding 600 μ l of 9M saturated lithium hydrosulfide after shearing, shaking for 30min at room temperature, then placing in a 60 ℃ oven for dissolving for more than 5h, centrifuging for 10min at 4 ℃ and 10000rpm, taking supernatant, and diluting with 8M urea by 10 times for dot hybridization experiment. As shown in FIG. 4, it can be seen from the results of the dot hybridization experiments that the antibodies of Fib-h can detect strong signals in silk products provided by cocoons, raw silk, boiled silk and Jiahua silk, but no signal is detected in silk fabrics purchased from Taobao, which indicates that the silk fabrics do not contain mulberry silk and are obvious mulberry silk imitation products.

Example 4

Detecting whether the mulberry silk facial mask base cloth contains mulberry silk fibers

The antibody in example 1 was used to detect the basic facial mask made of mulberry silk fiber on the market. The specific operation steps are as follows: firstly cutting various facial mask base fabrics, washing for 3 times, flattening and drying in an oven at 60 ℃, cutting the base fabrics into pieces in a centrifuge tube, adding 200uL CaCl ₂ And (3) vortexing the ternary solution by a vortexer for 10min, dissolving the ternary solution for 1h at 60 ℃, centrifuging the ternary solution for 10min at 4 ℃ and 10000rpm, taking supernatant, and diluting the supernatant by 10 times by using 8M urea to perform dot hybridization experiments, wherein the dot hybridization steps are the same as those of the antibody specificity detection part in example 1. In this example, cocoons of a large variety of silkworm were used as a positive control. As shown in FIG. 5, from the dot hybridization exposure chart, 4 antibodies of Fib-h detected obvious signals in the large-scale cocoon shell dissolution solution, and on each mask base clothIn the dissolving solution, signals are only provided in the silk mask for cleaning and cleaning the face and relieving and moisturizing the face, which indicates that the two mask base cloths contain mulberry silk components. No signal was detected for any of the other masks, even the base cloth identified as silk mask, indicating that none of these contained mulberry silk fibers. The results show that the antibody can sensitively detect whether the mask base cloth contains mulberry silk fibers.

Example 5

Detecting whether the facial mask liquid contains silk fibroin

The antibody in example 1 is used to detect whether the facial mask solution or other facial mask solutions with mulberry silk fibers as base cloth in the market contain silk fibroin. The anterior content of the middle silk gland of 7-day old 5 was diluted 25-fold with 1 XPBS (pH 7.4) and subjected to dot hybridization with 2-fold dilutions of 3 kinds of facial solutions of a clear facial silk mask, a soothing and moisturizing silk mask and a chitosan biomembrane. As shown in the figure 6 of the dot blot experiment result, the 4 antibodies of Fib-h detect obvious signals in the positive control DZ diluted solution with 25 times of silk gland tissue content, and detect weak signals in the silk mask base cloth mask solution, which indicates that the two silk mask base cloth mask solutions contain a trace amount of silk fibroin, and the fact that the silk fibroin in the silk mask base cloth is slightly dissolved in the mask solution is presumed. The other non-silk facial mask completely does not contain silk fibroin.

The above embodiments are exemplified by the present embodiment, but the present embodiment is not limited to the above optional embodiments, and persons skilled in the art can obtain other various embodiments by arbitrarily combining the above embodiments, and any person can obtain other various embodiments based on the teaching of the present embodiment. The above detailed description should not be construed as limiting the scope of the present embodiments, which should be defined in the claims, and the description should be used for interpreting the claims.

Sequence listing

　　SEQUENCE LISTING

<110> university of southwest

<120> mulberry silk fibroin heavy chain protein polyclonal antibody, preparation method and application thereof

　　<160> 4

　　<170> PatentIn version 3.3

　　<210> 1

　　<211> 11 bp

　　<212> DNA

<213> Artificial Synthesis

　　<220>

　　<223> Fib-h-1

　　<400> 1

GGYSRSDGYEY 11

　　<210> 2

　　<211> 10 bp

　　<212> DNA

<213> Artificial Synthesis

　　<220>

　　<223> Fib-h-2

　　<400> 2

AWSSESDFGT 10

<210> 3

<211> 14 bp

<212> DNA

<213> Artificial Synthesis

<220>

<223> Fib-h-3

<400> 3

SASSRSYDYSRRNV 14

　　<210> 4

　　<211> 17 bp

　　<212> DNA

<213> Artificial Synthesis

　　<220>

　　<223> Fib-h-4

　　<400> 4

QITTKKMQRKNK 12

Claims

1. A mulberry silk fibroin heavy chain protein polyclonal antibody is characterized in that: the antigen of the polypeptide Fib-h-1 polypeptide Fib-h-2 polypeptide Fib-h-3 polypeptide Fib-h-4 polypeptide Fib-h-1 amino acid sequence is shown in SEQ ID NO.1, Fib-h-2 amino acid sequence is shown in SEQ ID NO.2, Fib-h-3 amino acid sequence is shown in SEQ ID NO.3, and Fib-h-4 amino acid sequence is shown in SEQ ID NO. N.

2. The mulberry silk fibroin heavy chain polyclonal antibody of claim 1, which is characterized in that: the antigen comprises at least two of polypeptide Fib-h-1, polypeptide Fib-h-2, polypeptide Fib-h-3 or polypeptide Fib-h-4.

3. The mulberry silk fibroin heavy chain polyclonal antibody of claim 1, which is characterized in that: the antigen comprises at least three of polypeptide Fib-h-1, polypeptide Fib-h-2, polypeptide Fib-h-3 or polypeptide Fib-h-4.

4. The mulberry silk fibroin heavy chain polyclonal antibody of claim 1, which is characterized in that: the antigen comprises polypeptide Fib-h-1, polypeptide Fib-h-2, polypeptide Fib-h-3 and polypeptide Fib-h-4.

5. The method for preparing the mulberry silk fibroin heavy chain protein polyclonal antibody according to any one of claims 1-4, which is characterized in that: polypeptide antigen and KLH carrier protein are used, polypeptide-KLH is used for immunizing animals, blood collection is carried out after blood collection detection, antiserum is prepared, and then purification is carried out.

6. The method for preparing the mulberry silk fibroin heavy chain protein polyclonal antibody according to claim 5, which is characterized in that: the animal immunization is subcutaneous immunization, and is carried out once in 2-3 weeks and 4 times.

7. The method for preparing the mulberry silk fibroin heavy chain protein polyclonal antibody according to claim 5, which is characterized in that: the purification is that the polypeptide antigen and agarose medium are coupled to prepare an antigen affinity purification chromatographic column, the obtained antiserum and PBS are mixed in equal amount and then are slowly loaded, and after the antigen antibody is combined, glycine elution buffer solution is used for elution, thus obtaining the purified antibody.

8. The use of the mulberry silk fibroin heavy chain polyclonal antibody according to any one of claims 1-4, wherein: used for detecting whether silk fibroin is contained.

9. The use of the mulberry silk fibroin heavy chain polyclonal antibody according to claim 8, wherein: the application is to prepare a kit for detecting mulberry silk.

10. The use of the mulberry silk fibroin heavy chain polyclonal antibody according to claim 8, wherein: the method is used for molecular detection of whether the facial mask solution contains silk fibroin.