CN114805566B

CN114805566B - Mulberry silk fibroin heavy chain protein polyclonal antibody and preparation method and application thereof

Info

Publication number: CN114805566B
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: 2023-06-20
Anticipated expiration: 2042-05-16
Also published as: CN114805566A

Abstract

The invention relates to the technical field of biology, in particular to a mulberry silk fibroin heavy chain protein polyclonal antibody, a preparation method and application thereof, wherein an antigen 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 mulberry silk fibroin products and the detection method of the current mulberry silk products, the invention prepares the polyclonal antibody of the main component silk fibroin heavy chain of the mulberry silk fibroin by utilizing the principle that the mulberry silk fibroin fibers are proteins and the antigen-antibody affinity binding principle.

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, a preparation method and application thereof.

Background

The mulberry silk is a natural high molecular protein fiber and is mainly divided into two main types of mulberry silk and wild silkworm silk. China is the country which makes clothes by using wild silks at the earliest time and domesticates the wild silks into silkworms, and has culture history of cultivated silkworms for nearly 5000 years. The mulberry silk is a continuous long fiber formed by solidifying a silk secretion liquid during the cocoon formation of cooked silkworms, is a natural fiber, is one of the earliest animal fibers used by human beings, and is mainly used for manufacturing fiber clothing materials into clothing and home textile fabrics; at present, substances such as silk fibroin and sericin extracted from mulberry silk have anti-inflammatory, antibacterial and antioxidant activities and good biocompatibility, and are widely applied to the fields of medicines, chemical industry, foods, materials and the like.

The mulberry silk is a part formed by two parts, and the part formed by peripheral collagen is called sericin (sericin); the portion of the core consisting of the crystallized protein is called silk fibroin (silk); the silk glue wraps two silk fibroin fibers to form a silk. Sericin and silk fibroin belong to macromolecular 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% of the mass, sericin accounts for about 25%, and other mediums account for about 5%. The silk fibroin mainly comprises silk fibroin heavy chain (Fibroin heavy chain, fib-h), silk fibroin light chain (Fibroin light chain, fib-l) and P25 protein with molecular weight of 25kDa according to a 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 repeats and 11 non-crystalline regions and NC ends. Fib-l consists of a domain-free protein of 256 amino acid residues with a molecular weight of 25KDa, wherein the S-bond at the C-terminus is associated with the silk heavy chain, the heavy and light chains of 6 molecules form silk secretion vesicles with the P25 protein of 1 molecule, which are secreted into the silk gland lumen, then change from the gel state into the sol state in the silk gland lumen, and are secreted from the oral cavity in the form of fibers by the action of the anterior silk gland after mixing the middle silk gland with sericin.

Because of low immunogenicity of mulberry silk fibroin to human body, silk fibroin is prepared into various biological materials such as silk sponge, silk bracket, nano silk fibroin particles and the like which are used for drug slow release carriers, nerve and vessel regeneration brackets and the like through dissolution. With the wide application of silk fibroin, molecular techniques for qualitative detection of its components are urgently needed. Meanwhile, corresponding molecular detection means are also missing for silk products sold in the market at present, so that consumers are often deceived by silk counterfeit products to lose trust of the silk products. The current method for detecting mulberry silk is mainly a physical method, or is to use pasteurization liquid to dissolve the mulberry silk or use fire to detect whether the mulberry silk emits the taste of protein combustion or not to judge the mulberry silk. But these methods may be misdetected. Similar results are obtained with protein based fibers either using pasteurized or combustion methods. Therefore, development of a molecular diagnosis method capable of detecting mulberry silk fibroin 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 corresponding molecular detection means are lost due to silk products 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, wherein the amino acid sequence of Fib-h-1 is shown as SEQ ID NO.1, the amino acid sequence of Fib-h-2 is shown as SEQ ID NO.2, the amino acid sequence of Fib-h-3 is shown as SEQ ID NO.3, and the amino acid sequence of Fib-h-4 is shown as SEQ ID NO. N.

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

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

Further, the antigen includes polypeptide Fib-h-1, polypeptide Fib-h-2, polypeptide Fib-h-3 and polypeptide Fib-h-4.

The polypeptide sequences are shown below:

SEQ ID NO.1：GGYSRSDGYEY

SEQ ID NO.2：AWSSESDFGT

SEQ ID NO.3：SASSRSYDYSRRNV

SEQ ID NO.4：QITTKKMQRKNK

a method for preparing polyclonal antibody of heavy chain protein of mulberry silk fibroin comprises immunizing animals with polypeptide antigen and KLH carrier protein, collecting blood, detecting, collecting blood, preparing antiserum, and purifying.

Further, the animal is immunized subcutaneously, once every 2-3 weeks, 4 times.

And further, the purification is to couple the polypeptide antigen with agarose medium to prepare an antigen affinity purification chromatographic column, mix the obtained antiserum with PBS in equal quantity, slowly sample, and elute with glycine elution buffer after the antigen-antibody is combined, thus obtaining the required purified antibody.

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 disclosed, which is used for preparing a kit for detecting mulberry silk.

The application of the mulberry silk fibroin heavy chain protein polyclonal antibody is used for molecular detection of whether silk fibroin is contained in mask liquid.

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

1. in view of the wide application of mulberry silk fibroin products and the detection method of the current mulberry silk products, the invention prepares the polyclonal antibody of the main component silk fibroin heavy chain of the mulberry silk fibroin by utilizing the principle that the mulberry silk fibroin fibers are proteins and the antigen-antibody affinity binding principle.

2. The invention analyzes the immunocompetent polypeptide locus of the mulberry silk fibroin, artificially synthesizes the polypeptide of the silk fibroin heavy chain, then mixes the polypeptide with an adjuvant to be used as a complete antigen to be injected on living animals, performs animal immunization, and obtains the polyclonal antibody of 4 polypeptide fragments after purification. The WB experiment can qualitatively judge the silk fibroin of the mulberry silk; the antibody can be used for carrying out an immune experiment of a silk gland tissue section of a family to study the secretion and crystallization process of silk protein; meanwhile, biological products such as facial masks, cosmetics and medical dressings containing mulberry silk fibers can be identified, and whether the fibers contain mulberry silk can be also identified.

3. The antibody can be used for detecting the mask with mulberry silk fiber as the base cloth in the market at present; the method can also be used for molecular detection of whether the mask liquid contains silk fibroin.

Drawings

In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings that are needed in the embodiments will be briefly described below, it being understood that the following drawings only illustrate some embodiments of the present invention and therefore should not be considered limiting the scope, and other related drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.

FIG. 1 is a diagram showing the specificity detection 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 diagram of the mulberry silk fibroin heavy chain protein polypeptide antibody and the antigen in the silk gland cell of the present invention;

FIG. 3 is an immunoblotting detection chart of total protein extracted from mulberry silk fibroin heavy chain protein polypeptide antibody and silk gland cells;

FIG. 4 is a diagram showing the detection of cocoons of different species and different protein fibers by using antibodies, wherein A is a diagram showing the morphology of various cocoon sheets and protein fibers; b, rabbit Ig-G primary antibody is used as a WB negative control result; WB results of Ab-Fib-h antibody; d: the result of the examination and dyeing of the sample proteins of each sample;

FIG. 5 is a graph showing the result of detecting that silk mask base cloth contains mulberry fibroin by using an antibody;

FIG. 6 is a graph showing the results of detecting whether or not silk fibroin is contained in a mask liquid by using an antibody.

Detailed Description

For the purpose of making 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 clearly and completely described below with reference to the accompanying drawings in the embodiments of the present invention, and it is apparent that the described embodiments are some embodiments of the present invention, but not all embodiments.

Example 1

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

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

antibody purification

The polypeptide Fib-h-1, fib-h-2, fib-h-3 and Fib-h-4 are respectively coupled with agarose medium to prepare antigen affinity purification chromatographic column, the obtained antiserum is mixed with PBS in equal quantity and then is slowly loaded, after the antigen and antibody are combined, glycine elution buffer is used for eluting, the required purified antibody is obtained, 4-degree dialysis is immediately carried out overnight in PBS, and purity, concentration and titer are measured every other day;

antibody identification

The titers of the purified antibodies were measured by ELISA as Ab-Fib-h-1>1:512,000, respectively; ab-Fib-h-2>1:128,000; ab-Fib-h-3>1:512,000, ab-Fib-h-4>1:512,000, and measuring the concentration of the obtained antibody by using a BCA protein concentration measuring kit, wherein the concentration of the antibody is 5.4mg/ml respectively; ab-Fib-h-2 is 2.1mg/ml; ab-Fib-h-3 is 3.8mg/ml; ab-Fib-h-4 was 1.45mg/ml, and the results of dot hybridization of the antigen polypeptide and the antibody are shown in FIG. 1, which shows that each antibody recognizes only self antigen and has good antigen recognition.

Example 2

Antigen recognition of mulberry silk gland tissue silk fibroin heavy chain protein

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

Example 3

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

Cutting silkworm cocoons, silk and various fabrics into 5mg respectively in a centrifuge tube, adding 600 mul of 9M saturated lithium hydrosulfide after cutting, vibrating for 30min at room temperature, then placing into a 60 ℃ oven for dissolving for more than 5h, centrifuging at 4 ℃ for 10min at 10000rpm, taking the supernatant, and diluting 10 times by 8M urea to perform dot hybridization experiments. As shown in FIG. 4, from the results of the dot hybridization experiments, each antibody of Fib-h can detect a strong signal in silk products provided by cocoons, raw silk, cooked silk and Jiahua silk company, and no signal in silk fabrics purchased from Taobao net, indicating that it does not contain mulberry silk, and is an obvious counterfeit product of mulberry silk.

Example 4

Detecting whether mulberry silk mask base cloth contains mulberry silk fibers

The antibody of example 1 was used to detect that mulberry silk fiber is currently used as a basic mask in the market. The specific operation steps are as follows: firstly, cutting various mask base fabrics, washing for 3 times, flattening and drying at 60 ℃, cutting the base fabrics into pieces in a centrifuge tube, and adding 200uL CaCl ₂ The ternary solution was vortexed by a vortexing device for 10min, dissolved at 60℃for 1h, centrifuged at 10000rpm for 10min at 4℃to obtain a supernatant, which was subjected to 10-fold dilution with 8M urea for dot hybridization experiments, and the step of dot hybridization was the same as that of the antibody-specific detection portion of example 1. In this example, cocoons of a large silkworm breed were used as positive controls. As shown in fig. 5, from the dot hybridization exposure chart, 4 antibodies of Fib-h detected a clear signal in the large cut cocoon shell solution, while in each mask base cloth solution, only signals were present in the clear face and soothing and moisturizing silk mask, indicating that the two mask base cloths contained mulberry silk components. No signal was detected by any other mask, even the base cloth identified as silk mask, indicating that these did not contain mulberry silk fibers. The result shows that the antibody can sensitively detect whether the mask base cloth contains mulberry silk fibers.

Example 5

Detecting whether silk fibroin is contained in mask liquid

The antibody in example 1 was used to detect whether silk fibroin was contained in a mask solution or other mask solutions currently on the market using mulberry silk fiber as a base fabric. Taking the front content of middle silk gland of 7 days of 5 years old, diluting with 1 x PBS (PH=7.4) for 25 times, and performing dot hybridization experiment with 2 times of dilution liquid of 3 mask liquid of clear and clean silk mask, relaxing and moisturizing silk mask and chitosan biomembrane body. From the point hybridization experimental result, as shown in fig. 6, the 4 antibodies of Fib-h detect obvious signals in 25-fold dilution of the content of positive control DZ silk gland tissue, and weak signals are detected in the silk mask base cloth mask liquid, which indicates that the mask liquid of the two silk mask base cloths contains trace silk fibroin, and it is presumed that the silk fibroin in the mask base cloth is dissolved in the mask liquid in trace. Another non-silk mask is completely free of silk fibroin.

The above is an embodiment exemplified in this example, but this example is not limited to the above-described alternative embodiments, and a person skilled in the art may obtain various other embodiments by any combination of the above-described embodiments, and any person may obtain various other embodiments in the light of this example. The above detailed description should not be construed as limiting the scope of the present embodiments, which is defined in the claims and the description may be used to interpret the claims.

Sequence listing

　　SEQUENCE LISTING

<110> university of southwest

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

　　<160> 4

　　<170> PatentIn version 3.3

　　<210> 1

　　<211> 11 bp

　　<212> DNA

<213> Synthesis

　　<220>

　　<223> Fib-h-1

　　<400> 1

GGYSRSDGYEY 11

　　<210> 2

　　<211> 10 bp

　　<212> DNA

<213> Synthesis

　　<220>

　　<223> Fib-h-2

　　<400> 2

AWSSESDFGT 10

<210> 3

<211> 14 bp

<212> DNA

<213> Synthesis

<220>

<223> Fib-h-3

<400> 3

SASSRSYDYSRRNV 14

　　<210> 4

　　<211> 17 bp

　　<212> DNA

<213> 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 for preparing the antibody comprises a polypeptide Fib-h-4, and the amino acid sequence of the Fib-h-4 is shown as SEQ ID NO. 4.

2. A mulberry silk fibroin polyclonal antibody according to claim 1, wherein: the antigen for preparing the antibody also comprises at least one of polypeptide Fib-h-1, polypeptide Fib-h-2 or polypeptide Fib-h-3, wherein 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, and the amino acid sequence of the Fib-h-3 is shown as SEQ ID NO. 3.

3. A mulberry silk fibroin polyclonal antibody according to claim 2, wherein: the antigen for preparing the antibody also comprises at least two of polypeptide Fib-h-1, polypeptide Fib-h-2 or polypeptide Fib-h-3.

4. A mulberry silk fibroin polyclonal antibody according to claim 2, wherein: antigens from which the antibodies are made also include polypeptide Fib-h-1, polypeptide Fib-h-2 and polypeptide Fib-h-3.

5. The method for preparing the mulberry silk fibroin heavy chain protein polyclonal antibody according to any one of claims 1 to 4, wherein: coupling the polypeptide antigen with KLH carrier protein, immunizing animals by using the polypeptide-KLH, carrying out blood sampling detection, carrying out final blood sampling to prepare antiserum, and purifying.

6. The method for preparing the mulberry silk fibroin polyclonal antibody according to claim 5, wherein the method comprises the following steps: the animal immunization is subcutaneous immunization, and the immunization is performed once for 2-3 weeks and 4 times.

7. The method for preparing the mulberry silk fibroin polyclonal antibody according to claim 5, wherein the method comprises the following steps: the purification is to prepare an antigen affinity purification chromatographic column by coupling the polypeptide antigen with agarose medium, mix the obtained antiserum with PBS in equal quantity, slowly sample, and elute with glycine elution buffer after the antigen-antibody is combined, thus obtaining the required purified antibody.

8. Use of a mulberry silk fibroin heavy chain protein polyclonal antibody according to any one of claims 1-4, wherein: for detecting whether silk fibroin is contained.

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

10. The use of a mulberry silk fibroin polyclonal antibody according to claim 8, wherein: the method is used for molecular detection of whether silk fibroin is contained in the mask liquid.