CN115850448A - Lactoferrin polypeptide fragment, preparation method thereof, prepared antibody and application - Google Patents
Lactoferrin polypeptide fragment, preparation method thereof, prepared antibody and application Download PDFInfo
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- CN115850448A CN115850448A CN202210957805.XA CN202210957805A CN115850448A CN 115850448 A CN115850448 A CN 115850448A CN 202210957805 A CN202210957805 A CN 202210957805A CN 115850448 A CN115850448 A CN 115850448A
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- Peptides Or Proteins (AREA)
Abstract
In one aspect of the application, the application provides a lactoferrin polypeptide fragment, wherein an amino acid sequence table of the lactoferrin polypeptide fragment is shown as SEQ ID N0.1. In a second aspect, the present application provides a method for preparing the lactoferrin polypeptide fragment, wherein an Fmoc protecting group is used to protect amino acids, then HMP resin is used to sequentially couple alanine, leucine, cysteine, glutamic acid, threonine, asparagine, aspartic acid, asparagine, phenylalanine, leucine, asparagine, and lysine in this order, and finally the resin is separated to prepare the target polypeptide fragment. In a third aspect, the application also provides a Lactoferrin antibody prepared by using the Lactoferrin polypeptide fragment, and a technical scheme for applying the Lactoferrin polypeptide fragment in preparation of a Lactoferrin flow antibody and in preparation of a Lactoferrin detection product. The optimal epitope is screened out, the designed polypeptide fragment is easy to prepare, and the prepared antibody has strong specificity, high sensitivity and good stability.
Description
Technical Field
The application relates to the technical field of biomedicine, in particular to a lactoferrin polypeptide fragment, a preparation method thereof, a prepared antibody and application.
Background
Lactoferrin (Lactoferrin), a widely distributed iron-binding glycoprotein with a molecular weight of about 80kDa, is a member of the transferrin family, in human and mammalian milk and in a variety of other tissues and secretions. Lactoferrin has a high expression level in neutrophils, has wide biological activities including broad-spectrum antibacterial action, anti-inflammation, tumor cell growth inhibition, organism immune response regulation and the like, is considered to be a novel antibacterial and anticancer drug and a food and cosmetic additive with great development potential, for example, lactoferrin has been allowed by the U.S. food and drug administration as a food additive for sports and functional foods. Lactoferrin belongs to a component substance of the innate immune system and has functions and attributes of antiviral, antiparasitic, catalytic, antiallergic, and radioprotective, in addition to its primary function of being able to bind and transport iron ions. As the application of lactoferrin becomes more and more extensive, the demand for qualitative and quantitative detection of lactoferrin also becomes more and more vigorous.
Currently, immunological detection is the most common method for qualitatively and quantitatively detecting lactoferrin in a sample, and has been applied to some extent in the fields of scientific research and food processing. However, lactoferrin antibodies, which are the core components of immunological assays, often have problems such as low potency and poor sensitivity. In addition, most antibodies commonly used in flow cytometry are obtained by immunizing animals with cells or cell extracts, and although the antibodies can recognize the cells, the epitope is unclear, the theoretical basis of experimental data is inaccurate, the preparation method is complex, the repeatability is poor, and the artificial control is not easy, so that the generated antibodies have errors, and the nonspecific reaction of the antibodies can have adverse effects on later experiments.
Disclosure of Invention
In order to solve at least one technical problem, an immunogen is developed to provide a basis for preparing a lactoferrin specific antibody, and the application provides a lactoferrin polypeptide fragment, a preparation method thereof, a prepared antibody and application.
In one aspect, the application provides a lactoferrin polypeptide fragment, wherein an amino acid sequence table of the lactoferrin polypeptide fragment is shown as SEQ ID N0.1.
By adopting the technical scheme, the designed lactoferrin polypeptide fragment has strong hydrophilicity, is convenient to synthesize and purify, and is suitable for mass production. Moreover, the lactoferrin polypeptide fragment designed by the technical scheme has strong immunogenicity, and an antibody prepared by the lactoferrin polypeptide fragment has good specificity and high sensitivity, has clear and sharp WB bands, and is very suitable for being applied to Flow Cytometry (FC) detection of human tissue lymphoma cells (U-937).
In a second aspect, the present application provides a method for preparing the above lactoferrin polypeptide fragment, comprising the steps of:
s1, activation: connecting amino of the amino acid with Fmoc protecting group by using protecting group reaction to prepare amino acid protected by Fmoc protecting group, and activating carboxyl to obtain carboxyl-activated amino acid containing Fmoc protecting group;
s2, connecting resin: reacting the carboxyl activated amino acid containing the Fmoc protecting group obtained in the step S1 with HMP resin to obtain resin connected with the carboxyl activated amino acid containing the Fmoc protecting group;
s3, deprotection group: carrying out deprotection reaction on the resin connected with the amino acid containing the Fmoc protecting group activated by the carboxyl prepared in the step S2 to activate the amino group of the amino acid;
s4, coupling: repeating the step S1 to prepare new amino acid containing Fmoc protecting group activated by carboxyl, and coupling the amino activated resin prepared in the step S3 with the new amino acid containing Fmoc protecting group activated by carboxyl to prepare resin of the amino acid containing Fmoc protecting group for coupling the new amino acid;
s5, preparing polypeptide resin: repeating the operation of the step S3 to remove the protecting group of the resin of the Fmoc protecting group-containing amino acid coupled with the new amino acid prepared in the step S4, then repeating the operation of the step S4 to couple the new amino acid, and repeating the step until the resin connected with the target Lactoferrin polypeptide fragment is prepared;
s6, separation and purification: separating the resin connected with the target Lactoferrin polypeptide fragment prepared in the step S5 by using resin to prepare a Lactoferrin polypeptide fragment crude product, and purifying to prepare a Lactoferrin polypeptide fragment;
in the steps S1-S5, amino acids are added in the order of alanine, leucine, cysteine, glutamic acid, threonine, asparagine, aspartic acid, asparagine, phenylalanine, leucine, asparagine, and lysine.
By adopting the technical scheme, the Lactoferrin polypeptide fragment can be conveniently produced, and the prepared polypeptide fragment has the advantages of accurate structure, short preparation period and lower preparation cost.
Optionally, the synthesis process of steps S1 to S5 is completed by using an automated polypeptide synthesizer.
By adopting the technical scheme, the preparation process is simpler, the polypeptide fragment structure is more accurate, the coupling rate is high, the period is short, the automation degree is high, and the cost can be effectively reduced.
Optionally, in step S1, the activating step is to activate the carboxyl group by reacting Fmoc-AA with DCC and HOBT, and the specific reaction formula is as follows:
by adopting the technical scheme, the activation reaction condition is mild, the carboxyl activation is easy to realize, and the DCC and the HOBT are matched, so that the racemization effect can be effectively inhibited in the peptide synthesis.
Optionally, in step S2, the carboxyl-activated Fmoc-AA is reacted with an HMP resin, and the carboxyl-activated Fmoc-AA is reacted with the HMP resin under DMAP conditions, wherein the reaction formula is as follows:
by adopting the technical scheme, the reaction condition is mild, the reaction yield is high, the resin connection sites are accurate, the side reactions are few, and the purity of the finally prepared polypeptide fragments can be effectively improved.
Optionally, in step S3, the Fmoc removal reaction is performed under the action of Piperidine, and the specific reaction formula is as follows:
by adopting the technical scheme, the cutting effect is optimal by adopting the Piperidine to cut Fmoc, and redundant Piperidine and reaction byproducts can be removed by draining and washing the resin after reaction, so that the operation is easy.
Optionally, in step S4, the coupling adopts a coupling reaction, and the specific reaction formula is as follows:
by adopting the technical scheme, the coupling effect is good, the coupling rate is high, the side reactions are few, the reaction rate is high, the synthesis period can be effectively shortened, and the purity of the polypeptide fragment is improved.
Optionally, in step S6, the separation resin is TFA, and is combined with a scavenger containing EDT, a mixture of thioanisole and water, and reacts with the resin connected with the target Lactoferrin polypeptide fragment, so as to separate the target Lactoferrin polypeptide fragment from the resin.
By adopting the technical scheme, the method has the advantages of higher reaction yield, better separation effect, mild reaction conditions and easier removal of the used scavenging agent.
Optionally, after the target Lactoferrin polypeptide fragment is separated from the resin, the resin is further filtered and removed, and the scavenger is removed by reduced pressure distillation; and then dissolving the residue in water and extracting to obtain a Lactoferrin polypeptide fragment crude product.
By adopting the technical scheme, the separated resin and other reagents can be effectively removed, the crude Lactoferrin polypeptide fragment with lower impurity content can be obtained, the purification difficulty can be effectively reduced, and the purity of the Lactoferrin polypeptide fragment prepared after purification can be effectively improved.
Alternatively, the extracted extract is preferably diethyl ether.
By adopting the technical scheme, the cost can be effectively reduced by selecting the ether as the extracting agent.
Optionally, in step S6, the crude Lactoferrin polypeptide fragment is purified by high performance liquid chromatography.
By adopting the technical scheme and adopting high performance liquid chromatography, the purification effect is optimal, and the purity of the prepared Lactoferrin polypeptide fragment is high.
Optionally, the preferred operating conditions of the liquid chromatography are:
and (3) chromatographic column: c 18 25×250mm；
Mobile phase: a-0.1% aqueous TFA; b-0.1% TFA in 60% acetonitrile;
detection wavelength: 214nm;
flow rate: 10 ml/min;
elution gradient: 20 to 60% B, for 30min.
By adopting the technical scheme, an excellent purification effect can be obtained, and the purity of the Lactoferrin polypeptide fragment prepared after purification can reach more than 98%.
In a third aspect, the application also provides a Lactoferrin antibody prepared by using the Lactoferrin polypeptide fragment, the antibody is prepared by coupling the Lactoferrin polypeptide fragment with a carrier protein, immunizing a rabbit, taking blood when the specific IgG concentration in rabbit serum reaches a peak, and then separating the serum.
By adopting the technical scheme, the prepared rabbit antibody has strong specificity, high sensitivity and good stability; the Lactoferrin-containing cell line can also be efficiently combined with Lactoferrin-positive cells and can be used for sorting cells with Lactoferrin expression difference.
Optionally, the carrier protein is selected from hemocyanin or keyhole limpet hemocyanin.
By adopting the technical scheme, the complete immunogen can be prepared after the carrier protein is connected, and the immune effect is good.
Optionally, the rabbit immunization employs a basal immunization and/or multiple booster immunizations.
By adopting the technical scheme, a better immune effect can be obtained, and high-titer antibodies can be obtained.
In a fourth aspect, the application also provides a technical scheme for applying the Lactoferrin polypeptide fragment in preparation of a Lactoferrin flow antibody and a Lactoferrin detection product.
By adopting the technical scheme, the excellent characteristics of the Lactoferrin polypeptide fragment can be utilized, and when the Lactoferrin polypeptide fragment is applied to preparation of a Lactoferrin flow-type antibody, the prepared antibody has strong specificity, high sensitivity and good stability; when the method is applied to the preparation of lactoferrin detection products, WB strips are clear and sharp, and the detection sensitivity and the detection accuracy are high.
In summary, the invention includes at least one of the following beneficial technical effects:
1. the application designs a very ideal Lactoferrin immunogen polypeptide fragment and provides a linear epitope of Lactoferrin protein, so that an antibody prepared from the Lactoferrin polypeptide fragment can identify the surface protein of a living cell, can be used for a cell flow detection technology and WB (white cell) detection of the Lactoferrin protein, and has a wide application range.
2. According to the Lactoferrin protein structure and function, a plurality of antigen epitopes are designed, the preparation of antigens is completed by respectively synthesizing polypeptides and coupling carrier proteins by adopting a solid-phase polypeptide synthesis method, animals are immunized, the optimal antigen epitope is screened out through experiments, and the Lactoferrin antibody prepared by adopting the Lactoferrin immunogen polypeptide fragment has strong specificity, high sensitivity and good stability.
3. The preparation method is simple in preparation process, synthesis can be achieved by adopting an automatic polypeptide synthesizer, the prepared polypeptide fragment is accurate in structure and high in purity, the preparation period is short, and the preparation cost is low.
4. The Lactoferrin rabbit antibody prepared from the Lactoferrin immunogen polypeptide fragment can be efficiently combined with Lactoferrin positive cells, and can be used for sorting cells with Lactoferrin expression difference.
Drawings
FIG. 1 is a schematic diagram of high performance liquid chromatography analysis of a Lactoferrin-KA13 polypeptide according to an embodiment of the invention;
fig. 2 is a WB schematic diagram of breast milk detected by Lactoferrin antibody according to an embodiment of the present invention;
FIG. 3 is a WB diagram of a Lactoferrin antibody detecting recombinant protein according to an embodiment of the invention;
FIG. 4 is a schematic representation of the cell selection range assay for the FC assay of U-937 using Lactoferrin antibody and a control antibody according to an embodiment of the present invention;
FIG. 5 is a thermogram of the fluorescence distribution of Lactoferrin antibody and a control antibody in a FC assay of U-937 according to an embodiment of the present invention;
FIG. 6 is a graph of fluorescence intensity distribution of Lactoferrin antibody and a control antibody in the example of the invention for U-937 FC assay in each group of cells.
In fig. 1, HPLC Column: a high performance liquid chromatography column; detection wavelength: detecting the wavelength; gradient: a gradient; buffer A: a buffer solution A; buffer B: a buffer solution B;
in FIG. 2, breast milk: breast milk; in fig. 3, lactoferrin protein: a lactoferrin recombinant protein;
in fig. 4, 5 and 6, anti-Lactoferrin: a lactoferrin antibody; rabbit IGg: a rabbit antibody; blank: and (4) blank groups.
Detailed Description
The present application will be described in further detail with reference to the following examples and the accompanying drawings.
The noun explains: lactoferrin: lactoferrin, fmoc: 9-fluorenylmethoxycarbonyl, HMP resin: p-methylolphenoxymethyl polyethylene Resin, resin: resins (particularly HMP resins); DCC: dicyclohexylcarbodiimide, DMAP: 4-dimethylaminopyridine, HOBT: hydroxybenzotriazole, TFA: trifluoroacetic acid, EDT:1, 2-ethanedithiol, piperidine: piperidine, WB: enzyme-linked immunosorbent assay, U-937: human tissue lymphoma, FC: flow cytometry.
Experimental materials and reagents required for the examples of the present application:
HMP resin, available from CSBio Inc. in the United states.
Fmoc-AA (Fmoc protected amino acid), commercially available from Merck, was also prepared by reacting 9-fluorenylmethoxycarbonylsuccinimide (Fmoc-OSU) directly with the desired amino acid.
NMP (Nitrogen methyl pyrrolidone), available from Merck.
DCM (dichloromethane) was purchased from Merck.
MeOH (methanol), available from Merck.
Piperidine, available from Merck.
DMAP, purchased from Sigma.
HOBT, available from Sigma.
DCC, purchased from Sigma.
TFA, available from Sigma.
EDT, available from Sigma.
Thioanisole, available from Sigma.
Crystalline phenol, available from Beijing Chemicals, inc.
Acetonitrile, from Merck.
The experimental apparatus required by the embodiment of the application:
polypeptide automatic synthesizer: an American CSBio model 336 multichannel polypeptide automatic synthesizer;
rotating the evaporator: japanese YAMATO CE50 rotary evaporator;
high performance liquid chromatograph: waters type 600E;
a freeze dryer: labbcano, usa;
a vacuum circulating water pump: zheng Changcheng Kogyo SH-B type;
a centrifuge: SIGMA corporation, usa.
The application designs a lactoferrin polypeptide fragment, and an amino acid sequence table of the lactoferrin polypeptide fragment is shown as SEQ ID N0.1.
Firstly, according to the structure and the function of Lactoferrin protein, a plurality of antigen epitopes are designed for the Lactoferrin polypeptide fragment. Then, the application adopts a solid phase polypeptide synthesis method, respectively synthesizes polypeptides according to designed antigen epitopes, couples with carrier protein to complete the preparation of the antigen, and screens out the optimal antigen epitope through experiments after immunizing animals. Finally, according to the screened optimal epitope, a polypeptide fragment is designed, the synthesis feasibility and the synthesis difficulty of the polypeptide fragment are verified by a solid-phase polypeptide synthesis method, and the Lactoferrin polypeptide fragment is finally screened and designed.
Compared with the prior art that animals are directly immunized by the Lactoferrin protein antigen to prepare a monoclonal or polyclonal antibody of Lactoferrin and the Lactoferrin antibody is prepared by a hybridoma technology, the technical scheme of the application adopts the optimal epitope obtained by an optimization and screening method, and the Lactoferrin polypeptide fragment designed according to the epitope is a more excellent immunogen polypeptide.
The Lactoferrin polypeptide fragment of the present application can be prepared by the following steps:
s1, activation: connecting amino of the amino acid with Fmoc protecting group by using protecting group reaction to prepare amino acid protected by Fmoc protecting group, and activating carboxyl to obtain carboxyl-activated amino acid containing Fmoc protecting group;
s2, connecting resin: reacting the carboxyl activated amino acid containing the Fmoc protecting group obtained in the step S1 with HMP resin to obtain resin connected with the carboxyl activated amino acid containing the Fmoc protecting group;
s3, deprotection group: carrying out deprotection reaction on the resin connected with the amino acid containing the Fmoc protecting group activated by carboxyl prepared in the step S2 to activate the amino group of the amino acid;
s4, coupling: repeating the step S1 to prepare new amino acid containing Fmoc protecting group activated by carboxyl, and coupling the amino activated resin prepared in the step S3 with the new amino acid containing Fmoc protecting group activated by carboxyl to prepare resin of the amino acid containing Fmoc protecting group for coupling the new amino acid;
s5, preparing polypeptide resin: repeating the operation of the step S3 to remove the protecting group of the resin of the Fmoc protecting group-containing amino acid coupled with the new amino acid prepared in the step S4, then repeating the operation of the step S4 to couple the new amino acid, and repeating the step until the resin connected with the target Lactoferrin polypeptide fragment is prepared;
s6, separation and purification: separating the resin connected with the target Lactoferrin polypeptide fragment prepared in the step S5 by using resin to prepare a Lactoferrin polypeptide fragment crude product, and purifying to prepare a Lactoferrin polypeptide fragment;
in the steps S1-S5, amino acids are added in the order of alanine, leucine, cysteine, glutamic acid, threonine, asparagine, aspartic acid, asparagine, phenylalanine, leucine, asparagine, and lysine.
The preparation method has simple preparation process, can realize synthesis by adopting an automatic polypeptide synthesizer, and has the advantages of accurate structure of the prepared polypeptide fragment, higher purity, short preparation period and lower preparation cost.
The following are specific examples of the present application.
Example 1 preparation of Lactoferrin polypeptide fragments
The Lactoferrin polypeptide fragment of this example was prepared by the following steps:
a) Activation of Fmoc-AA
The structural formula of Fmoc-AA is shown as follows:
can also be expressed as: fmoc-AA-COOH.
The Fmoc-AA is activated by reaction with DCC and HOBT to produce carboxyl activated Fmoc-AA-COOBP having the formula:
b) Linking activated amino acids to resins
Reacting the activated cysteine with a HMP resin under DMAP conditions to obtain a resin connected with amino acid, wherein the reaction formula is as follows:
c) Fmoc-protecting group removal from amino acid-linked resins
Under the action of Piperidine, removing the Fmoc protecting group of the resin connected with the amino acid, wherein the specific reaction formula is as follows:
d) Coupling Fmoc-deprotected resin with excess carboxy-activated Fmoc-AA
The reaction process of Fmoc-AA carboxyl activation is the same as that of the step a), and the activated Fmoc-AA is further coupled with the resin obtained in the step c), and the specific reaction formula is as follows:
e) Repeating the steps c) to d) for 13 times to obtain the polypeptide resin, wherein the adding sequence of the carboxyl activated amino acid is as follows: alanine, leucine, cysteine, glutamic acid, threonine, asparagine, aspartic acid, asparagine, phenylalanine, leucine, asparagine, and lysine.
f) Isolation of Lactoferrin polypeptides
Reacting the polypeptide resin with TFA, in combination with a scavenger mixed with EDT, thioanisole and water, cleaves the Lactoferrin polypeptide fragment from the peptide resin. After the reaction, the resin was filtered off and the scavenger was removed by distillation under reduced pressure. And then dissolving the Lactoferrin polypeptide fragment in water, and extracting with diethyl ether to obtain a Lactoferrin polypeptide compound crude product.
g) Separation, purification and analysis of Lactoferrin-KA13 polypeptide
And (3) separating and purifying the crude Lactoferrin polypeptide compound by adopting high performance liquid chromatography.
The operating conditions specifically adopted are:
and (3) chromatographic column: c 18 25×250mm；
Mobile phase: a-0.1% aqueous TFA; b-0.1% TFA in 60% acetonitrile;
detection wavelength: 214nm;
flow rate: 10 ml/min;
elution gradient: 20 to 60% by volume of B, for 30min.
Therefore, the pure Lactoferrin polypeptide fragment with the purity of more than 98 percent can be obtained.
And (3) carrying out high performance liquid chromatography analysis on the pure Lactoferrin polypeptide fragment obtained after separation and purification, and detecting the purity of the pure Lactoferrin polypeptide fragment. The specific analysis conditions were:
a chromatographic column: c 18 4.6×150mm；
Mobile phase: a-0.1% aqueous TFA; b-0.1% TFA in 60% acetonitrile;
detection wavelength: 214nm;
flow rate: 1 ml/min;
elution gradient: 20 to 60% by volume of B, for 30min.
The analysis result is shown in fig. 1, and the analysis result shows that the purity of the Lactoferrin polypeptide fragment can reach more than 98%.
Example 2 preparation and detection of Lactoferrin antibodies
1. Material
Example 1 preparation of the resulting Lactoferrin polypeptide fragment;
new Zealand big ear white rabbits were used for the experiments.
2. Antibody preparation procedure
Since Lactoferrin polypeptide fragments prepared in example 1 are not fully immunogenic, coupling to a carrier protein is required to prepare a complete immunogen. Thus, lactoferrin polypeptide fragments are first coupled to a protein carrier (hemocyanin or keyhole limpet hemocyanin) to prepare the complete immunogen.
After basic immunization (complete adjuvant and polypeptide antigen full emulsification) and multiple times of boosting immunization (incomplete adjuvant and polypeptide antigen full emulsification) of the New Zealand big ear white rabbits for experiments are subjected to back multipoint immunization, when the concentration of specific IgG in serum reaches the peak, the ear veins are used for taking blood, and then the serum is separated to obtain the Lactoferrin rabbit antibody.
Antibody titers and specificities were assessed using ELISA and WB methods.
After IgG was purified, fluorescein PE was labeled, and U-937 cells were detected by flow cytometry. Specifically, the flow of the detection operation is as follows: after fixing the U-937 cell suspension with 50% ethanol, PE-labeled Lactoferrin antibody was added, 2. Mu.L each of PE-labeled isotype control IgG, and a blank control was added to the solution at PE concentration. After incubation, detection was performed by flow cytometry.
3. Test results
Sensitivity of antibody detection by ELISA method: the detection result shows that the Lactoferrin antibody titer is superior to 1:32000 the antibody has high sensitivity.
The specificity of the antibody was measured by the WB method, and the results are shown in FIGS. 2 and 3.
As shown in fig. 2, lactoferrin protein has a molecular weight of about 80kDa. As shown in FIG. 3, when recombinant protein or breast milk samples are detected by a WB method with the Lactoferrin antibody prepared by the method, signal bands are all located at 75 to 100KDa, the theoretical molecular weight is consistent, and no impurity band exists, so that the specificity of the antibody is proved to be excellent.
The performance of the labeled cells of Lactoferrin antibody was measured by FC. The results are shown in FIGS. 4 to 6.
FIG. 4 shows the range of cell selection in the experimental group and isotype control group
X coordinate axis: FSC-H is a forward angle assay used to select cells based on their size.
Y coordinate axis: SSC-H is a lateral angle assay used to select cells based on intracellular granules.
FIG. 5 shows a thermal map of the fluorescence distribution of cells from the experimental and isotype control groups.
X coordinate axis: fluorescence intensity of PE.
Y coordinate axis: number of cells at a particular fluorescence intensity.
The results show that 84.1% of cells can be marked as positive by the PE-marked Lactoferrin antibody, and the positive rate of the same type control under the same condition is about 0.1%. Therefore, the Lactoferrin antibody can be used for FC detection, and has stable antigen-antibody binding and excellent labeling effect.
FIG. 6 shows the fluorescence intensity distribution curves for each group of cells, each curve representing a grouping as shown in the legend.
X coordinate axis: fluorescence intensity of PE.
Y coordinate axis: counting of cells within a specific fluorescence intensity range.
The results show that the addition of the PE-labeled Lactoferrin antibody causes the peak of the cell number to appear at a position where the fluorescence intensity is higher. Compared with the same type of control, the peak value of the curve of the experimental group is shifted to the right by about 2 orders of magnitude, which indicates that the Lactoferrin antibody can be efficiently combined with Lactoferrin positive cells and can be used for sorting cells with Lactoferrin expression difference.
As can be seen from the above examples of the present invention, the present invention establishes a highly desirable immunogenic polypeptide and successfully produces antibodies useful for Lactoferrin detection, namely: according to the structure and the function of the Lactoferrin protein, a plurality of antigen epitopes are analyzed and designed by means of protein database analysis software, polypeptides are respectively synthesized by adopting a solid-phase polypeptide synthesis method and are coupled with carrier protein to complete the preparation of the antigen, animals are immunized, the optimal antigen epitope is screened out through experiments, and the rabbit anti-Lactoferrin polyclonal flow detection antibody with strong specificity, high sensitivity and good stability is successfully prepared. The invention provides high-quality antibodies for the vast scientific research personnel and opens up a new idea for the research and development of the flow type antibodies.
The above embodiments are preferred embodiments of the present application, and the protection scope of the present application is not limited by the above embodiments, so: all equivalent changes made according to the structure, shape and principle of the present application shall be covered by the protection scope of the present application.
Claims (17)
1. A lactoferrin polypeptide fragment, characterized in that the amino acid sequence table of the lactoferrin polypeptide fragment is shown as SEQ ID N0.1.
2. A method for preparing a lactoferrin polypeptide fragment of claim 1, comprising the steps of:
s1, activation: obtaining Fmoc-protected amino acid Fmoc-AA, and activating carboxyl of the Fmoc-AA to obtain carboxyl activated Fmoc-AA;
s2, connecting resin: reacting the Fmoc-AA activated by the carboxyl obtained in the step S1 with HMP resin to obtain resin connected with the Fmoc-AA;
s3, deprotection group: removing Fmoc from the resin connected with Fmoc-AA prepared in the step S2 through deprotection reaction to activate amino groups of amino acids;
s4, coupling: repeating the step S1 to prepare new carboxyl activated Fmoc-AA, and coupling the amino activated resin prepared in the step S3 with the new carboxyl activated Fmoc-AA to prepare resin of the Fmoc-containing polypeptide fragment for coupling new amino acid;
s5, preparing polypeptide resin: repeating the operation of the step S3 to remove Fmoc of the resin containing the Fmoc polypeptide fragment coupled with the new amino acid prepared in the step S4, then repeating the operation of the step S4 to couple the new amino acid, repeating the step, and finally repeating the operation of the step S3 to remove Fmoc until the resin connected with the target Lactoferrin polypeptide fragment is prepared;
s6, separation and purification: separating the resin connected with the target Lactoferrin polypeptide fragment prepared in the step S5 by using resin to prepare a Lactoferrin polypeptide fragment crude product, and purifying to prepare a Lactoferrin polypeptide fragment;
in the steps S1-S5, amino acids are added in the order of cysteine, alanine, leucine, cysteine, glutamic acid, threonine, asparagine, aspartic acid, asparagine, phenylalanine, leucine, asparagine, and lysine.
3. The method for preparing a lactoferrin polypeptide fragment according to claim 2, wherein the synthesis process of steps S1 to S5 is performed by using an automated polypeptide synthesizer.
4. The method for preparing the polypeptide fragment of lactoferrin according to claim 2, wherein in step S1, fmoc-AA is prepared after the amino acids are Fmoc-linked by protecting group reaction, and the specific structural formula is as follows:
5. the method for preparing the polypeptide fragment of lactoferrin according to claim 4, wherein the activation of carboxyl group in step S1 is performed by reacting Fmoc-AA with DCC and HOBT, and the specific reaction formula is as follows:
6. the method for preparing the polypeptide fragment of lactoferrin according to claim 2, wherein the carboxyl group activated Fmoc-AA is reacted with HMP resin in step S2 by reacting the carboxyl group activated Fmoc-AA with HMP resin under DMAP condition, the specific reaction formula is as follows:
7. the method for preparing the polypeptide fragment of lactoferrin according to claim 2, wherein in step S3, fmoc removal is performed by using picoridine to remove Fmoc, and the specific reaction formula is as follows:
8. the method for preparing the polypeptide fragment of lactoferrin according to claim 2, wherein the coupling in step S4 is coupling reaction, and the specific reaction formula is as follows:
9. the method for preparing the Lactoferrin polypeptide fragment according to claim 2, wherein in the step S6, the separation resin is reacted with the resin to which the target Lactoferrin polypeptide fragment is linked using TFA in combination with a scavenger comprising EDT, thioanisole and water mixture, to separate the target Lactoferrin polypeptide fragment from the resin.
10. The method for preparing a Lactoferrin polypeptide fragment, according to claim 9, wherein the separation of the target Lactoferrin polypeptide fragment from the resin is followed by removing the resin by filtration and removing the scavenger by distillation under reduced pressure; and then dissolving the residue in water and extracting to obtain a crude Lactoferrin polypeptide fragment.
11. The method for the preparation of a polypeptide fragment of lactoferrin according to claim 10, wherein the extracted extract is ether.
12. The method for preparing a Lactoferrin polypeptide fragment as claimed in claim 2, wherein in step S6, the Lactoferrin polypeptide fragment crude product is purified by high performance liquid chromatography.
13. The method for the preparation of a polypeptide fragment of lactoferrin according to claim 12, wherein the liquid chromatography operating conditions are:
a chromatographic column: c 18 25×250mm；
Mobile phase: a-0.1% aqueous TFA; b-0.1% TFA in 60% acetonitrile;
detection wavelength: 214nm;
flow rate: 10 ml/min;
elution gradient: 20 to 60% by volume of B, for 30min.
14. The Lactoferrin antibody produced from the polypeptide fragment of Lactoferrin according to claim 1, wherein the antibody is produced by coupling Lactoferrin polypeptide fragment and carrier protein, immunizing rabbit, collecting blood at the time of peak concentration of specific IgG in rabbit serum, and separating the serum.
15. The Lactoferrin antibody according to claim 14, wherein said carrier protein is selected from the group consisting of hemocyanin and keyhole limpet hemocyanin.
16. The Lactoferrin antibody according to claim 14, wherein said rabbit immunization employs a basal immunization and/or multiple booster immunizations.
17. Use of a lactoferrin polypeptide fragment of claim 1 in the preparation of a lactoferrin flow antibody and in the preparation of a lactoferrin detection product.
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| CN117534753A (en) * | 2023-12-07 | 2024-02-09 | 北京博奥森生物技术有限公司 | Polypeptide for inducing high-performance NF- κB polyclonal antibody and application thereof |
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| CN117534753A (en) * | 2023-12-07 | 2024-02-09 | 北京博奥森生物技术有限公司 | Polypeptide for inducing high-performance NF- κB polyclonal antibody and application thereof |
| CN117534753B (en) * | 2023-12-07 | 2024-05-10 | 北京博奥森生物技术有限公司 | Polypeptide for inducing high-performance NF- κB polyclonal antibody and application thereof |
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