LT6770B - Nilaparvata lugens pce3 polypeptide and method for preparing polyclonal antibody thereof - Google Patents

Abstract

The present invention discloses a Nilaparvata lugens PCE3 polypeptide and a method for preparing polyclonal antibody thereof. The amino acid sequence of the PCE3 polypeptide is RRALPKENSKEANP. The preparation of the polyclonal antibody includes the following steps: firstly synthesizing the Nilaparvata lugens PCE3 polypeptide; crosslinking the synthesized polypeptide with a carrier protein to form a compound protein, immunizing animals with the compound protein, preparing antiserum with the blood of immunized animals, and isolating and purifying antibody from the antiserum, to obtain the antibody against the Nilaparvata lugens PCE3 polypeptide. The polyclonal antibody against the Nilaparvata lugens PCE3 polypeptide obtained in the present invention has good specificity and high purity, and can specifically bind to Nilaparvata lugens PCE3 protein to have reactions and can be used in related studies on the Nilaparvata lugens PCE3 protein.

Description

FIELD OF THE INVENTION

The present invention relates to a polypeptide and a method of producing an antibody specific therefor, which is mainly used to detect a natural protein antigen, in particular a method of producing a rabbit polyclonal antibody against a Nilaparvata lugens PCE3 polypeptide.

BACKGROUND OF THE INVENTION

PCE3, called the inactive form of coagulation-promoting enzyme 3 (en. Proclotting enzyme 3), plays a vital role in the process of insect reproduction, such as egg development and others. Nilaparvata lugens is an important agricultural pest. Research and detection of Nilaparvata lugens PCE3 is of great importance for the prevention and control of Nilaparvata lugens.

CN104974245A describes a Nilaparvata lugens VgR polypeptide and a method for producing a polyclonal antibody thereof.

SUMMARY OF THE INVENTION

It is an object of the present invention to provide a Nilaparvata lugens PCE3 polypeptide having the amino acid sequence RRALPKENSKEANP.

Another object of the present invention is to provide a method of producing a rabbit antibody against Nilaparvata lugens PCE3 polypeptide. The antibody obtained by this method has high specificity and high purity and can specifically recognize the native PCE3 protein in tissues or cells.

A method of producing a rabbit antibody against a Nilaparvata lugens PCE3 polypeptide, wherein a Nilaparvata lugens PCE3 polypeptide having the amino acid sequence RRALPKENSKEANP is first synthesized; the synthesized modified polypeptide is cross-linked to a carrier protein to form a composite protein, the animals are immunized with the composite protein, an antiserum is prepared from the blood of the immunized animals, and the antibody is isolated and purified from the antiserum.

Further, the carrier protein is mollusc lymphocytin (KLH) or bovine serum albumin (BSA); the sulfhydryl group and the amino group of the protein carrier of the modified polypeptide are covalently linked by a cross-linking agent to form a composite protein.

Subsequently, the composite protein, after mixing with the immunoadjuvant and preparing the emulsion, is injected subcutaneously into the back of the rabbit, followed by repeated immunizations three times after the initial immunization to obtain the antiserum.

In addition, the antiserum is purified by peptide affinity chromatography to obtain a high purity rabbit antibody against the Nilaparvata lugens PCE3 polypeptide.

More specifically, the preparation method described above involves the following steps:

Step 2: Attaching the cysteine to the C-terminus to facilitate binding to the carrier protein when synthesizing the Nilaparvata lugens PCE3 polypeptide sequence, synthesizing the PCE3-modified polypeptide by a polypeptide autosynthesis device, attaching the PCE3-modified polypeptide to the unbranched protein after purification. PCE3 modified polypeptide-KLH composite protein;

Step 2: Injection of the emulsified PCE3 modified polypeptide-KLH composite protein into the rabbit subcutaneous tissue, followed by three re-immunizations after the primary immunization;

step 2: collecting and isolating serum containing a rabbit antibody against the Nilaparvata lugens PCE3 modified polypeptide;

Step 2: Performing antiserum-purified peptide affinity using a PCE3 peptide affinity chromatography column to obtain a PCE3 polypeptide antibody.

Step 2: Perform a PCE3 polypeptide antibody titration assay;

Step 2: Determine the specificity of the PCE3 polypeptide antibody by immunoblotting.

The polyclonal antibody of the present invention has good specificity and high purity, and can also specifically recognize the native PCE3 protein in tissues or cells.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the result of immunoblotting. The target band on the immunoblotting membrane coincides with a size of about 50 kDa and corresponds to the theoretical molecular weight of the PCE3 protein (52.2 kDa).

DETAILED DESCRIPTION OF THE INVENTION

The present invention will be further described below with specific embodiments. It is to be understood that these embodiments are used only to illustrate the present invention and are not intended to limit the scope of the present invention. Without departing from the technical solution of the present invention, any modification or alteration of the present invention that could be readily made by one skilled in the art is within the scope of the definition of the present invention.

example. Nilaparvata lugens PCE3 sequence analysis, and PCE3 polypeptide modeling and synthesis

According to the Nilaparvata lugens PCE3 sequence (access number MN586815) in the GenBank database, the Nilaparvata lugens PCE3 protein sequence consists of 460 amino acids. Nilaparvata lugens PCE3 protein characteristics were analyzed using the Protean software module in error-free DNA software to obtain a protein with a molecular weight (52.2 KDa). The amino acid sequence properties of the protein, such as antigenicity, hydrophilicity, surface probability, and the polypeptide sequence RRALPKENSKEANP were selected and used as an antigen, were further analyzed. To facilitate binding to the protein carrier and increase the immunogenicity of the polypeptide, cysteine C was attached to the C-terminus of the aforementioned polypeptide, so that the final sequence of the polypeptide to be synthesized was RRALPKENSKEANPC. Polypeptide synthesis was performed by an automated peptide synthesizer. The purity of the synthesized polypeptide was determined by HPLC and the result was 94.97%. The molecular weight of the polypeptide was determined by mass spectrometry, and the result obtained was 1712.70.

example. Coupling of a polypeptide to a carrier protein

Protein-carrier KLH was cross-linked to the synthesized PCE3 polypeptide using MBS as a cross-linking agent: KLH was dissolved in up to 10 mg / ml in cross-linking buffer; MBS was dissolved in DMF to 10 mg / ml; the dissolved KLH solution and the MBS solution were mixed in a ratio of 10: 1 (w / w), and the KLH was activated for 30 minutes at room temperature; the activated KLH solution was purified with Sephadex G-25; the activated KLH solution was mixed with the PCE3 polypeptide solution in a ratio of 1: 1 (w / w) to react for 3 hours at room temperature; the above reaction solution was dialyzed against PBS overnight at 4 ° C to obtain the PCE3 polypeptide-KLH composite protein.

example. Immunization of experimental animals

Male New Zealand rabbits of the appropriate age were selected for immunization. Prior to immunization, 2-3 ml of blood was collected from rabbit ear veins and used as a negative control for subsequent ELISA. During the first immunization, 0.5 mg of PCE3 polypeptide-KLH composite protein was dissolved in 0.5 ml of PBS solution and emulsified using the same amount of Freund's complete adjuvant, followed by subcutaneous injection at multiple sites in the rabbit body. The first booster immunization was given 2 weeks later. 0.5 mg of the PCE3 polypeptide-KLH composite protein was dissolved in 0.5 ml of PBS solution, mixed with the same amount of Freund's incomplete adjuvant, and an emulsion was prepared, followed by subcutaneous injection at multiple sites in the body. Thereafter, the same revaccination was given every 3 weeks, for a total of 3 times. One week after each revaccination, a small amount of blood was drawn from the ear veins of the rabbits and the immune serum titre was determined by indirect ELISA. When the titer was greater than 1: 20,000, rabbit blood was taken from the carotid artery to prepare serum.

example. Purification of Antibodies On a PCE3 polypeptide affinity column, 1 ml of activated gel suspension was injected onto the chromatography column. After the liquid was drained from the column, 5 ml of coupling buffer was added to wash the column. The synthesized PCE3 polypeptide was dissolved in 1 ml of coupling buffer and applied to a chromatography column, then 1 ml of coupling buffer was added to the column, spun and stirred at 4 ° C overnight. The chromatography column was washed with 8 ml of coupling buffer, then blocked with 3 ml of blocking solution at room temperature for 1 hour. To prepare a chromatographic column for PCE3 polypeptides, the chromatography column was washed 3 times with binding buffer until liquid was removed from the column. Binding buffer containing PCE3 antibody serum was added to the column, stirred for 1 hour at room temperature, and then the column was washed with 30 ml of wash buffer until the absorbance peak of the effluent stabilized at 280 nm. The column was washed with 15 ml of wash buffer to obtain purified PCE3 antibody.

example. Determination of antibody titer by indirect ELISA

The ELISA plate was coated with PCE3 polypeptide-KLH at 4 ° C overnight, and blocked with 5% skim milk powder for 2 hours at room temperature; PCE3 antibody was diluted in different ratios of 1: 1000, 1: 2000, 1: 4000, 1: 8000, 1: 16000, 1: 32000, 1: 64000, 1: 128000, 1: 256000, 1: 512000, and incubated at room temperature. 2 hours or 4 ° C overnight; and then horseradish peroxidase-labeled goat anti-rabbit secondary antibody is added, and incubated at room temperature for 2 hours. TMB was added to the chromogenic reaction, after quenching with sulfuric acid, the absorbance at 450 nm was measured. When the ratio of the absorbance of the sample to the negative serum was greater than 2.1, the antibody titer was calculated. The titre of purified PCE3 antibody after the assay was 1: 512000.

example. Determination of PCE3 antibody specificity by immunoblotting

SDS-PAGE gel was prepared by a standard method. 10 μl of tissue lysate with a protein concentration of 5 mg / ml was added to the sample wells of the vertical electrophoresis tank and electrophoresed. Electrophoresis was stopped when the bromophenol blue indicator was completely removed from the separating gel. The protein was transferred to an NC membrane using the wet transfer method at a constant voltage of 100 V for 90 minutes. The PCE3 antibody obtained in Example 4 was used as a primary antibody diluted 1: 500, hybridized to a wet transfer NC membrane at room temperature for 2 hours, and then hybridized with HRP-labeled goat anti-rabbit antibody at room temperature for 2 hours. The experimental results of this example showed that PCE3 was universally expressed in the ovaries of Nilaparvata lugens.

Claims (5)

1 A synthetic Nilaparvata lugens PCE3 polypeptide having the amino acid sequence RRALPKENSKEANP. A method of producing rabbit antibodies to a Nilaparvata lugens PCE3 polypeptide, comprising first synthesizing a Nilaparvata lugens PCE3 polypeptide having the amino acid sequence RRALPKENSKEANP; the synthesized modified polypeptide is conjugated to a carrier protein to form a composite protein, the animals are immunized with the composite protein, an antiserum is prepared from the blood of the immunized animals, and the antibody is isolated and purified from the antiserum. The method of claim 2, wherein the carrier protein is mollusc lymph hemocyanin (KLH) or bovine serum albumin (BSA); the sulfhydryl group and the protein-carrier amino group of the modified polypeptide are covalently linked by a cross-linking agent to form a composite protein. The method of claim 3, wherein after mixing the composite protein with the immunoadjuvant and preparing the emulsion, the composite protein is injected subcutaneously into the back of the rabbit; after the primary immunization, the immunization is repeated three times to obtain the antiserum. The method of claim 4, wherein the antiserum is purified using peptide affinity chromatography to obtain a purified rabbit antibody against Nilaparvata lugens PCE3 polypeptide.