阅读说明：本技术 一种弓形虫Actin蛋白的表位多克隆抗体及其应用 (Epitope polyclonal antibody of toxoplasma gondii Actin protein and application thereof ) 是由 余莉 朱进进 沈继龙 罗庆礼 于 2019-10-14 设计创作，主要内容包括：本发明公开了一种弓形虫Actin蛋白的表位多克隆抗体及其应用,其中弓形虫Actin蛋白的表位多克隆抗体,是以弓形虫Actin氨基酸序列中C端227-237为特异性较好的抗原决定簇肽段,获得弓形虫抗原决定簇多肽,进而制备的弓形虫Actin蛋白的表位多克隆抗体。本发明设计的多肽抗原表位成分简单,引发的免疫反应针对性强,能特异性检测识别弓形虫Actin蛋白,主要在检测弓形虫蛋白的表达水平方面作为内参对照,能较为准确地确定目的蛋白的相对表达量；也可以通过免疫荧光对入侵细胞中的弓形虫进行定位,对其分子作用机制的进一步研究提供很大的帮助。(The invention discloses an epitope polyclonal antibody of toxoplasma gondii Actin protein and application thereof, wherein the epitope polyclonal antibody of the toxoplasma gondii Actin protein takes C end 227-237 in a toxoplasma gondii Actin amino acid sequence as an antigenic determinant peptide segment with better specificity to obtain toxoplasma gondii antigenic determinant polypeptide, and then the epitope polyclonal antibody of the toxoplasma gondii Actin protein is prepared. The polypeptide epitope designed by the invention has simple components and strong pertinence of the initiated immunoreaction, can specifically detect and identify the toxoplasma gondii Actin protein, is mainly used as an internal reference in the aspect of detecting the expression level of the toxoplasma gondii protein, and can more accurately determine the relative expression quantity of the target protein; the toxoplasma in invading cells can also be localized by immunofluorescence, and further research on the molecular action mechanism of the toxoplasma also provides great help.)

1. An epitope polyclonal antibody of toxoplasma gondii Actin protein, which is characterized in that:

the C end 227-237 in the toxoplasma gondii Actin amino acid sequence is used as an antigenic determinant peptide segment with better specificity to obtain the toxoplasma gondii antigenic determinant polypeptide, thereby preparing the epitope polyclonal antibody of the toxoplasma gondii Actin protein.

2. The polyclonal antibody against epitopes of toxoplasma gondii Actin protein as claimed in claim 1, characterized in that it is prepared by a method comprising the following steps:

step 1: by analyzing the epitope in the Actin amino acid sequence of the Toxoplasma gondii, homology comparison is carried out between the epitope and the Actin amino acid sequence of human, mouse, monkey, chicken and other species, the C end 227-237 is determined as an antigenic determinant peptide segment with better specificity, a cysteine C is added in front of the sequence, and KLH is coupled to obtain the Toxoplasma gondii antigenic determinant polypeptide;

step 2: dissolving 1mg toxoplasma gondii antigenic determinant polypeptide in 1ml of sterile PBS buffer solution with the pH value of 7.4 to prepare a polypeptide solution; adding 1ml of polypeptide solution into a 2.5ml sterile injection syringe, adding 1ml Freund's complete adjuvant, and repeatedly pushing and pulling on ice for 30min for emulsification until water-in-oil drops are formed; taking two 2-2.5kg New Zealand white rabbits, performing subcutaneous immunization for 400 mug/time, performing immunization for 2-3 weeks for one time, performing blood sampling detection after 3-4 times of immunization, determining the titer of antiserum against polypeptide by an indirect ELISA method, and when the titer is more than 1: 50000 performing final blood collection to prepare antiserum;

and step 3: and (3) purifying by adopting Protein G, mixing the antiserum obtained in the step (2) with PBS buffer solution with the pH value of 7.4 in an equivalent manner, slowly loading the mixture, and eluting by using glycine buffer solution with the pH value of 7.4 after the antibody is combined to obtain the purified antibody.

3. The epitope polyclonal antibody of toxoplasma gondii Actin protein as claimed in claim 2, wherein:

in step 1, the amino acid sequence of the toxoplasma gondii epitope polypeptide is shown as SEQ ID NO: 1 is shown.

4. Use of an epitope polyclonal antibody of toxoplasma Actin, as claimed in claim 1, 2 or 3, wherein: the Toxoplasma gondii Actin epitope polyclonal antibody is used as a detection reagent in the process of specifically detecting and identifying Toxoplasma gondii Actin protein.

Technical Field

The invention relates to an epitope polyclonal antibody of toxoplasma gondii Actin protein and application thereof, belonging to the field of immunology.

Background

Toxoplasma is a widespread intracellular parasitic protozoa between humans and animals, and its infection seriously jeopardizes human health and the development of animal husbandry. The existing research results show that a plurality of molecules of toxoplasma gondii (rhomoboids, ROMs), Actin (Actin), microline proteins (MICs) and the like participate in the invasion process.

Actin is an important skeleton protein and can be roughly divided into six types, wherein beta-Actin is widely distributed in various tissues, the size of beta-Actin is 42-43 kDa, the expression quantity is rich and relatively constant, the beta-Actin is a common internal reference for PCR, and an antibody of the beta-Actin is a good internal reference index for Western Blot.

Unfortunately, because Actin is highly conserved among different species, it is difficult to obtain a good Actin antiserum for a specific species, no commercial toxoplasma Actin antibody is available so far, an internal reference control is lacked in detecting the expression level of toxoplasma protein, the relative expression amount of the target protein cannot be accurately determined, and further research on the molecular action mechanism of the toxoplasma Actin antibody is limited.

Disclosure of Invention

The invention aims to provide an epitope polyclonal antibody of toxoplasma Actin and application thereof, and lays a foundation for research on an invasion mechanism of toxoplasma and an action relationship between invasion molecules.

The epitope polyclonal antibody of toxoplasma gondii Actin takes C end 227-237 in an amino acid sequence of toxoplasma gondii Actin as an antigenic determinant peptide segment with better specificity to obtain toxoplasma gondii antigenic determinant polypeptide, thereby preparing the epitope polyclonal antibody of toxoplasma gondii Actin protein. The method specifically comprises the following steps:

step 1: by analyzing the epitope in the Actin amino acid sequence of the Toxoplasma gondii, homology comparison is carried out between the epitope and the Actin amino acid sequence of human, mouse, monkey, chicken and other species, the C end 227-237 is determined as an antigenic determinant peptide segment with better specificity, a cysteine C is added in front of the sequence, and KLH is coupled to obtain the Toxoplasma gondii antigenic determinant polypeptide;

step 2: dissolving 1mg of toxoplasma gondii epitope polypeptide in 1ml of sterile PBS buffer (pH 7.4) to prepare a polypeptide solution; adding 1ml polypeptide solution into 2.5ml aseptic injection syringe, adding 1ml Freund's complete adjuvant (second and third immunization are replaced by Freund's incomplete adjuvant), repeatedly pushing and pulling on ice for 30min for emulsification to form water-in-oil drops (dropping into water surface and not immediately dispersing); taking two New Zealand white rabbits (2-2.5kg), performing subcutaneous immunization for 400 mug/time, performing immunization for 2-3 weeks once, performing blood sampling detection after 3-4 times of immunization, determining the titer of antiserum against polypeptide by an indirect ELISA method, and when the titer is more than 1: 50000 performing final blood collection to prepare antiserum;

and step 3: and (3) purifying by adopting Protein G, mixing the antiserum obtained in the step (2) with a PBS (phosphate buffer solution) (pH value of 7.4) in equal amount, slowly loading the mixture, and eluting by using a glycine buffer solution (pH value of 7.4) after the antibody is combined to obtain the purified antibody.

In step 1, the amino acid sequence of the toxoplasma gondii epitope polypeptide is shown as SEQ ID NO: 1 is shown.

The application of the epitope polyclonal antibody of the toxoplasma gondii Actin protein is to be used as a detection reagent in the process of specifically detecting and identifying the toxoplasma gondii Actin protein.

The invention has the beneficial effects that:

the invention adopts biological information software, deduces the sequence of the toxoplasma gondii Actin protein, and designs a polypeptide epitope component which is simple and has strong pertinence to the induced immune response according to the result of multi-parameter prediction of hydrophilicity, plasticity, antigenicity and the like of the toxoplasma gondii Actin protein, and can specifically detect and recognize the toxoplasma gondii Actin protein. The method is mainly used as an internal reference control in the aspect of detecting the expression level of the toxoplasma gondii protein, and can accurately determine the relative expression quantity of the target protein; the toxoplasma in invading cells can also be localized by immunofluorescence, and further research on the molecular action mechanism of the toxoplasma also provides great help.

Drawings

FIG. 1 is an epitope homology alignment. From FIG. 1, it can be seen that the epitope of the fragment has low homology with human Actin.

Figure 2 is an antibody titer assay. From FIG. 2, it can be seen that a high titer monoclonal antibody was obtained.

FIG. 3 is an antibody SDS-PAGE analysis. It can be seen from FIG. 3 that the purity of the purified antibody was above 85%.

FIG. 4 shows Western Blot identification of antibody. The left panel is Toxoplasma gondii Actin polyclonal antibody, and the right panel is mouse Actin monoclonal antibody. From FIG. 4, it can be seen that Toxoplasma gondii Actin epitope polyclonal antibody can specifically recognize Toxoplasma gondii Actin protein, and has no cross reaction with human, mouse, monkey, chicken and other species Actin.

Figure 5 is an antibody immunofluoresence identification. It can be seen from FIG. 5 that Toxoplasma gondii Actin polyclonal antibodies specifically recognize Toxoplasma gondii.

Detailed Description

1. Analyzing the epitope in the Actin amino acid sequence by using an online software Bepipred 1.0 Server, determining the antigenic determinant peptide segment of C end 227-.

2. Animal immunization

A polypeptide solution was prepared by dissolving 1mg of Toxoplasma gondii ROP18 epitope polypeptide (linked to KLH) in 1ml of sterile PBS buffer. Taking two 2.5ml sterile syringes, removing the needle heads, connecting the lower end outlet with a liquid conveying pipe, then adding 1ml prepared polypeptide solution into the needle cylinder, adding 1ml Freund's complete adjuvant (the second immunization and the third immunization are replaced by Freund's incomplete adjuvant), repeatedly pushing and pulling on ice for 30min for emulsification until water-in-oil drops are formed (the drops are dropped on the water surface and do not immediately disperse). Two New Zealand white rabbits (2-2.5kg) were immunized subcutaneously at 400. mu.g/time for 2-3 weeks. After 3-4 times of immunization, blood sampling detection is carried out, the titer of antiserum against the polypeptide is determined by an indirect ELISA method, and when the titer is more than 1: 50000 antiserum is prepared from the final blood sample and is ready for purification.

3. Antibody purification

And (3) purifying by adopting Protein G, mixing the obtained antiserum and PBS in equal amount, slowly loading the mixture, eluting by using a glycine elution buffer solution after the antibody is combined to obtain the required purified antibody, immediately dialyzing in the PBS at 4 ℃ overnight, and measuring the purity, the concentration and the titer every other day.

4. Antibody identification

4.1 Indirect ELISA Titer detection of purified antibodies

(1) The coated plate is designed according to the experimental requirements, and the plate strips are marked.

(2) The polypeptides were diluted to the desired concentration with PBS coating, mixed well and added to the strips at 100. mu.l per well overnight in a refrigerator at 4 ℃.

Coating antigen; polypeptides

Coating concentration; 5. mu.g/ml, 100. mu.l/well

Coating buffer solution; phosphate buffer (PBS, pH7.4)

(3) After coating, the coating solution was discarded, the plate was washed 3 times, 200. mu.l of blocking solution was added to each well, and the plate was incubated at 37 ℃ for 1 hour. The ELISA plate was removed, the internal solution was discarded, and the plate was washed 1 time.

(4) Purified antibodies were purified as described in 1:500, 2-fold dilution, 200. mu.l of blocking solution per well, and 1h in a 37 ℃ incubator.

(5) Taking out the enzyme label plate, discarding the internal liquid, washing the plate for 3 times, adding 100 mul of diluted enzyme-labeled secondary antibody and enzyme-labeled secondary antibody into each hole: goat anti-rabbit-HRP, 1: 5000. An incubator at 37 ℃ for 1 h.

(6) Taking out the enzyme label plate, discarding the inner liquid, washing the plate for 4 times, adding 100 μ l TMB color developing solution into each hole, and determining the color developing time according to the color depth, generally 37 deg.C, 15 min.

(7) The reaction was stopped by adding 100. mu.l of 1M HCl solution to each well. Immediately reading on a microplate reader at 450nm, and determining the titer of the sample according to the dilution corresponding to the well with the OD value being greater than 2.1 times of the set negative control OD value. The antibody titer was found to be 1: 12800.

4.2 concentration determination of the resulting antibody Using BCA protein concentration determination kit

Antibody concentration: 3.20mg/ml

Volume: 10ml of

Buffer solution: phosphate buffer (PBS, pH7.4)

4.3 antibody purity characterization

After purification, the antibody was subjected to SDS-PAGE and stained with Coomassie Brilliant blue. The purity of the purified antibody is more than 85%.

4.4 identification of antibody specificity

Toxoplasma gondii (TgWH6), Human (HFF), monkey (Vero), chicken (DF-1) and mouse (RAW) cell proteins are extracted to carry out Western Blot, and toxoplasma gondii Actin multi-antibody and mouse Actin monoclonal antibody are respectively used as primary antibodies for incubation, and the specificity of the toxoplasma gondii Actin multi-antibody is identified by comparison.

5. Use of antibodies in immunofluorescence

(1) HFF cell count was plated at approximately 1 × 10 per well⁴And (4) respectively.

(2) After 4h, the cells were observed to adhere to approximately 70% under the mirror, and freshly obtained GFP-RH tachyzoites were counted at approximately 1X10 per well of tachyzoite⁴And (4) respectively.

(3) After 24h, the tachyzoites enter the cells under the observation of the observation under the mirror and form nauplius vesicles, the culture solution in the wells is sucked away, washed 3 times with PBS, 200. mu.l of 4% paraformaldehyde is added to each well, and after fixation at 4 ℃ for 30min, washed three times with PBS, 3min each time.

(4) Mu.l of 0.2% TritonX-100 was added to each well, and after membrane permeation, the wells were washed 3 times with PBS, 3min each time.

(5) Add 200. mu.l of 5% goat serum to each well, block at 37 ℃ for 1h and wash 3 times with PBS, 5min each time.

(6) The primary antibody was diluted 1:200 with 1% BSA and 200. mu.l was added to each well, and after overnight incubation at 4 ℃ PBS was washed 5 times for 3min each.

(7) The secondary antibody was diluted 1:100 with 1% BSA and 200. mu.l/well incubated at 37 ℃ for 1h and washed 5 times with PBS for 3min each.

(8) Nuclei were stained with 100. mu.l of DAPI for 5min per well and washed 5 times with PBS for 3min each.

(9) And (5) slicing and observing.

As can be seen from the immunofluorescence results in FIG. 5, toxoplasma gondii is stained red, and the antibody can specifically react with toxoplasma gondii Actin protein without obvious cross reaction with host cells.

SEQUENCE LISTING

<110> university of medical in Anhui

<120> epitope polyclonal antibody of toxoplasma gondii Actin protein and application thereof

<130> 2019.09.04

<160> 1

<170> PatentIn version 3.1

<210> 1

<211> 11

<212> PRT

<213> Toxoplasma gondii

<400> 1

Glu Met Lys Ala Ala Glu Asp Ser Ser Asp Ile

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