阅读说明：本技术 抗f4/80的多克隆抗体及制备方法 (Polyclonal antibody against F4/80 and preparation method thereof ) 是由 刘志强 杨锡琴 袁增强 王树坤 朱晓明 于 2019-10-18 设计创作，主要内容包括：本发明属于生物技术领域,本发明公开了一种抗F4/80的多克隆抗体及制备方法,其中制备方法为：所述的抗F4/80的多克隆抗体通过F4/80蛋白片段作为抗原免疫获得。本发明的多克隆抗体对巨噬细胞具有显著的免疫荧光染色性能。(The invention belongs to the technical field of biology, and discloses polyclonal antibodies against F4/80 and a preparation method thereof, wherein the preparation method comprises the step of immunizing the polyclonal antibody against F4/80 by taking an F4/80 protein fragment as an antigen.)

1, polyclonal antibody against F4/80 and the preparation method is characterized in that the polyclonal antibody against F4/80 is obtained by immunizing F4/80 protein fragment as antigen.

2. The method for preparing an anti-F4/80 polyclonal antibody according to claim 1, wherein the F4/80 protein fragment comprises the amino acid sequence of F4/80 protein 1 to 179.

3. The method for preparing an anti-F4/80 polyclonal antibody according to claim 2, wherein the antigen is obtained by fusion expression of the F4/80 protein fragment and GST tag.

4. The method of claim 3, wherein the polyclonal antibody against F4/80 is an anti-rabbit polyclonal antibody obtained from an immunized rabbit.

5, polyclonal antibodies against F4/80, wherein the polyclonal antibody against F4/80 is prepared by the preparation method of any one of claims 1-4 to .

Technical Field

The invention belongs to the technical field of biology, and particularly relates to anti-F4/80 polyclonal antibodies and a preparation method thereof.

Background

The macrophage is immune cells with various physiological functions, and plays an important role in the physiological and pathological processes of infection response, body immunity, tumor, inflammatory reaction and the like.

In recent years, research on macrophage surface markers is continuously advanced, and a plurality of special protein and polysaccharide antigens expressed on the surfaces of macrophages are disclosed and are expected to be used as targeted markers of the macrophages, wherein F4/80 is of macrophage markers of interest, F4/80 is of special glycoprotein expressed on the surfaces of the macrophages and has higher expression in the macrophages of intestines, lungs and other parts, Laroui and the like [17] in a enteritis mouse model, the nanoparticle modified by the F4/80 antibody carries nucleic acid molecules to carry out intestinal macrophage targeted drug delivery research, the treatment effect is remarkably improved, and the F4/80 is used as a receptor molecule to have important prospects in targeted intervention research of related diseases.

The F4/80 protein contains 931 amino acids (aa), wherein 645-931aa are 7 transmembrane and intracellular sequences which cannot serve as a surface receptor, and 1-644aa are extracellular sequences which are sequences recognized by antibodies like . the conventional anti-F4/80 antibody is generally prepared from a specific extracellular sequence fragment serving as an antigen immunized animal, however, since the F4/80 extracellular sequence is longer (644aa), most fragments are ineffective fragments without immunological activity, when animals are immunized with the full-length extracellular sequence, the fragments without immunological activity may shield or interfere the immunologically active fragment to influence the generation of the antibody, when animals are immunized with the shorter immunologically active fragment, the shorter antigen sequence may cause a significant reduction in the immunostimulating effect, even cannot induce the generation of the antibody.

Disclosure of Invention

The invention aims to provide anti-F4/80 polyclonal antibodies and a preparation method thereof.

The above purpose of the invention is realized by the following technical scheme:

the embodiment of the aspect of the invention provides a preparation method of polyclonal antibodies against F4/80, wherein the polyclonal antibodies against F4/80 are obtained by immunizing F4/80 protein fragments as antigens.

Further , the F4/80 protein fragment contains the amino acid sequence of F4/80 protein No.1 to 179.

, the antigen is obtained by fusion expression of the F4/80 protein fragment and GST tag.

, the polyclonal antibody against F4/80 is the polyclonal antibody against rabbit obtained by immunizing rabbit.

The example of the second aspect of the invention provides polyclonal antibodies against F4/80, wherein the polyclonal antibody against F4/80 is prepared by the preparation method.

The invention has the advantages that the used F4/80 nitrogen end 1-179aa polypeptide has good immunogenicity, and is a sequence with dominant immune epitopes in an extracellular sequence of F4/80 protein; compared with the existing commercialized rabbit polyclonal antibody similar to the rabbit obtained by using other nitrogen-terminal polypeptides, the anti-F4/80 polyclonal antibody obtained by using the polypeptide as the antigen for immunizing rabbits has obviously better affinity performance and high sensitivity of immunofluorescence staining macrophages.

Drawings

FIG. 1 shows the distribution of immune epitopes of F4/80 protein predicted by BioSun in example;

FIG. 2 is a graph of serum antibody titer measurements at various time points in an example of the present invention;

FIG. 3 shows the purified electrophoresis test of an embodiment of the present invention;

FIG. 4 shows the comparison between the performance of the antibody of example of the present invention and that of the same type of antibody currently commercialized.

Detailed Description

The invention is further described in by the following examples to provide those skilled in the art with an understanding of the invention at , but not to limit the invention in any way.

polyclonal antibody against F4/80 and the preparation method thereof:

(1) predicting the distribution of immune epitopes of an extracellular section (1-644aa) of F4/80 by Biosun epitope prediction software, and selecting a 1-179aa fragment as a dominant antigen according to a prediction curve;

(2) collecting mouse macrophage system Raw264.7, extracting genome mRNA, and performing reverse transcription by using an Oliga primer to obtain whole genome cDNA;

(3) designing and synthesizing primers at two ends of a 1-179aa fragment, respectively adding xhol and xbal enzyme cutting sites at the two ends, (an upstream primer: GCCTCGAGATGTGGGGCTTTTGGCTGCTCCTC; a downstream primer: GCTCTAGAAGTCACACATTCATCTTCATC) gene library), and obtaining a DNA sequence corresponding to the 1-179aa polypeptide through PCR amplification;

(4) the PCR product was purified by agarose gel electrophoresis and gel recovery, then ligated to T-vector, and transformed into HB101 competent cells, and applied to LB solid medium (AMP +), and cultured overnight;

(5) selecting 10 single colonies from a plurality of single colonies growing in a solid culture medium, inoculating the single colonies into an LB liquid culture medium (AMP +), carrying out shaking amplification at 37 ℃, selecting 5 single colonies from the amplified colonies, carrying out sequencing by a Tianhui remote company, selecting strains with correct sequencing, amplifying and extracting plasmids;

(6) the plasmid is double-digested by using xhol and xbal restriction enzymes, the digested product is subjected to agarose gel electrophoresis and gel cutting according to the length of the fragment to recover and purify the DNA sequence of the F4/80 protein 1-179aa fragment, and then the DNA sequence is connected to pBV/IL1 and pBV/GST vectors, the product HB101 competent bacteria is connected, and the LB solid medium (Amp +) is plated;

(7) selecting 10 single colonies growing in a solid culture medium, inoculating the single colonies into 3mL LB liquid culture medium, amplifying in a shaking table at 37 ℃, identifying the successful insertion of gene fragments by colony PCR, selecting 5 single colonies, sending the samples to sequence, firstly inoculating strains of bacterial liquid with correct sequence into 40mL LB liquid culture medium, and culturing for 6 hours at 37 ℃;

(8) taking 10mL of the strain to be transferred into 200mL of LB culture medium, culturing for 3 hours at 37 ℃, and transferring the strain to be cultured in a water bath at 42 ℃ for overnight induction; collecting bacterial liquid, suspending 25mM Tis-HCl with pH8.5, diluting to 100mL, adding 1mL lysozyme, ultrasonically crushing, centrifuging at 4 ℃ and 12000rpm for 10min, removing supernatant, and repeating for three times to obtain inclusion bodies;

(9) diluting the inclusion body with 25mM Tis-HCl pH8.5 to a constant volume of 60ml, adding urea to 90ml, adding 0.9ml mercaptoethanol after dissolving, boiling for 5min, filtering with cotton, purifying antigen by Ni column, eluting with 250mM imidazole 6M urea 25mM Tis-HCl 0.1% B-ME pH8.5; carrying out SDS-PAGE electrophoretic identification;

(10) taking 1mg of the cloned pBV/GST/F4/8 protein 1-179aa as an antigen, diluting the antigen to 1mL by sterile water, adding 1mL of Freund's complete adjuvant, mixing, stirring at a high speed for emulsification, immunizing adult New Zealand big ear white rabbits, females and backs by subcutaneous multi-point injection (no less than 6 points), immunizing times per month, continuously immunizing for 3 times, and using Freund's incomplete adjuvant for the second and third immunizations;

(11) collecting blood from ear vein 2 weeks, 1 month, 2 months and 2.5 months after times of immunization, measuring serum antibody titer by Elisa, collecting animal whole blood from heart 2 weeks after the third immunization for antibody extraction, centrifuging, and collecting supernatant;

(12) by using an octanoic acid-ammonium sulfate precipitation method, firstly mixing serum and normal saline at a ratio of 1: 1, adding 2 times volume of acetic acid (0.01M, pH 4.0), adjusting pH to 4.8 with 6N HCl, slowly dripping octanoic acid according to 25uL of octanoic acid per 1mL of serum under magnetic stirring, and stirring at room temperature for 30 min;

(13) centrifuging (12000r/min, 30min, 4 deg.C), collecting supernatant, adding 1/10 volume PBS (0.2M, pH7.2), adjusting pH to 7.6 with 5N NaOH, adding ammonium sulfate under magnetic stirring to give final concentration of 0.23g/mL, and standing at 4 deg.C overnight;

(14) centrifuging (12000r/min, 30min, 4 deg.C), discarding supernatant, dissolving precipitate with PBS (0.2M, pH7.2), and dialyzing in dialysis bag for over 24 hr;

(15) centrifuging (12000r/min, 20min, 4 deg.C), removing insoluble substances, and storing at-20 deg.C to obtain purified IgG;

(16) the protein content of the samples was determined by UV-754 ultraviolet spectrophotometer.

The F4/80 nitrogen end 1-179aa polypeptide used in the embodiment of the invention has good immunogenicity, and is a sequence with dominant immune epitopes in an extracellular sequence of F4/80 protein predicted by Biosun software; compared with the existing commercialized rabbit polyclonal antibody similar to the rabbit obtained by using other nitrogen-terminal polypeptides, the anti-F4/80 polyclonal antibody obtained by using the polypeptide as the antigen for immunizing rabbits has obviously better affinity performance and high sensitivity of immunofluorescence staining macrophages.