Polypeptide with SEQ ID NO.2 sequence, antibody with strong ADCC effect and application
阅读说明：本技术 一种具有seq id no.2序列的多肽以及具有强adcc效应的抗体和应用 (Polypeptide with SEQ ID NO.2 sequence, antibody with strong ADCC effect and application ) 是由 张科 罗语思 徐瑞雪 孙泽华 于 2019-11-08 设计创作，主要内容包括：本发明公开了一种具有SEQ ID NO.2序列的多肽以及具有强ADCC效应的抗体和应用,涉及生物医药技术领域。具体地,该多肽的氨基酸序列如SEQ ID No.2所示,该多肽能够作为抗原,用于诱导产生具有强ADCC效应的HIV抗体,能够为有效预防、诊断或治疗HIV提供一种途径。(The invention discloses a polypeptide with an SEQ ID NO.2 sequence, an antibody with strong ADCC effect and application, and relates to the technical field of biological medicine. Specifically, the amino acid sequence of the polypeptide is shown as SEQ ID No.2, the polypeptide can be used as an antigen for inducing and generating an HIV antibody with strong ADCC effect, and a way can be provided for effectively preventing, diagnosing or treating HIV.)
1. A polypeptide, characterized in that the amino acid sequence of the polypeptide is shown as SEQ ID No. 2.
2. Use of a polypeptide according to claim 1 as antigen for screening of HIV antibodies with a strong ADCC effect.
Technical Field
The invention relates to the technical field of biological medicines, in particular to a polypeptide with an SEQ ID NO.2 sequence, an antibody with strong ADCC effect and application.
Background
AIDS is a very harmful infectious disease caused by infection with the HIV virus. HIV is a virus that attacks the human immune system. It takes the most important CD4T lymphocyte in human immune system as the main target of attack, largely destroys the cell, and makes human body lose immune function.
Therefore, the human body is easy to be infected with various diseases, malignant tumors can occur, and the fatality rate is high. The incubation period of HIV in human body is 8-9 years on average, and before AIDS, the HIV can live and work for many years without any symptoms.
Currently, there is no effective HIV-1 vaccine.
In view of this, the invention is particularly proposed.
Disclosure of Invention
The invention aims to provide a polypeptide, application of the polypeptide as an antigen in inducing generation of an HIV antibody with strong ADCC effect, a method for preparing the HIV antibody with strong ADCC effect, the antibody with strong ADCC effect, a CAR-T cell targeting HIV, a reagent or a kit for detecting HIV virus and a medicine for treating AIDS. The polypeptide can be used as an antigen for inducing the generation of HIV antibodies with strong ADCC effect.
The invention is realized by the following steps:
in a first aspect, the embodiments provide a polypeptide having an amino acid sequence at least 69% identical to any one of SEQ ID nos. 1-3. The polypeptide can be used as an antigen for inducing the generation of HIV antibodies with strong ADCC effect.
In alternative embodiments, the amino acid sequence of the polypeptide is as set forth in any one of (a) - (c) below:
(a) a sequence having at least 69% identity to SEQ ID No. 1;
(b) a sequence having at least 80% identity to SEQ ID No. 2;
(c) a sequence having at least 87.5% identity to SEQ ID No. 3. Sequence conservation of SEQ ID Nos. 1-3 with Induction reference is made in particular to FIG. 1 (NCBI blast, based on the results of HIV database analysis). In FIG. 1, D1 corresponds to the sequence shown in SEQ ID No.1, D2 corresponds to the sequence shown in SEQ ID No.2, and D3 corresponds to the sequence shown in SEQ ID No. 3.
In an alternative embodiment, the amino acid sequence of the polypeptide is as shown in (d):
(d) as shown in formula 1;
formula 1: ACVPTDP-X1-PQEVVLVNV; in formula 1, X1 is P or N;
preferably, X1 is P.
In an alternative embodiment, the amino acid sequence of the polypeptide is as set forth in any one of SEQ ID Nos. 1-3.
In a second aspect, the embodiments of the present invention provide the use of a polypeptide as provided in the above embodiments as an antigen for inducing the production of HIV antibodies with a strong ADCC effect.
In a third aspect, the embodiments of the present invention provide a method for preparing an HIV antibody having a strong ADCC effect, comprising: the polypeptides provided in the above embodiments are used as antigens to immunize animals.
In alternative embodiments, the animal is selected from one or more of a rabbit, a mouse, an alpaca, a cow, a horse and a camel.
In a fourth aspect, the embodiments of the present invention provide an antibody with strong ADCC effect, which is prepared by the method or epitope thereof, which is the polypeptide provided in the above embodiments.
In a fifth aspect, embodiments of the invention provide an HIV-targeted CAR-T cell having the antibody or scFV fragment thereof provided in the above embodiments on the cell membrane surface.
In a sixth aspect, the embodiments of the present invention provide a reagent or a kit for detecting HIV virus, which contains the antibody provided in the above embodiments or the polypeptide provided in any of the above embodiments.
In a seventh aspect, the embodiments of the present invention provide a medicament for treating aids, which comprises the antibody provided in the above embodiments or the CAR-T cell provided in the above embodiments.
The invention has the following beneficial effects:
the application provides a polypeptide, the amino acid sequence of the polypeptide has at least 69% of identity with any one sequence in SEQ ID No.1-3, the polypeptide can be used as an antigen for inducing the generation of HIV antibodies with strong ADCC effect, and a way for effectively preventing, diagnosing or treating HIV is provided.
In addition, the embodiment of the invention also provides application of the polypeptide as an antigen in inducing generation of an HIV antibody with strong ADCC effect, a method for preparing the HIV antibody with strong ADCC effect, the antibody with strong ADCC effect, a CAR-T cell targeting HIV, a reagent or a kit for detecting HIV virus and a medicine for treating AIDS.
Drawings
In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings needed to be used in the embodiments will be briefly described below, it should be understood that the following drawings only illustrate some embodiments of the present invention and therefore should not be considered as limiting the scope, and for those skilled in the art, other related drawings can be obtained according to the drawings without inventive efforts.
FIG. 1 shows the conservation of the sequences shown in SEQ ID No.1 to SEQ ID No.3 in the summary of the present invention;
FIG. 2 is a flow chart of the construction of a yeast-displayed HIV-1HXB2 fragment library as provided in example 4 of the present invention;
FIG. 3 shows the result 1 of the measurement of recombinant yeast cells by the flow cytometer provided in example 4 of the present invention;
FIG. 4 shows the result 2 of the measurement of recombinant yeast cells by the flow cytometer provided in example 4 of the present invention;
FIG. 5 shows the measurement result 3 of recombinant yeast cells by the flow cytometer provided in example 4 of the present invention;
FIG. 6 is a depiction of the IgG sequence of a positive yeast clone provided in example 4 of the present invention used to map the epitope to full length HIV-1HXB2gp 160;
FIG. 7 shows the results of experiments for eliminating the antibody absorption of the epitope provided in test example 1 of the present invention;
FIG. 8 shows the results of screening for memory B cells using epitopes provided in test example 2 of the present invention.
Detailed Description
In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially.
The features and properties of the present invention are described in further detail below with reference to examples.
Example 1
A polypeptide, the polypeptide is an antigen, and the antigen epitope of the antigen comprises a first epitope shown as SEQ ID No. 1.
Example 2
A polypeptide, the polypeptide is an antigen, and the antigen epitope of the antigen comprises a second epitope shown as SEQ ID No. 2.
Example 3
A polypeptide, the polypeptide is an antigen, and the antigen epitope of the antigen comprises a third epitope shown as SEQ ID No. 3.
Example 4
(1) Purification of serum IgG antibodies
Materials and methods:
9 plasma samples from HIV-1 infected subjects. PBMC samples were provided by the chinese centers for disease prevention and control. PBMCs from sample donors were provided by the hong cross of hong kong. This study was approved by the institutional review board of hong kong university and the academic ethics committee.
Plasma samples (hong Kong cross) were sterilized at 56 ℃ for 45 minutes and then centrifuged at 10000g for 10 minutes. The supernatant was filtered through a 0.2 μm microporous membrane and after filtration the antibody protein was loaded onto a G-Sepharose column, which had been equilibrated with Phosphate Buffered Saline (PBS) before use. After washing with 25 column volumes of PBS, IgG antibodies were eluted with 0.5M acetic acid (pH 3.0) and immediately neutralized with 3M Tris-HCL (pH 9.0) and the buffer was changed to PBS. The purity of the polyclonal IgG antibodies was confirmed by performing reducing and non-reducing SDS-PAGE gels.
(2) Construction method of yeast-displayed HIV-1HXB2 fragment library
Referring to FIG. 2, DNA encoding full length HIV-1HXB2gp160 was amplified by PCR using a recombinant plasmid containing the full length HIV-1HXB2gp160 gene as a template and a pair of primers P-ENV-F and P-ENV-R. The primer is also used for amplifying other HIV-1gp160 isolates, including 89.6, 92HT, 92V5037, Bal, GXC44-2, GXE44-2, JRCSF, JRFL, R2 and Z2Z 6-2.
The PCR-amplified HIV-1HXB2gp160 DNA was gel purified, and then the purified HIV-1HXB2gp160 DNA (2. mu.g) was gel-purified with a total volume of 20. mu.l of digestion buffer (50mM Tris-HCl, pH7.5, 10mM MnCl) containing 1 unit DNase I (Roche)2) Digestion was carried out at 15 ℃ for 15 minutes. After digestion, the reaction was stopped by addition of EDTA to a final concentration of 50 mM. The digestion products were analyzed on an agarose gel, and fragments ranging in size from 100bp to 500bp were purified from the gel and blunt-ended with T4 DNA polymerase (New England BioLabs). The blunt-ended DNA fragment was ligated to pComb3X vector, purified and electroporated into TG1 competent cells to construct a library of bacterial HIV-1HXB2gp160 epitope fragments.
The obtained HIV-1HXB2gp160 epitope fragment library was gel purified and PCR re-amplified using high fidelity DNA polymerase (Invitrogen) and a pair of primers YDRDF and YDRDR to add two overhang regions for homologous recombination with the linearized yeast display plasmid pYD 7. Specifically, the PCR system for PCR re-amplification is: denaturation at 95 ℃ for 3 min; 30 seconds at 95 ℃, 30 seconds at 58 ℃ and 30 seconds at 72 ℃ for 20 cycles; followed by extension at 72 ℃ for 10 min.
The reamplified PCR product with the protruding region inserted and the linearized PYD7 plasmid DNA were purified in 3: 1 (molar mass ratio) and electroporated into competent yeast EBY100 cells according to the lithium acetate method to obtain a yeast display library.
Specifically, the method of electroporation comprises: 10ml of the yeast strain EBY100 in the stationary growth phase was inoculated into 100ml of YPD medium and cultured at 37 ℃ and 250rpm for 45 hours until OD600 nm reached 1.6-1.8. Cells were centrifuged at 2500g for 3 min at 4 ℃, washed three times with sterile deionized water, and 20ml of electroporation buffer (1M Sorbital ═ 1mM CaCl)2) And cleaning once. Subsequently, the yeast cells were resuspended in 20ml LiAc buffer (0.1M LiAc +10mM DTT) and incubated at 30 ℃ for 30 minutes without shaking. The LiAc buffer was discarded and the cell was washed with cold electroporation buffer.
1ml of electroporation buffer is used to resuspend the cells at a cell density of 1.6-2.0 hundred million/ml. Transformation by electric shock was performed in 2mm cuvettes containing DNA/cell mixtures with parameters of electric shock: voltage 2.5KV, electric pulse 25uf, resistance 200 Ω. After 1 hour recovery in YPD medium at 30 deg.C, resuspension cells were cultured in SDCAA culture and shaken overnight at 30 deg.C for recombinant selection. After expansion, the recombinant yeast cells were aliquoted and stored in SDCAA medium (yeast nitrogen-based Casamino Acids medium containing 20 g/l glucose) supplemented with 15% glycerol at-80 ℃. Each aliquot contained 1 million recombinant yeast cells in 1ml of freezing medium.
(3) Screening of Yeast libraries
Induction of expression of HIV-1HXB2gp160 epitope fragment on yeast cell surface: the recombinant yeast cells obtained in step (2) were grown in SDCAA medium at 30 ℃ for one day and passaged once with fresh medium to eliminate dead cells. Then, the yeast cells are resuspended in SGCAA medium to an optical density (OD600) of 0.5-1.0 at 600nm, and then induced at 20 ℃ for 36-48 hours.
Sorting against serum purified IgG antibodies: induced recombinant yeast cells (obtained in step (2)) were first stained with purified IgG antibody (obtained in step (1)) at a final concentration of 500nM by incubation at 4 ℃ for 3-4 hours.
After washing 3 times with cold PBS, recombinant yeast cells were treated with goat anti-human IgG [ F (ab')2Specificity of]PE and FITC conjugated to a mouse anti-c-myc antibody were stained at 4 ℃ for 1 hour. After three washes with cold PBS, the stained recombinant yeast cells were assayed by using FACSAria III flow cytometer (BD Biosciences), and the double positive yeast population was classified and recovered.
PE-and/or FITC-labeled beads and unstained yeast were used to set up the compensation prior to sorting.
Referring to fig. 3 to 5, the measurement results were divided into a High ADCC group (fig. 3), a Low ADCC group (fig. 4) and a healthy control group (fig. 5) in this order, and the results of the High ADCC group were all significant, in which Q2 is the percentage of positive yeast expression antigen epitope and positive binding to the patient serum. The Low ADCC group had 2 poor specificities and were not suitable for use as antigens.
To sort recombinant yeast libraries stained with polyclonal IgG antibodies purified from different sera, the same gates were used. For each polyclonal IgG sample, sort 5X 105And (4) yeast cells.
(4) Identification of epitopes
From each of the sorted recombinant yeast libraries of step (2), recombinant yeast plasmids were extracted using a yeast cell plasmid extraction kit (Omega Bio-Tek) and electroporated into E.coli TG1 electrocompetent cells. From each sorted library, more than 200 ampicillin resistant clones were picked for sequencing. The insert in yeast cells (HIV-1DNA sequence insert) was translated by website software (http:// in silica. ehu. es/translate /). Only recombinant clones with HIV-1DNA sequence insert and correct open reading frame were considered positive.
The IgG sequence of the positive yeast clones was used to map against the epitope of full-length HIV-1HXB2gp160 (HIV database accession No. b.fr.83.HXB2_ LAI _ IIIB _ BRU _ K03455). Inserts less than 4aa in length or less than 75% identical were not considered in the analysis. For each serum purified IgG sample, about 150 valid positive clones were identified.
The frequency of each amino acid present in the positive clones was calculated and the same total number (total 5000 amino acids per sample) was calibrated in each serum purified IgG sample, see figure 6. In FIG. 6, the Y-axis represents the copy number, the X-axis represents the amino acid position of the HIV-1HXB2gp 140 protein (total 800+), and the right label represents the sample name, such as Polyclonal IgGs MY05, which represents Polyclonal antibodies in the serum of patient MY 05.
Variable loops indicate the bottom several letters of the X-axis, e.g., V1-V5 represent functional domains of HIV antigen, V1 is loop1, V2 is loop2, V4 is loop4, V5 is loop5, MR is two domains on gp41, MR1 is MHC related protein1, and MR1 is MHC related protein 2. CD4bs is CD4 binding site.
Test example 1
The function of the epitope provided by the invention is verified.
Antibody uptake experiments: the recombinant yeast cells expressing the epitope fragment of HIV-1HXB2gp160 obtained in example 4 were taken (10)8) Washed twice with PBS, and the washed recombinant yeast cells were incubated with 500. mu.g IgG of antibody purified from serum of volunteers in PBS in a total volume of 1ml at 4 ℃ overnight by centrifugation at 2000 Xg for 5 minutes.
The supernatant was transferred to 10 by centrifugation at 2000 Xg for 5 minutes to pellet the recombinant yeast cells8Recombinant yeast cell surface, and the mixture at room temperature 1h incubation. Yeast cells were pelleted by centrifugation at 2000 × g for 5 minutes and the supernatant was collected in a new tube.
Continuous elimination of antibodies is performed by switching the recombinant yeast expressing the epitope in a second round of consumption, followed by another round of consumption with the same recombinant yeast. OD280 was then measured using a NanoDrop device to determine the antibody concentration in the depleted IgG sample, see figure 7.
In FIG. 7, the X-axis is the name of the sample, the first is patient 18, IgG18-D1 is the serum left after antibody absorption by patient 18D 1 for ADCC, IgG7944-D1D2D3 is the serum left after antibody absorption by patients 7944D 1-D3 for ADCC, and so on. Here a total of 3 patients' sera were used, 10,7944 and P10 respectively. In this example, D1 is the polypeptide shown in SEQ ID No.1, D2 is the polypeptide shown in SEQ ID No.2, and D3 is the polypeptide shown in SEQ ID No. 3.
As is clear from FIG. 7, the serum adsorbed by the antigen motif no longer has the function of inducing high ADCC effect. That is, yeast expressing the antigen is mixed with serum to adsorb the antibody binding to the motif, and then the yeast is removed, so that the antibody having no motif binding ability remains in the serum.
Test example 2
Use of an epitope for screening antibodies for inducing a strong ADCC effect.
Labeling of antigen: expression of plasmid containing Fc fragment-linked epitope motifs (pcDNA) from 293F (Thermal, R79007) cellsTM3.1(+)), the epitopes comprise the first epitope (D1), the second epitope (D2) and the third epitope (D3) provided in examples 1-3, respectively.
Then, the protein in the 293F supernatant is purified by a nickel column purification method, the protein is diluted to 0.5-2 mg/ml by I3BS, and then biotin (EZ-Link) of the Thermal company is utilizedTMSulf good component-NHS-LC-Biotin, 21335) according to the kit process, labeling the epitope protein, and using the labeled protein molecule for screening antigen-specific memory B cells, with the screening results shown in FIG. 8.
In summary, the present application provides a polypeptide, the amino acid sequence of which has at least 69% identity with any one of SEQ ID nos. 1-3, which can be used as an antigen for inducing the production of HIV antibodies with strong ADCC effect, and which can provide a way for the effective prevention, diagnosis or treatment of HIV.
In addition, the embodiment of the invention also provides application of the polypeptide as an antigen in inducing generation of an HIV antibody with strong ADCC effect, a method for preparing the HIV antibody with strong ADCC effect, the antibody with strong ADCC effect, a CAR-T cell targeting HIV, a reagent or a kit for detecting HIV virus and a medicine for treating AIDS.
The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
SEQUENCE LISTING
<110> Guizhou medical university
<120> polypeptide with SEQ ID NO.2 sequence, antibody with strong ADCC effect and application
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