CN114316057B - Nano antibody targeting mitochondrial outer membrane translocation factor complex TOM20 subunit and application thereof - Google Patents

Abstract

The invention discloses a nano antibody of targeted mitochondrial outer membrane transposition factor complex TOM20 subunit and application thereof, wherein the complementary determining region of the nano antibody comprises an amino acid sequence CDR1 shown in SEQ ID NO.1, an amino acid sequence CDR2 shown in SEQ ID NO.2 and an amino acid sequence CDR3 shown in SEQ ID NO. 3. The invention also discloses any one of the following applications of the nano antibody: 1) used for detecting the TOM20 subunit of the mitochondrial outer membrane translocation factor complex; 2) the kit is used for preparing a mitochondrial outer membrane translocation factor complex TOM20 subunit detection reagent or kit; 3) used for enriching and purifying the subunit of the mitochondrial outer membrane translocation factor complex TOM 20; 4) the enrichment and purification reagent is used for preparing the subunit TOM20 of the mitochondrial outer membrane translocation factor complex. The invention proves the binding condition and specificity of the nano antibody and the TOM20 protein.

Description

Nano antibody targeting mitochondrial outer membrane translocation factor complex TOM20 subunit and application thereof

Technical Field

The invention relates to the technical field of antibodies, in particular to a nano antibody targeting a mitochondrial outer membrane translocation factor complex TOM20 subunit and application thereof.

Background

Mitochondria contain about 1000-1500 proteins, 99% of which are encoded by nuclear genes, and most of which are transported into mitochondria after intracytoplasmic translation by The mitochondrial transit enzyme complex (TOM) for corresponding functions. TOM is a membrane protein complex composed of 7 subunits, in which TOM20, the most important receptor subunit in the TOM complex, is responsible for the first step of mitochondrial transport proteins, and directly recognizes a precursor protein having an N-terminal leader sequence from many proteins, and then delivers it to TOM40 protein complex. TOM20 is composed of three domains, the mitochondrial membrane gap region (1-6), the transmembrane region (7-24), and the cytoplasmic region (25-145). Since TOM20 is localized only to the outer mitochondrial membrane, it generally acts as a marker for mitochondria.

Clinical and genetic heterogeneous diseases caused by genetic oxidative phosphorylation disorders are called mitochondrial diseases. Mutations in mitochondrial DNA and nuclear genes encoding mitochondrial proteins are major factors contributing to mitochondrial disease. Over the last three decades, mutations that cause these diseases have been found in nearly 290 genes, but many patients have not yet been molecularly diagnosed. The complexity of mitochondrial biology and genetics remains a difficult point in understanding the molecular mechanisms of mitochondrial disease, and so to date there has been no effective way to treat mitochondrial disease.

In view of the importance of mitochondria, studies on the structure, function and physicochemical properties of components such as mitochondrial membranes, respiratory chain enzymes and mitochondrial DNA have become important subjects in cell biology studies, and therefore techniques for extracting mitochondria have become an essential means in mitochondrial studies. The current methods of mitochondrial refining are mainly by density gradient centrifugation, which takes up to several hours. However, many physiological and biochemical reactions in cells are very rapid, and half-lives of some proteins or other molecules are very short, so that the proteins or other molecules are difficult to capture, and the research on molecular mechanisms of mitochondrial diseases is not facilitated. Therefore, it is hoped that a novel method for rapidly extracting mitochondria can be developed, namely, the mitochondria can be extracted by the co-immunoprecipitation method by using the nanobody of TOM 20.

In addition, studies have shown that mitochondria play an important role in cell signaling, apoptosis regulation, and energy metabolism in drug-induced cancer cell death. Therefore, mitochondria are also considered to be important targets for cancer chemotherapy. Macroautophagy is a degradation pathway in cells that removes proteins, as well as larger organelles, such as dysfunctional organelles and intracellular pathogens. The nano antibody of TOM20 can specifically target mitochondria, and can deliver mitochondria to lysosomes by using an autophagy-targeting chimera (AUTAC) technology, thereby eliminating mitochondria to achieve the purpose of treating tumors and other mitochondria-related diseases.

Phage display screening is a high throughput method for rapidly identifying polypeptides or antibody fragments that bind to a particular target molecule. By applying conventional molecular biotechnology, large-capacity polypeptide or protein/antibody fragment genes can be constructed into a phage genome, and the polypeptide and protein/antibody fragments corresponding to the genes are expressed on phage coat proteins by utilizing the direct correspondence between the phage gene phenotype and the protein phenotype. Researchers can obtain a polypeptide, protein or antibody fragment that binds to a given target molecule in a short period of time through several rounds of biological screening of binding-rinsing-elution-amplification. Therefore, if a researcher uses target proteins of various diseases as research objects, the purified proteins are coated in a solid medium (such as an ELISA plate), a phage polypeptide or antibody library is screened, and three to four rounds of screening are performed, so that a polypeptide or an antibody specifically binding to the target protein can be obtained, and the obtained polypeptide or antibody 1) can be used as a carrier for labeling a contrast agent for molecular image diagnosis of the diseases; 2) can be coupled with various chemical drugs, biological drugs (cell factors, proteins, immunotoxins) and the like to perform targeted therapy on diseases, so as to reduce the usage amount and side effects of the drugs; 3) certain polypeptides, or antibodies, themselves have the effect of agonizing or antagonizing the function of the target protein. To date, a large number of polypeptides or antibodies identified by phage screening have entered clinical or preclinical testing for the treatment of various diseases or as vectors for targeted molecular diagnostics and targeted therapy.

Single-domain antibodies (sdabs) are a class of antibodies that consist of heavy chains only, a form of antibody that occurs naturally in camelids and cartilaginous fish. Compared to traditional antibodies, single domain antibodies lack the CH1 domain as well as the light chain; its function relies solely on the heavy chain variable region to recognize the antigen. The single domain antibody with the Fc segment removed is also called a nano antibody, the crystal width of the single domain antibody is 2.5nm, the length of the single domain antibody is 4nm, the molecular weight of the single domain antibody is only 1/10 (about 15 kDa) of the traditional whole antibody, and the single domain antibody still has the complete antigen recognition capability. The nanobody is not very immunogenic to human body as a therapeutic antibody because of its high similarity to the sequence of VH of human antibody. The nanobody has the advantages of easy expression and purification by using a prokaryotic system, strong tissue penetrability, and good solubility and stability to a certain range of pH and temperature due to the small molecular weight. Meanwhile, the nano antibody is easy to modify or carry out various chemical markers through genetic engineering, and a functional antibody preparation can be generated in a short time.

The phage nano antibody library is widely used for screening and identifying nano antibodies of various targets, and the nano antibodies combined with target proteins can be obtained within two to three weeks or two to three months by screening a constructed natural nano antibody library or an immune phage nano antibody library prepared after immune camel.

There are few reports on nanobodies targeting mitochondrial TOM complexes.

Disclosure of Invention

The invention screens the nano antibody combined with the outer membrane region structure of TOM20 mitochondria by using a natural high-capacity phage nano antibody library constructed by more than 103 alpacas peripheral blood, after three rounds of screening, randomly picks 200 phage clones to perform phage ELISA for preliminary verification, performs sequencing analysis on the preliminarily verified positive clones, and finally picks six sequences thereof for verification, so that the P2B7 nano antibody has higher affinity with TOM20, stronger stability, easy preparation and high yield.

Based on the above research results, the present invention claims the following technical solutions:

a nanobody targeting a subunit of the mitochondrial outer membrane transposable factor complex TOM20, the complementarity determining region of which comprises the amino acid sequence CDR1 shown in SEQ ID No.1, the amino acid sequence CDR2 shown in SEQ ID No.2 and the amino acid sequence CDR3 shown in SEQ ID No. 3.

The framework region of the nano antibody comprises an amino acid sequence FR1 shown in SEQ ID NO.4, an amino acid sequence FR2 shown in SEQ ID NO.5, an amino acid sequence FR3 shown in SEQ ID NO.6 and an amino acid sequence FR4 shown in SEQ ID NO. 7.

The amino acid sequence of the nano antibody is shown in SEQ ID NO. 8.

The invention also claims a polynucleotide encoding any of the nanobodies described above.

The invention also claims an expression vector containing the polynucleotide.

Preferably, the expression vector is PET-14B or PET28 a-sumo.

The invention also claims a recombinant cell comprising the aforementioned expression vector or having the aforementioned polynucleotide integrated into its genome.

The invention also claims a nanobody phage, the surface of which includes any of the nanobodies described above.

The invention also claims a kit for detecting the TOM20 subunit of the mitochondrial outer membrane translocation factor complex, which comprises any one of the nanobodies or the nanobody phage.

The invention also claims any one of the following applications of the nano-antibody described in any one of the above:

1) used for detecting mitochondrial outer membrane translocation factor complex TOM20 subunit;

2) the kit is used for preparing a mitochondrial outer membrane translocation factor complex TOM20 subunit detection reagent or kit;

3) used for enriching and purifying the subunit of the mitochondrial outer membrane translocation factor complex TOM 20;

4) the enrichment and purification reagent is used for preparing the subunit TOM20 of the mitochondrial outer membrane translocation factor complex.

The invention has the following beneficial effects:

through using natural high-capacity phage nano-antibody library constructed by more than 103 alpaca peripheral blood, screening nano-antibodies combined with TOM20 mitochondrial outer membrane region, after three rounds of screening, randomly picking 200 phage clones for phage ELISA to perform preliminary verification, performing sequencing analysis on the preliminarily verified positive clones, and finally picking six sequences for verification, the P2B7 nano-antibody is found to have higher affinity with TOM20, and has stronger stability, easy preparation and high yield, and the combination condition and specificity with TOM20 protein are verified at the cell level.

The P2B7 nanometer antibody can be applied to:

1) the nanometer antibody can be used for rapidly purifying cell mitochondria in vitro by targeting TOM20 so as to research the molecular mechanism of mitochondrial diseases; the method is expected to develop a novel method for rapidly extracting mitochondria, namely, the mitochondria are extracted by using a nano antibody of TOM20 through a co-immunoprecipitation method.

2) The nano antibody of the targeting mitochondrion TOM20 can be fused with a nano antibody of autophagy protein LC3 and the like, and an autophagy-targeting chimera (AUTAC) technology is utilized to deliver the mitochondrion to the lysosome to eliminate the mitochondrion in the disease cell, so that the degradation of the mitochondrion in the disease cell is induced, and the aim of treating tumors and other related diseases of the mitochondrion is fulfilled.

3) Various diagnostic and therapeutic strategies targeting mitochondrial TOM 20.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, it is obvious that the drawings in the following description are only some embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to the drawings without creative efforts.

Wherein:

FIG. 1A is a schematic representation of the cytoplasmic domain of TOM 20; FIG. 1B shows electrophoretic Coomassie blue staining after purification of ToM20 (human and murine) cytoplasmic domain expression; FIG. 1C shows the TOM20 nano antibody library screening titers.

FIG. 2A shows the number of repetitions of ELISA screening>2 and TOM20/BSA ratio>3, a nanobody sequence; FIG. 2B is an SDS-PAGE identification; FIG. 2C shows the results of HIS-TAG western blot identification; FIG. 2D shows the primary ELISA of purified nanobody with TOM20-Human, showing that P2B7 binds strongly with TOM 20-Human; FIG. 2E shows the control protein GFP and TOM20-Human ELISA validation, showing that GFP has no binding activity to TOM 20-Human.

FIG. 3A shows the results of the binding ELISA assay of P2B7 with TOM 20-Human; FIGS. 3B and 3C are the results of Biacore testing the affinity of P2B7 nanobody to TOM20-Human and TOM20-Mouse antigen proteins, respectively, and show that P2B7 can bind to 2 antigens; fig. 3D is a result of co-localization experiment of P2B7 and mitochondria in Hela cells observed by confocal laser microscopy, and it was revealed that P2B7 can be localized on mitochondria.

Detailed Description

The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention. The experimental method steps in the examples are, unless otherwise specified, conventional method steps in the art; all biological and chemical reagents, unless otherwise specified, are conventional in the art and are commercially available.

Example 1

First, TOM20 cytoplasmic domain expression purification and nano antibody screening

The size of TOM20 mitochondrion cytoplasmic domain protein (25-145) is about 14 kD, the structural domain schematic diagram is shown in figure 1A, and the protein is suitable for serving as a target protein for nano antibody screening. The TOM20 cytoplasmic domain gene is designed and synthesized, optimized and synthesized, the nucleotide sequence of the TOM20 cytoplasmic domain gene is shown as SEQ ID NO.9, and the TOM20 cytoplasmic domain gene is transformed into escherichia coli for induced expression and purification and is used for screening nano antibodies. The expression purification steps are as follows: a) in order to prevent the formation of inclusion bodies and the degradation of proteins, the induction conditions were investigated at a low temperature of 16 ℃ by IPTG (isopropyl-. beta. -D-thiogalactoside) at different concentrations (0.2 mM, 0.4 mM, 1 mM) and finally determined to be induced overnight at a low temperature of 16 ℃ with 0.4 mM IPTG; b) carrying out a large amount of induction expression according to the induction conditions of the preliminary experiment, and carrying out bacterium breaking under the working condition of a high-pressure bacterium breaking instrument of 1000W; c) 17000 g, centrifuging at 4 ℃ for 30 min, taking the supernatant and incubating with Ni filler at 4 ℃ for 1 hour; d) eluting target protein by imidazole with gradient concentration; e) after Ni column purification, molecular sieve separation was performed to remove contaminating proteins, and AKTA parameters were set at 0.5 mL flow rate/min, collected every 1 mL. f) And determining the purity of the target protein according to the electrophoresis result, and determining the protein concentration by using a BCA method.

Screening natural high-capacity phage display nano antibody library constructed by more than 103 alpaca peripheral blood by adopting an immune tube method, wherein the capacity of the selected phage display library is 2 multiplied by 10⁹. The screening steps are as follows: a) coating the target protein on an immune tube according to the concentration of 50 mu g/mL, and carrying out 3 rounds of enrichment screening; b) the genes encoding the antibodies of the library were sequenced using a third round of phage eluents.

Finally, after the protein is purified by a Ni column and a molecular sieve, the high-purity target protein is obtained and is used for the subsequent nano antibody screening (figure 1B). Coating TOM20 cytoplasm domain to perform three rounds of natural alpaca nano antibody library screening, after three rounds of screening, phage titer shows that the library is highly enriched, which indicates that the nano antibody combined with TOM20 is amplified, after three rounds of phage nano antibody library screening, the library screening titer is 4 × 10⁷pfu/ml to 4.4X 10⁹pfu/ml, enriched by 110-fold (FIG. 1C).

Second, phage cloning ELISA preliminary verification of positive clones

After three rounds of screening, randomly picking 200 phage clones, expanding and inducing the expression of the nano-antibody, using crude phage nano-antibody extract to incubate with an ELISA plate coated by TOM20, amplifying the signal of the phage antibody, and defining the positive clone as the light absorption value combined with TOM20 is 2.5 times greater than the BSA control; and then sequencing and analyzing the positive clone extracted plasmid, extracting a sequence to obtain a nano antibody protein sequence, and carrying out comparative analysis on the sequence to obtain the distribution frequency of the positive sequence.

Through ELISA monoclonal verification technology, 88 positive clones are obtained in total, sequencing is carried out, and sequencing results show that 8 non-signals exist and 80 normal clones exist. According to the sequence of the nano antibody, the sequence of the nano antibody can be obtained from the sequencing result, 80 sequences are translated into amino acid and then sequenced, and multiple sequences are compared, so that 27 different sequences of the nano antibody are obtained. The clones with the repetition times more than 2 times total 12 clones (figure 2A), and the sequence of 6 nanobodies with higher repetition times and stronger binding than three times of BSA is expressed and purified, and the corresponding nanobody numbers are P2G2, P2G4, P2F4, P2H6, P2B7 and P2F5 (figure 2A); further expressing and purifying the six nano antibodies (fig. 2B and 2C) respectively, except that the protein is not obtained from P2G4, all the other proteins obtain high-purity recombinant protein; using the purified nanobody, ELISA preliminary validation revealed that P2B7 bound significantly to TOM20-Human, but not to GFP, a control protein (fig. 2D and 2E), and the amino acid sequence of nanobody P2B7 was as follows (SEQ ID No. 8):

MAVQLVESGGGLVQAGGSLRLSCVASGSTFSNFVMGWYRQAPGKQRVLVATISSGGSTNYADSVKGRFTISRDNTKNTVYLQMNSLKPEDTSVYYCNARRGSVIVPTLAYDYWGQGTQVTVSS。

purification expression of three, nanometer antibody

The gene sequence of the nano antibody is cloned into a PET-14B (Youbaobao) or PET28a-sumo (Youbao) vector, and a hemagglutinin tag (hemagglutinin HA tag) is fused and expressed for subsequent detection. The expression purification steps are as follows: a) to prevent the formation of inclusion bodies and protein degradation, induction was performed at low temperature of 16 ℃ using IPTG at a concentration of 0.2 mM; b) carrying out a large amount of induction expression according to the induction conditions of the preliminary experiment, and carrying out bacterium breaking under the working condition of a high-pressure bacterium breaking instrument of 1000W; c) 17000 g, centrifuging at 4 ℃ for 30 min, taking the supernatant and incubating with Ni filler at 4 ℃ for 1 hour; d) eluting target protein by imidazole with gradient concentration; e) after Ni column purification, molecular sieve separation was performed to remove contaminating proteins, and AKTA parameters were set at 0.5 mL flow rate/min, collected every 1 mL.

Affinity detection of four-TOM 20 nano antibody

1. The experimental procedure was as follows:

(1) ELISA experiments

This experiment was used to verify whether the in vitro expression of purified nanobodies could interact directly with the in vitro purified antigenic proteins. The method comprises the following steps: a) diluting antigen protein with PBS to 10ug/ml, plating at 100 ul/well, coating the well plate, and standing overnight at 4 deg.C; b) sealing for 2h at room temperature; c) incubating the nano antibodies with different concentrations for 1h at room temperature; d) incubating a second antibody anti-HA HRP (Beijing Yinqiao Shenzhou) at room temperature for 1 h; e) TMB color development; e) stopping the reaction by using a stop solution; g) and measuring the light absorption value at 450 nm by using a microplate reader, and establishing a curve according to the light absorption value.

(2) Surface Plasmon Resonance (SPR) experiment

This experiment was used to further verify the binding of antigen and nanobody and calculate the equilibrium constant for both. Purified antigen protein is fixed on a chip, nano antibodies with different concentrations are sequentially added to analyze the affinity with the antigen protein, reaction signals within 360 seconds are recorded, a kinetic curve is made, and each relevant parameter is calculated.

(3) Co-localization experiment of nano antibody and cell mitochondria

Hela cells were revived and after 3 passages the cells were seeded in 24-well plates containing cell-slides for subsequent experiments. The method comprises the following specific steps: a) cells were washed 3 times with PBS and fixed in 4% paraformaldehyde for 20 minutes; b) PBS washing 3 times, FDB (Fluorescence Dilution Buffer: PBS conjugation 5% total bone serum, 2% BSA, 0.1% Saponin) Dilution of the nanobody, the final concentration of 2 uM, room temperature and cell incubation for 1 h; c) washing with PBS for 3 times, diluting anti-HA and mitochondrial marking antibody COXIV with FDB at a ratio of 1:100, and incubating with cells for 1h at room temperature; d) PBS was washed 3 times, Alexa 488 and 594 conjugated secondary antibodies were diluted 1:100 with FDB and incubated with cells for 1h at room temperature; f) washed 3 times with PBS, mounted, and photographed after drying.

2. Results of the experiment

The binding of P2B7 nanobody to TOM20 cytoplasmic domain was further examined by ELISA, and the results (fig. 3A) showed that P2B7 and TOM20 showed higher binding activity, but had substantially no binding activity to the control GFP protein. Surface plasmon resonance experiments further found that the P2B7 nanobody can bind not only to TOM20-Human but also to TOM20-Mouse, and the binding affinity constants KD of P2B7 to TOM20-Human and TOM20-Mouse are 44.8 nM and 412.6 nM, respectively (FIGS. 3B and 3C). The immunofluorescence result of the nano antibody shows that the P2B7 can be co-localized with a cell mitochondrial marker COXIV (FIG. 3D), and the result shows that the P2B7 nano antibody can specifically recognize mitochondria and has the potential of separating and purifying the mitochondria.

While the invention has been described in connection with what is presently considered to be the most practical and preferred embodiment, it is to be understood that the invention is not to be limited to the disclosed embodiment, but on the contrary, is intended to cover various modifications and equivalent arrangements included within the spirit and scope of the appended claims.

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Claims (10)

1. A nanobody targeting the TOM20 subunit of the mitochondrial outer membrane translocator complex, characterized by: the complementarity determining region of the nano antibody comprises an amino acid sequence CDR1 shown in SEQ ID NO.1, an amino acid sequence CDR2 shown in SEQ ID NO.2 and an amino acid sequence CDR3 shown in SEQ ID NO. 3.

2. The nanobody of claim 1, wherein: the framework region of the nano antibody comprises an amino acid sequence FR1 shown in SEQ ID NO.4, an amino acid sequence FR2 shown in SEQ ID NO.5, an amino acid sequence FR3 shown in SEQ ID NO.6 and an amino acid sequence FR4 shown in SEQ ID NO. 7.

3. The nanobody of claim 2, wherein: the amino acid sequence of the nano antibody is shown in SEQ ID NO. 8.

4. A polynucleotide, wherein: the polynucleotide encodes the nanobody of any one of claims 1 to 3.

5. An expression vector, characterized in that: the expression vector comprising the polynucleotide of claim 4.

6. The expression vector of claim 5, wherein: the expression vector is PET-14B or PET28 a-sumo.

7. A recombinant cell, wherein: the recombinant cell comprises the expression vector of claim 5 or has the polynucleotide of claim 4 integrated into its genome.

8. A nanobody phage, characterized by: the surface of the bacteriophage comprises a nanobody according to any one of claims 1 to 3.

9. A kit for detecting mitochondrial outer membrane translocation factor complex TOM20 subunit, comprising: comprising the nanobody of any one of claims 1 to 3 or the nanobody phage of claim 8.

10. Use of the nanobody of any one of claims 1 to 3 for any one of the following applications:

1) used for detecting mitochondrial outer membrane translocation factor complex TOM20 subunit;

2) the kit is used for preparing a mitochondrial outer membrane translocation factor complex TOM20 subunit detection reagent or kit;

3) used for enriching and purifying the subunit of the mitochondrial outer membrane translocation factor complex TOM 20;

4) the enrichment and purification reagent is used for preparing the subunit TOM20 of the mitochondrial outer membrane translocation factor complex.

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