CN112812189B

CN112812189B - Nano antibody for resisting glycosyltransferase A subunit and application thereof

Info

Publication number: CN112812189B
Application number: CN202110077974.XA
Authority: CN
Inventors: 郭刚; 刘宇; 刘开云; 舒月力; 李彦; 王宁
Original assignee: West China Hospital of Sichuan University
Current assignee: West China Hospital of Sichuan University
Priority date: 2020-11-30
Filing date: 2021-01-20
Publication date: 2022-05-17
Anticipated expiration: 2041-01-20
Also published as: CN114853895A; CN114853896B; CN114853895B; CN112812189A; CN114853896A

Abstract

The invention discloses a nano antibody of an anti-glycosyltransferase A subunit and application thereof, wherein a variable region of the nano antibody is provided with 3 complementarity determining regions CDR-1, CDR-2 and CDR-3, the CDR-1 consists of an amino acid sequence shown in SEQ ID No.1, the CDR-2 consists of an amino acid sequence shown in SEQ ID No.2, and the CDR-3 consists of an amino acid sequence shown in SEQ ID No. 3. The nano antibody for resisting the glycosyltransferase A subunit has the activity of specifically binding with the glycosyltransferase A subunit, neutralizes the activity of the glycosyltransferase A, can be used for detecting clostridium difficile, and can also be used for preparing a medicament for treating the clostridium difficile; the nano antibody also has the characteristics of small molecular weight, high affinity, stable structure and performance and the like; can resist the acid environment of the stomach and is not easily degraded by pepsin, and the like.

Description

Nano antibody for resisting glycosyltransferase A subunit and application thereof

Technical Field

The invention belongs to the technical field of antibodies, relates to a nano antibody technology, and particularly relates to a nano antibody for resisting glycosyltransferase A (TcdA) subunit and application thereof.

Background

Clostridium Difficile (CD) is a normal flora in the intestinal tract of animals and humans, widely distributed in the natural environment, and can live in water, soil, vegetables, dogs, cats and in hospital environments. The antibiotic is used in an irregular way, so that intestinal flora is disordered, and drug-resistant CD can grow and reproduce in a large quantity, which causes diseases such as antibiotic-associated diarrhea, pseudomembranous enteritis and the like and is a main pathogen of the antibiotic-associated diarrhea. The spore resistance of the CD is strong and difficult to eliminate, and the CD can exist in the hospital environment for a long time, so that the CD becomes one of the main pathogenic bacteria of acquired diarrhea in the hospital.

Infection and pathogenesis of CD related diseases are mainly caused by excessive proliferation of toxigenic CD in intestinal tracts and massive release of toxin. Under normal conditions, the CD in the intestinal tract is inhibited by dominant flora such as bifidobacterium, bacteroides, eubacterium and the like, is in disadvantage and is not pathogenic; after receiving a large amount of broad-spectrum antibacterial drugs, immunosuppressants or chemotherapy drugs for a long time, the intestinal microecological balance is broken, the intestinal flora is disordered, CD is excessively propagated and releases toxin, and diseases related to Clostridium Difficile Infection (CDI) are caused.

When CD is propagated in large quantities, it destroys the normal flora of the colon by surface proteins of the thallus or spores, then colonizes, enters the mucus layer with the aid of flagella and proteases, adheres to intestinal epithelial cells, releases associated toxins, destroys the intestinal cells, changes the cytoskeleton, releases mucus and inflammatory products, and causes intestinal infectious diseases. After infection, high fever, watery stool, abdominal distension, toxic megacolon and other manifestations may appear. CD can produce 6 kinds of toxin, and the main pathogenic factors are toxin A, toxin B and binary toxin. The toxin A is enterotoxin which can be combined with a toxin receptor on a brush border cell of an intestinal mucosa, chemotaxis leucocyte, activate macrophage, mast cell and neutrophil, and release potent inflammatory transmitter and cytokine, thereby causing permeability increase of local mucosal blood vessels, villus damage, mucosal hemorrhage and necrosis; in pseudomembranous enteritis, the most direct role of toxins a and B is to disrupt the tight junctions of the epithelial barrier, promoting the release of inflammatory factors, the migration of neutrophils and the formation of pseudomembranes. Since the C-terminal of C-difficile toxin a and B has a functional region for toxin recognition and binding to a cell receptor, called receptor-binding domain (RBD), if an antibody against this region can be developed, and the RBD is covered, the binding of the toxin can be directly blocked, and the toxin cannot exert the toxin effect, thereby achieving the immune defense effect.

Because the infection rate and infection degree of CD become more serious, the development of new prevention and treatment methods is particularly important. In view of the important role of the toxin a, glycosyltransferase a, in CD infection, development of specific antibodies directed against glycosyltransferase a can also play a major role in combating CDI. Currently, there is no report on specific antibodies against glycosyltransferase a RBD.

Disclosure of Invention

Aiming at the current technical situation that the clostridium difficile is lack of effective treatment drugs, the invention aims to provide a nanobody of an anti-glycosyltransferase A subunit (TcdA), which has the activity of specifically binding with RBD of the glycosyltransferase A subunit, can be used for detecting clostridium difficile and inhibiting the activity of clostridium difficile, and has good clinical application prospect.

The variable region of the nano antibody for resisting the glycosyltransferase A subunit provided by the invention is provided with 3 complementarity determining regions CDR-1, CDR-2 and CDR-3, wherein the CDR-1 consists of an amino acid sequence shown in SEQ ID NO.1, the CDR-2 consists of an amino acid sequence shown in SEQ ID NO.2, and the CDR-3 consists of an amino acid sequence shown in SEQ ID NO. 3.

In a preferred implementation mode, the variable region sequence of the nanobody consists of the amino acid sequence shown in SEQ ID No. 4-6. Of course, the sequence of the variable region of the nanobody of the present invention is not limited to the amino acid sequence shown in SEQ ID No.4-6, but may be a derivative sequence obtained by substitution and/or deletion of one or more amino acid residues in the sequence shown, and having the same biological activity as SEQ ID No.4-6 (i.e., having three complementarity determining regions CDR-1, CDR-2 and CDR-3, and having an activity of specifically binding to glycosyltransferase A subunit), or having an enhanced activity or a reduced activity. Such derivative sequences are also within the scope of the present invention.

The invention further provides a nucleotide coding sequence for coding the nano antibody containing the variable region, and the coding sequence is shown by SEQ ID NO.7, SEQ ID NO.8 and SEQ ID NO. 9.

The invention further provides application of the nano antibody in preparation of clostridium difficile treatment medicines.

The invention further provides application of the nano antibody in preparation of a clostridium difficile detection reagent.

Compared with the prior art, the invention has the following beneficial effects:

1. the nano antibody for resisting the glycosyltransferase A subunit has the activity of specifically binding with the glycosyltransferase A subunit, neutralizes the activity of the glycosyltransferase A, can be used for detecting clostridium difficile, and can also be used for preparing a medicament for treating the clostridium difficile;

2. the nano antibody for resisting the glycosyltransferase A subunit has the characteristics of small molecular weight, high affinity, stable structure and performance and the like; the stomach acid environment can be resisted, and the stomach acid environment is not easily degraded by pepsin;

3. the preparation cost of the nanometer antibody for resisting the glycosyltransferase A subunit provided by the invention is low, and the production cost of the antibody can be greatly reduced.

Drawings

FIG. 1 is a diagram showing the results of screening nanobodies against glycosyltransferase A subunits;

FIG. 2 is a diagram showing the result of SDS-PAGE analysis after the purification of nanobodies against glycosyltransferase A subunits;

FIG. 3 is a diagram showing the results of Elisa detection of the specificity of the nanobody.

Detailed Description

So that the technical solutions of the embodiments of the present invention will be clearly and completely described in conjunction with the accompanying drawings, 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, belong to the present invention.

Examples

One antibody that naturally lacks a light chain, namely the heavy chain antibody (hcAb), is present in alpaca serum. Single domain heavy chain antibodies (sdabs) refer to genetically engineered antibodies consisting of only heavy chain antibody Variable regions (Variable regions), also known as VHH antibodies (VHH antibodies) or nanobodies (Nb). Compared with the traditional antibody, the single-domain heavy chain antibody has small molecular weight, strong stress resistance and high activity under the strong acid condition, so that the antibody is very suitable for entering the intestinal tract through gastric delivery, and simultaneously keeps good activity to participate in inhibiting clostridium difficile. The invention utilizes phage display technology to screen out phage positive clones which can be combined with a target molecule glycosyltransferase A subunit from an alpaca phage library, thereby obtaining a nano antibody nucleic acid sequence of the glycosyltransferase A subunit, and can be efficiently expressed in escherichia coli, thereby obtaining a target antibody, namely the nano antibody of the glycosyltransferase A subunit through purification.

The specific operations are described in detail below with reference to specific examples.

(I) screening of Nanobodies against glycosyltransferase A subunits

(1) Glycosyltransferase A subunit protein with RBD amino acid sequence is diluted to 10 mu g/mL by 10mM PBS, added into an enzyme label plate according to 100 mu l per hole, and coated for 2h at 37 ℃; the glycosyltransferase A subunit protein can be prepared as described in Clostridium differential viral toxin receptor binding domain peptide differential toxin in active and passive challenge modules, Jing-Hui Tian et al. Vaccine 35(2017) 4079-.

(2) Discarding the coating solution, and adding 300. mu.L 10mM PBS per well to wash for 3 times; adding 300 mu L of 10mM PBS containing 5% skimmed milk powder into each well and sealing for 1 h;

(3) the blocking solution was discarded, and the wells were washed 3 times with 300. mu.L of 10mM PBST (containing 0.1% Tween by volume) and 3 times with 300. mu.L of 10mM PBS per well;

(4) add 50 μ L of alpaca phage library (originally used) purchased from Bio-engineering Co., Ltd, King-Kuehui) and 50 μ L of 10mM PBS containing 5% skim milk powder, incubate for 2h at 37 ℃;

(5) discard the supernatant, wash 6 times with 300. mu.L PBST, 1min intervals each time, then wash 3 times with 300. mu.L 10mM PBS;

(6) discarding the supernatant, adding Gly-HCl eluent with pH =2.2, standing and eluting for 8 min; adding a pH =9.1 Tris-HCl buffer solution for neutralization; when the absorbance of the resulting eluate was about 0.6 at OD600nm, it was infected with Escherichia coli TG1(Lucigen, 60502-1) and infected at 37 ℃ for 1 hour at 220rpm, after which 10. mu.L of the infected bacterial suspension was diluted 10-fold in 2YT medium (containing 1.6% (W/V) Tryptone, 1% (W/V) Yeast Extract, 0.5% (W/V) NaCl), 10. mu.L of each gradient was titrated by streamline, and the remaining bacterial suspension was applied to a large ampicillin plate and cultured overnight at 37 ℃;

(7) the overnight scraped cells were collected in EP tubes using 2YT medium and used for the next round of panning with the alpaca phage library.

The steps (1) to (7) are the first round of panning, and then the second round of panning and the third round of panning are carried out according to the same steps; except that in the second panning run, 12 wash passes of 10mM PBST were involved per site; in the third panning, each 10mM PBST wash pass involved was 18.

The results of three rounds of panning are shown in fig. 1, and the titer gradually increases with the increase of the number of screening rounds, which indicates that the sequence is enriched and the screening process is normal in the screening process.

(II) identification of specific phage-positive clones

(1) After the third round of screening is finished, the eluate (OD 600nm with the absorbance value of about 0.6) obtained in the third round of step (6) is infected with Escherichia coli TG1 (purchased from Lucigen, 60502-1) at 37 ℃ and 220rpm for 1h, then 10 mul of bacterial liquid is diluted by 10 times and coated on a flat plate, and the plate is subjected to inverted culture at 37 ℃ overnight;

(2) preparing a 2YT + glucose + ampicillin culture medium according to 2YT, 0.2% glucose and 100 mug/ml ampicillin, and then flatly paving the culture medium in a 96-well plate; picking the monoclonal in the flat plate in the step (1) by using a gun head, placing the monoclonal in a 96-well plate paved with a culture medium, and culturing for 4 hours at 37 ℃ and at the rotating speed of 220 rpm; then 100. mu.L of the bacterial liquid in the 96-well plate is put into a new 96-well plate, 50. mu.L of helper phage M13K07 (purchased from Biolabs, N03155) is added into the bacterial liquid, and the bacterial liquid is cultured for 1h at 37 ℃ and the rotating speed of 220 rpm;

(3) centrifuging the cultured bacterial liquid at 4000rpm for 8min, removing supernatant, changing into A + K culture medium (containing 1.6% (W/V) Tryptone, 1% (W/V) Yeast Extract, 0.5% (W/V) NaCl, 100 μ g/ml Amp, 50 μ g/ml Kana), and culturing at 37 deg.C and 220rpm overnight;

(4) centrifuging the cultured bacteria liquid at 4000rmp for 8min, taking the supernatant, adding the supernatant into an enzyme label plate (see the relevant glycosyltransferase A subunit protein coating operation in the step one) coated with TcdA protein, and incubating for 1h at 37 ℃;

(5) washing with 10mM PBST for 3 times and then 10mM PBS for 3 times according to 400 mu L of each well, and drying after washing each time;

(6) to 100. mu.L per well, horseradish peroxidase-labeled anti-M13 antibody (purchased from Cassia Proteus) diluted with 10mM PBS containing 5% skimmed milk powder was added and incubated at 37 ℃ for 40 min;

(7) according to 400 mu L of each well, washing for 3 times by using 10mM PBST and then washing for 3 times by using 10mM PBS, and drying after washing each time;

(8) adding TMB developing solution (from Solarbio, PR 1210) at 100 μ L per well, and reacting at 37 deg.C in dark for 15 min; then 50. mu.L of 2M H was added to each well₂The reaction was terminated by stopping the reaction with SO4, and the absorbance of each well was measured at 450 nm.

The phages obtained in the second round of panning (i.e., the eluate obtained in the second round of step (6) (OD 600nm shows an absorbance of about 0.6)) and the phages obtained in the third round of selection were enriched according to steps (1) to (3), and tested for ELISA titer according to steps (4) to (8) (i.e., absorbance was measured at 450nm in each well), and the results of the measurements are shown in Table 1.

As can be seen from Table 1, after the third round of enrichment, both the positive rate and the binding titer were improved.

TABLE 1 identification of specific phage positive clones

Selecting positive clone to analyze, and determining the codes as shown in SEQ ID NO.7, SEQ ID NO.8 and SEQ ID NO. 9; the amino acid sequence of the variable region is shown as SEQ number 4-SEQ number 6; wherein the 101-position 115 amino acid sequence of SEQ number 4 is CDR-1 shown in SEQ number 1, the 101-position 115 amino acid sequence of SEQ number 5 is CDR-2 shown in SEQ number 2, and the 101-position 115 amino acid sequence of SEQ number 6 is CDR-3 shown in SEQ number 3.

(III) expression of Nanobodies against glycosyltransferase A subunits

(1) Carrying out double enzyme digestion on the obtained nucleotide sequence SEQ number 7-SEQ number 9 by adopting NcoI/Not I, connecting the double enzyme digestion with a pEt 25b (+) vector subjected to the same enzyme digestion, electrically transforming the connected plasmid into an escherichia coli Rosetta DE3 expression strain, selecting a monoclonal strain, inoculating the monoclonal strain into 4mL of LB-Amp culture medium (containing 1% of Tryptone, 0.5% of Yeast Extract, 1% of NaCl and 100 mug/mL of Amp deionized water), and culturing for 5h at 37 ℃ and at the rotating speed of 220 rpm;

(2) 100mL of LB-Amp-Glu medium (containing 1% Tryptone, 0.5% Yeast Extract, 1% NaCl, 0.2% Glu, 100. mu.g/mL Amp) was inoculated with 1% (V/V) and cultured at 37 ℃ and 220rpm to an absorbance of about 0.5 at OD600 nm; thereafter, IPTG (from Amresco) was added to a final concentration of 0.1mM and induced at 30 ℃ overnight at 220 rpm;

(3) centrifuging the product obtained by induction at 12000rpm for 10min, then removing the supernatant, and collecting thalli;

(4) every 1g of the bacterial liquid is metered by 30mLBuffer A, and Buffer A (50mM PB, 300mM NaCl) is added into the collected thalli to resuspend the thalli;

(5) carrying out ultrasonic crushing on the resuspended thalli; the ultrasonic conditions are as follows: 25-35 min, 5s ultrasonic interval of 7s and 35% power;

(6) centrifuging the ultrasonically-broken bacterial liquid at 4 ℃ and 12000rpm for 20min, taking the supernatant, filtering the supernatant through a 0.45-micron filter membrane, and collecting the bacterial liquid for purifying the nano antibody of the anti-glycosyltransferase A subunit.

Purification of Nanobodies directed against glycosyltransferase A subunits

Purifying the bacteria liquid after ultrasonic crushing and filtering by adopting a chromatographic column, wherein the specific operation is as follows:

(1) cleaning: starting up the machine, cleaning each chromatography column passage, and setting the column pressure limit to 0.45 MPa;

(2) balancing: washing with first-grade water according to 8mL/min, and cleaning the filler until the baseline is stable; then, the Buffer B (50mM PB, 300mM NaCl, 500mM imidazole) solution is changed according to 8mL/min until the baseline is stable; then, the Buffer A (50mM PB, 300mM NaCl) solution was changed to flush until the baseline was stable at 8 mL/min.

(3) Sampling: adding the bacterial liquid into a chromatographic column according to the concentration of 8 mL/min;

(4) and (3) re-balancing: after the sample loading is finished, re-balancing the chromatographic column by Buffer A liquid according to 8mL/min, and washing until the baseline is completely stable;

(5) and (3) elution: according to 8mL/min, washing the chromatographic column by 90% Buffer A and 10% Buffer B, and collecting the eluent with the 280nm absorption value higher than 100 mAU;

further, according to 8mL/min, washing the chromatographic column by using 80% Buffer A and 20% Buffer B, and collecting the eluent with the 280nm absorption value higher than 100 mAU;

further, according to 8mL/min, washing the chromatographic column by using 70% Buffer A and 30% Buffer B, and collecting eluent with the 280nm absorption value higher than 100 mAU;

further, according to 8mL/min, washing the chromatographic column by using 60% Buffer A and 40% Buffer B, and collecting eluent with the 280nm absorption value higher than 100 mAU;

further, according to 8mL/min, washing the chromatographic column by using 50% Buffer A and 50% Buffer B, and collecting the eluent with the 280nm absorption value higher than 100 mAU;

further, according to 8mL/min, using 100% Buffer B to wash the chromatographic column, and collecting the eluent with the 280nm absorption value higher than 100 mAU;

completing the purification of the expressed nano antibody of the anti-glycosyltransferase A subunit;

(6) cleaning: washing the chromatographic column by using 100 percent Buffer B until the baseline is stable, and replacing the first-level water to wash the chromatographic column until the baseline is stable; and washing the chromatographic column with 20% ethanol water solution until the baseline is stable, taking down the chromatographic column, and storing at 4 deg.C.

SDS-PAGE analysis was performed on the purified nanobody against glycosyltransferase A subunit, and lanes 1-3 are purified nanobodies, as shown in FIG. 2. The result shows that the relative molecular mass of the nano antibody of the glycosyltransferase A subunit is about 15KD and is consistent with the theoretical molecular mass, and the purified band has no obvious miscellaneous band, which shows that the purity of the nano antibody is higher,

(V) Elisa detection of the specificity of the nano antibody

(1) Taking a purified nano antibody sample of the anti-glycosyltransferase A subunit, respectively adding the sample to an enzyme label plate (related to glycosyltransferase A subunit protein coating operation in the step (I)) coated by the glycosyltransferase A subunit (TcdA protein), a BSA coated enzyme label plate (same as the preparation method of the TcdA protein coated enzyme label plate) and an ovalbumin coated enzyme label plate (same as the preparation method of the TcdA protein coated enzyme label plate) according to 100 microlitres per hole, and placing for 60min at 37 ℃;

(2) PBST was washed 3 times per 300 ul/well; washing with PBS for 3 times according to 300 ul/hole, and thoroughly drying the liquid in the plate after the last washing;

(3) adding antibody (antibody is a second antibody diluted with 1:5000 antibody diluent (10 mM PBS, 5% skimmed milk powder)) into the ELISA plate at 100 ul/well, and standing at 37 deg.C for 60 min;

(4) PBST was washed 3 times per 300 ul/well; washing with PBS for 3 times according to 300 ul/hole, and thoroughly drying the liquid in the plate after the last washing;

(5) adding TMB developing solution (from Solarbio, PR 1210) at 100 μ l/well, and developing at 37 deg.C in dark for 15 min; then 50. mu.l/well of 2M H was added₂The reaction was stopped with SO4 and the absorbance of each well was measured at 450nm, the results are shown in FIG. 3.

As can be seen from the figure, when three kinds of nanobodies are used to detect three kinds of antigen-coated plates, namely, glycosyltransferase a subunit, BSA and ovalbumin (i.e., OVA), the nanobody obtained in this example can specifically bind to the glycosyltransferase a subunit.

It will be appreciated by those of ordinary skill in the art that the embodiments described herein are intended to assist the reader in understanding the principles of the invention and are to be construed as being without limitation to such specifically recited embodiments and examples. Those skilled in the art can make various other specific changes and combinations based on the teachings of the present invention without departing from the spirit of the invention, and these changes and combinations are within the scope of the invention.

SEQUENCE LISTING

<110> Sichuan university Hospital in Huaxi

<120> nano antibody for resisting glycosyltransferase A subunit and application thereof

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Asp Lys Tyr Val Thr Ser Ala Arg Leu Leu Ala Ser Asn Leu Tyr

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<211> 15

<212> PRT

<213> Lama pacos

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His Tyr Tyr Arg Tyr Asp Gly Ser Gly Leu Tyr Leu Tyr Asp Tyr

1 5 10 15

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<211> 15

<212> PRT

<213> Lama pacos

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Arg Arg Pro Phe Ile Thr Gly Ser Thr Arg Arg Pro Ser Asp Arg

1 5 10 15

<210> 4

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<212> PRT

<213> Lama pacos

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Met Ala Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Ala

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Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Arg Thr Phe Ser

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Ser Asn Ala Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu

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Phe Val Ala Ala Ile Ser Ser Ser Asp Asp Ser Thr Asn Tyr Ala Asp

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Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr

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Val Tyr Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr

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Tyr Cys Ala Ala Asp Lys Tyr Val Thr Ser Ala Arg Leu Leu Ala Ser

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Asn Leu Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser Ala Ala

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atggcccagg tgcagctgca ggagtctgga ggaggcttgg tgcaggctgg agggtctctg 60

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caggctccag ggaaggagcg tgagttcgta gcagcgattt cttctgacgg ttcttctact 180

aactatgcag actccgtgaa gggccgattc accatctcca gagacaacgc caagaacacg 240

gtgtatcttc aaatgaacag cctgaaacct gaggacacgg ccgtttatta ctgtgcagcc 300

aggcggccgt ttattactgg ttctacacgg agaccatctg accggtgggg tcagggtact 360

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Claims

1. A nanobody against glycosyltransferase A subunit, characterized in that it has complementarity determining regions CDR-1, CDR-2, CDR-3 in the variable region shown in SEQ ID NO. 4.

2. The nanobody against glycosyltransferase a subunit of claim 1, characterized in that the variable region sequence of the nanobody consists of the amino acid sequence shown in SEQ ID No. 4.

3. A polynucleotide encoding the nanobody of claim 2, wherein the sequence of the polynucleotide is represented by SEQ ID No. 7.

4. Use of the nanobody against glycosyltransferase a subunit of claim 2 in the preparation of a clostridium difficile detection reagent.