CN114853895B - A kind of nanobody and application thereof against glycosyltransferase A subunit - Google Patents

Abstract

The invention discloses a nanometer antibody of an anti-glycosyltransferase A subunit and application thereof, wherein a variable region of the nanometer antibody has complementarity determining regions CDR-1, CDR-2 and CDR-3 in the variable region shown in SEQ ID NO. 5. The nanometer antibody of the anti-glycosyltransferase A subunit has the activity of specifically combining with the glycosyltransferase A subunit and neutralizing the activity of glycosyltransferase A, can be used for detecting clostridium difficile and can also be used for preparing medicines for treating 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 stomach acid environment, is not easy to be degraded by pepsin, and the like.

Description

A kind of anti-glycosyltransferase A subunit nanobody and its application

This application is a divisional application with application number 202110077974.X, application date January 20, 2021, and the title of the invention "a nanobody against glycosyltransferase A subunit and its application".

technical field

The invention belongs to the technical field of antibodies and relates to nanobody technology, in particular to a nanobody against glycosyltransferase A (TcdA) subunit and its application.

Background technique

Clostridium difficile (CD) is widely distributed in the natural environment and can survive in water, soil, vegetables, dogs, cats and hospital environments, and is a normal flora in the intestinal tract of animals and humans. Irregular use of antibiotics will cause intestinal flora imbalance, and drug-resistant CD will grow and multiply in large numbers, leading to diseases such as antibiotic-associated diarrhea and pseudomembranous enteritis, which are the main pathogens of antibiotic-associated diarrheal diseases. The spores of CD have strong resistance and are difficult to eliminate. They can exist in the hospital environment for a long time, making CD one of the main pathogenic bacteria of hospital-acquired diarrhea.

The infection and pathogenesis of CD-related diseases are mainly caused by the excessive proliferation of toxigenic CD in the intestinal tract and the release of a large number of toxins. Under normal circumstances, CD in the intestinal tract is inhibited by dominant flora such as Bifidobacterium, Bacteroides, and Eubacterium, and is at a disadvantage and does not cause disease. The balance of intestinal microecology is broken, the intestinal flora is out of balance, and CD overgrows and releases toxins, causing Clostridium difficile infection (Clostridi μm difficile infection, CDI) related diseases.

When CD proliferates in large quantities, it destroys the normal flora of the colon through the surface proteins of the bacteria or spores, and then colonizes. With the assistance of flagella and proteases, it enters the mucus layer, adheres to the intestinal epithelial cells, releases related toxins, and destroys the intestinal tract. cells, change the cytoskeleton, release mucus and inflammatory products, and cause intestinal infectious diseases. After infection, symptoms such as high fever, bloody stools, abdominal distension, and toxic megacolon may appear. CD can produce 6 kinds of toxins, the main pathogenic factors are toxin A, toxin B and binary toxin. Among them, toxin A is enterotoxin, which can bind to toxin receptors on intestinal mucosal brush border cells, chemoattract leukocytes, activate macrophages, mast cells and neutrophils, and release potent inflammatory mediators and cytokines , resulting in increased permeability of local mucosal vessels, villi damage, mucosal hemorrhage, and necrosis; in pseudomembranous colitis, the most direct effect of toxins A and B is to destroy the tight junction of the epithelial barrier, promote the release of inflammatory factors, mediate The movement of granulocytes and the formation of pseudomembranes. Since the C-terminals of Clostridium difficile toxin A and toxin B both have a functional region for toxin recognition and binding to cell receptors, called the receptor-binding domain (RBD), if a target for this region can be developed Antibodies, covering RBD, can directly block the binding of toxins, making it impossible to exert the effect of toxins, thereby achieving the role of immune defense.

Due to the increasing prevalence and severity of CD infection, it is particularly important to develop new methods of prevention and treatment. In view of the important role of toxin A, namely glycosyltransferase A, in CD infection, the development of specific antibodies against glycosyltransferase A can also play a major role in resisting CDI. At present, there is no report on the specific antibody against glycosyltransferase A RBD.

Contents of the invention

Aiming at the lack of current technical status of Clostridium difficile effective therapeutic drugs, the object of the present invention is to provide a nanobody against glycosyltransferase A subunit (TcdA), which has an RBD with glycosyltransferase A subunit The specific binding activity can be used to detect Clostridium difficile and inhibit the activity of Clostridium difficile, and has good clinical application prospect.

The nanobody of the anti-glycosyltransferase A subunit provided by the present invention has three complementarity determining regions CDR-1, CDR-2, and CDR-3 in its variable region, and CDR-1 is represented by SEQ ID NO.1 Composed of amino acid sequences, CDR-2 is composed of the amino acid sequence shown in SEQ ID NO.2, and CDR-3 is composed of the amino acid sequence shown in SEQ ID NO.3.

In a preferred implementation, the variable region sequence of the Nanobody consists of the amino acid sequences shown in SEQ ID NO.4-6. Of course, the variable region sequence of the Nanobody of the present invention is not limited to the amino acid sequence shown in SEQ ID NO.4-6, it can also be replaced and/or deleted by one or more amino acid residues on the shown sequence Obtained, and has the same biological activity as SEQ ID NO.4-6 (that is, has three complementarity determining regions of CDR-1, CDR-2, and CDR-3, and can specifically bind to the activity of glycosyltransferase A subunit ), or activity-enhancing, or activity-weakening derivative sequences. Such derivative sequences also belong to the protection scope of the present invention.

The present invention further provides a nucleotide coding sequence encoding the above-mentioned Nanobody containing the variable region, the coding sequence is shown by SEQ ID NO.7, SEQ ID NO.8 and SEQ ID NO.9.

The present invention further provides the application of the above nanobody in the preparation of Clostridium difficile therapeutic drug.

The present invention further provides the application of the above nanobody in the preparation of Clostridium difficile detection reagent.

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

1. The nanobody against glycosyltransferase A subunit provided by the present invention has the activity of specifically binding to glycosyltransferase A subunit, and neutralizes the activity of glycosyltransferase A, and can be used to detect Clostridium difficile , can also be used for the preparation of drugs for the treatment of Clostridium difficile;

2. The anti-glycosyltransferase A subunit nanobody provided by the present invention also has the characteristics of small molecular weight, high affinity, stable structure and performance; it can withstand the acidic environment of the stomach and is not easily degraded by pepsin;

3. The preparation cost of the anti-glycosyltransferase A subunit nanobody provided by the present invention is low, which can greatly reduce the production cost of the antibody.

Description of drawings

Fig. 1 is the schematic diagram of the screening result of the nanobody of anti-glycosyltransferase A subunit;

Figure 2 is a schematic diagram of the SDS-PAGE analysis results after purification of the nanobody against glycosyltransferase A subunit;

Fig. 3 is a schematic diagram of the Elisa detection results of Nanobody specificity.

Detailed ways

In order to clearly and completely describe the technical solutions of the various embodiments of the present invention with reference to the accompanying drawings, obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by persons of ordinary skill in the art without making creative efforts belong to the present invention.

Example

There is an antibody in the serum of alpaca that naturally lacks the light chain, that is, the heavy-chain antibody (hcAb). Single domain heavy chain antibody (single domain antibody, sdAb) refers to a genetically engineered antibody composed only of the heavy chain antibody variable region (Variable region), also known as VHH antibody (variable domain of heavychain of heavy-chain antibody, VHH antibody ) or nanobody (Nanobody, Nb). Compared with traditional antibodies, single-domain heavy chain antibodies have a small molecular weight, strong stress resistance, and high activity under strong acid conditions, so this is very suitable for delivery into the intestinal tract through the stomach, while maintaining good activity involved in the inhibition of the difficile shuttle bacteria. The present invention uses phage display technology to screen out phage-positive clones capable of binding to the target molecule glycosyltransferase A subunit from an alpaca phage library, thereby obtaining a nanobody nucleic acid sequence against the glycosyltransferase A subunit, and It can be highly expressed in Escherichia coli, thereby purifying and obtaining the target antibody, that is, the nanobody against the subunit of glycosyltransferase A.

The specific operations are described in detail below in conjunction with specific embodiments.

(1) Screening of nanobodies against glycosyltransferase A subunit

(1) Dilute the glycosyltransferase A subunit protein with RBD amino acid sequence to a final concentration of 10 μg/mL with 10 mM PBS, add 100 μl per well to the microtiter plate, and coat at 37 ° C for 2 hours; the glycosyltransferase The preparation of A subunit protein can be found in Clostridium difficile chimeric toxin receptor binding domain vaccine induced protection against different strains in active and passive challenge models, Jing-Hui Tian et al. Vaccine 35(2017) 4079-4087.

(2) Discard the coating solution, add 300 μL 10mM PBS to each well to wash 3 times; then add 300 μL 10mM PBS containing 5% skimmed milk powder to each well to block for 1 hour;

(3) Discard the blocking solution, add 300 μL 10mM PBST (containing Tween with a volume concentration of 0.1%) to each well to wash 3 times, and then add 300 μL 10mM PBS to each well to wash 3 times;

(4) Add 50 μL alpaca phage library (the original alpaca phage library was purchased from Jinkairui Bioengineering Co., Ltd.) and 50 μL 10 mM PBS containing 5% skimmed milk powder, and incubate at 37°C for 2 hours;

(5) Discard the supernatant, wash 6 times with 300 μL PBST, with an interval of 1 min each time, and then wash 3 times with 300 μL 10mM PBS;

(6) Discard the supernatant, add Gly-HCl eluent with pH=2.2 and let stand for elution for 8min; then add Tris-HCl buffer with pH=9.1 to neutralize; At 0.6, it was infected with Escherichia coli TG1 (Lucigen, 60502-1), and infected at 37°C and 220rpm for 1h, and then 10 μL of infected bacterial liquid was taken, and 2YT medium (containing 1.6% (W/V) Tryptone, 1% (W/V) Yeast Extract, 0.5% (W/V) NaCl) were diluted in a 10-fold gradient, and then 10 μL was taken from each gradient to measure the titer through the streamline, and all the remaining bacterial solution was applied to a large On the ampicillin plate, cultivate overnight at 37°C;

(7) Scrape overnight bacterial cells with 2YT medium and collect them into EP tubes for the alpaca phage library used in the next round of panning.

The above steps (1)-(7) are the first round of panning, and then follow the same steps to carry out the second round of panning and the third round of panning; the difference is that in the second round of panning, all The number of 10mM PBST washing times involved is 12 times; in the third round of panning process, the number of 10mM PBST washing times involved is 18 times.

The results of the three rounds of panning are shown in Figure 1. With the increase of the number of rounds of screening, the titer gradually increased, indicating that during the screening process, there was sequence enrichment and the screening process was normal.

(2) Identification of specific phage-positive clones

(1) After the third round of screening, the eluate obtained in the third round of step (6) (the absorbance value at OD600nm is about 0.6) was infected with Escherichia coli TG1 (purchased from Lucigen, 60502-1), and the Infect at 220rpm for 1 hour, then take 10 μL of the bacterial solution to 10-fold dilution and spread it on the plate, then incubate it upside down at 37°C overnight;

(2) Prepare 2YT + glucose + ampicillin medium according to 2YT, 0.2% glucose, and 100 μg/ml ampicillin, and spread it in a 96-well plate; In a 96-well plate covered with culture medium, culture it at 37°C and 220rpm for 4h; then take 100 μL of the bacterial solution in the above 96-well plate and place it in a new 96-well plate, and add 50 μL of helper phage M13K07 (purchased (from Biolabs, N03155), cultivated at 37°C and 220rpm for 1h;

(3) discard the supernatant after centrifuging the cultured bacterial solution at 4000rpm for 8min, and replace it with A+K medium (containing 1.6% (W/V) Tryptone, 1% (W/V) Yeast Extract, 0.5% ( W/V) NaCl, 100 μg/ml Amp, 50 μg/ml Kana), cultivate overnight at 37° C. and 220 rpm;

(4) Centrifuge the cultured bacterium solution at 4000rmp for 8min, take the supernatant and add it to the microtiter plate coated with TcdA protein (see (1) about the glycosyltransferase A subunit protein coating operation), at 37 Incubate at ℃ for 1h;

(5) Wash 3 times with 10mM PBST and 3 times with 10mM PBS according to 400μL per well, and pat dry after each wash;

(6) According to 100 μL per well, add horseradish peroxidase-labeled anti-M13 antibody (purchased from Yiqiao Shenzhou Company) diluted in 10 mM PBS containing 5% skimmed milk powder, and incubate at 37 ° C for 40 min;

(7) According to 400 μL per well, first wash with 10mM PBST for 3 times and then with 10mM PBS for 3 times, and pat dry after each washing;

(8) According to 100 μL per well, add TMB chromogenic solution (purchased from Solarbio, PR1210), and react in the dark at 37°C for 15 minutes; then add 50 μL per well, add 2M H ₂ SO4 stop solution to stop the reaction, and measure at 450 nm Absorbance of each well.

The phage obtained in the second round of panning (i.e. the eluate obtained in the second round of step (6) (absorbance value at OD600nm is about 0.6)) and the phage obtained in the third round of selection were carried out according to steps (1)-(3). For enrichment, according to steps (4)-(8), the ELISA titer was detected (that is, the absorbance value of each plate well was measured at 450 nm), and the measurement results are shown in Table 1.

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

Table 1 Identification table of specific phage positive clones

Pick positive clones for sequencing analysis, and it can be determined that the codes are 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 in SEQ NO.4-SEQ NO.6; wherein, The 101-115th amino acid sequence of SEQ NO.4 is CDR-1 shown in SEQ NO.1, the 101-115th amino acid sequence of SEQ NO.5 is CDR-2 shown in SEQ NO.2, and SEQ NO.6 The 101-115 amino acid sequence is CDR-3 shown in SEQ NO.3.

(3) Expression of nanobodies against glycosyltransferase A subunit

(1) Use NcoI/Not I to perform double enzyme digestion on the obtained nucleotide sequence SEQ NO.7-SEQ NO.9, connect it to the pEt 25b(+) vector that has undergone the same digestion, and electrotransform the connected plasmid into Escherichia coli Rosetta DE3 expression strains, and pick monoclonal strains and inoculate them in 4mL LB-Amp medium (deionized water containing 1% Tryptone, 0.5% YeastExtract, 1% NaCl, 100μg/ml Amp), at 37°C , Cultivate for 5 hours at a rotating speed of 220rpm;

(2) Inoculate 100mL LB-Amp-Glu medium (containing 1% Tryptone, 0.5% YeastExtract, 1% NaCl, 0.2% Glu, 100μg/ml Amp) according to 1% (V/V), at 37°C, 220rpm Cultivate at a rotating speed until the absorbance value at OD600nm is about 0.5; after that, add IPTG (purchased from Amresco) at a final concentration of 0.1 mM, and induce overnight at 30° C. and 220 rpm;

(3) Centrifuge the induced product at 12000rpm for 10min, then discard the supernatant and collect the bacteria;

(4) Measure with 30mL Buffer A per 1g of bacterial liquid, add Buffer A (50mM PB, 300mMNaCl) to the collected bacterial cells to resuspend the bacterial cells;

(5) Ultrasonic disruption of the resuspended bacteria; ultrasonic conditions: 25 to 35 minutes, ultrasonic 5s interval 7s, power 35%;

(6) The sonicated bacterial solution was centrifuged at 4°C and 12,000 rpm for 20 min, the supernatant was taken, passed through a 0.45 μm filter membrane, and the bacterial solution was collected for purification of nanobodies against glycosyltransferase A subunit.

(4) Purification of nanobodies against glycosyltransferase A subunit

Use a chromatographic column to purify the ultrasonically crushed and filtered bacterial liquid, and the specific operations are as follows:

(1) Cleaning: start the machine, clean the channels of each chromatography column, and set the column pressure limit to 0.45MPa;

(2) Equilibrium: Rinse the filler with primary water at 8mL/min until the baseline is stable; then, change Buffer B (50mM PB, 300mM NaCl, 500mM imidazole) at 8mL/min to rinse until the baseline is stable; 8mL/min, change Buffer A (50mM PB, 300mM NaCl) solution to wash until the baseline is stable.

(3) Sample loading: Add the bacterial solution to the chromatography column at 8mL/min;

(4) Rebalance: After loading the sample, rebalance the chromatography column with Buffer A solution at 8mL/min, and wash until the baseline is completely stable;

(5) Elution: Wash the column with 90% Buffer A+10% Buffer B at 8mL/min, and collect the eluate with an absorbance at 280nm higher than 100mAU;

Further, according to 8mL/min, use 80% Buffer A+20% Buffer B to wash the chromatography column, and collect the eluate whose 280nm absorption value is higher than 100mAU;

Further, according to 8mL/min, use 70% Buffer A+30% Buffer B to wash the chromatography column, and collect the eluate whose 280nm absorption value is higher than 100mAU;

Further, according to 8mL/min, use 60% Buffer A+40% Buffer B to wash the chromatography column, and collect the eluate whose 280nm absorption value is higher than 100mAU;

Further, according to 8mL/min, use 50% Buffer A+50% Buffer B to wash the chromatography column, and collect the eluate whose 280nm absorption value is higher than 100mAU;

Further, according to 8mL/min, use 100% Buffer B to wash the chromatography column, and collect the eluate whose 280nm absorption value is higher than 100mAU;

Complete the purification of the expressed nanobody against the subunit of glycosyltransferase A;

(6) Cleaning: Use 100% Buffer B to wash the column until the baseline is stable, replace the primary water to wash the column until the baseline is stable; then use an aqueous solution containing 20% ethanol to wash the column until the baseline is stable, remove the chromatographic column column and stored at 4°C.

SDS-PAGE analysis was performed on the purified nanobody against glycosyltransferase A subunit, as shown in FIG. 2 , lanes 1-3 are purified nanobodies. The results showed that the relative molecular mass of the nanobody against glycosyltransferase A subunit was about 15KD, which was consistent with the theoretical molecular mass, and the purified band had no obvious impurities, indicating that the nanobody had a high purity.

(5) Elisa detects the specificity of the above nanobodies

(1) Take the purified nanobody samples against glycosyltransferase A subunit, and put them on glycosyltransferase A subunit (TcdA protein)-coated microtiter plates (see (1)) at 100 μl/well. For glycosyltransferase A subunit protein coating operation), BSA-coated microtiter plate (the same method as TcdA protein-coated microtiter plate) and ovalbumin-coated microtiter plate (similar to TcdA protein-coated microtiter plate) The preparation method of the ELISA plate is the same) add the sample and place it at 37°C for 60min;

(2) Wash 3 times with PBST according to 300ul/well; wash 3 times with PBS according to 300ul/well, and thoroughly pat dry the liquid in the plate after the last wash;

(3) Add antibody (the antibody is the secondary antibody diluted at 1:5000 with antibody diluent (10mM PBS, 5% skimmed milk powder)) to the microtiter plate at 100ul/well, and place it at 37°C for 60min;

(4) According to 300ul/well, wash with PBST 3 times first; according to 300ul/well, then wash with PBS 3 times, and thoroughly pat dry the liquid in the plate after the last wash;

(5) Add TMB chromogenic solution (purchased from Solarbio, PR1210) at 100 μl/well, and develop color at 37°C in the dark for 15 minutes; then add 2M H ₂ SO4 at 50 μl/well to terminate the reaction, and measure each well at 450 nm Absorbance, the results are shown in Figure 3.

As can be seen from the figure, three kinds of nanobodies were used to detect three kinds of antigen-coated plates of glycosyltransferase A subunit, BSA, and ovalbumin (OVA), and the nanobodies obtained in this example can specifically The binding glycosyltransferase A subunit.

Those skilled in the art will appreciate that the embodiments described here are to help readers understand the principles of the present invention, and it should be understood that the protection scope of the present invention is not limited to such specific statements and embodiments. Those skilled in the art can make various other specific modifications and combinations based on the technical revelations disclosed in the present invention without departing from the essence of the present invention, and these modifications and combinations are still within the protection scope of the present invention.

sequence listing

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Claims (4)

1. A nanobody against a subunit of glycosyltransferase a, characterized in that it has the heavy chain variable region shown in SEQ ID No. 5.

2. The nanobody against glycosyltransferase a subunit according to claim 1, wherein the heavy chain variable region sequence of the nanobody consists of the amino acid sequence shown in SEQ ID No. 5.

3. A nucleic acid encoding the nanobody of claim 2, wherein the sequence of the nucleic acid is set forth in SEQ ID No. 8.

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

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