EXPERIMENTS

Experimental objective：
To formulate a functional and nutritive liquid and solid culture medium for bacterial growth. Liquid media serve various purposes, including microbial propagation and testing.

Equipment list：
1)Tryptone
2)Yeast extract
3)Sodium chloride
4)Distilled water
5)Sterilized conical flask, cap
6)Autoclave
7)Lab refrigerator
8)Measuring cylinder
9)Weighing scale

Method:
1)Weigh out 2.50 g of premix LB broth powder by weighing scale (including 1.00 g tryptone, 0.50 g yeast extract and 1.00 g sodium chloride)
2)Transfer the LB broth powder into 250 mL conical flask
3)Add distilled water up to total volume of 100 mL by 100 mL measuring cylinder
4)Close the cap and autoclave it for 20 min at least
5)Wait until it cools

2.1 pET28a-Sumo-Nb-human, pET28a-Sumo-Nb-SCFV, pET28a-Sumo-UreB-Nb6, pET28a-UreB

Extraction of plasmid DNA
Experimental objective：
To extract and purify plasmid DNA from cultured bacteria for use in downstream applications such as PCR.the target gene fragments.

Equipment list:
1)Benchtop microcentrifuge (capable of 12,000× g)
2)Centrifuge tubes (1.5 mL)
3)Collection tubes for spin columns
4)Adjustable-volume micropipettes
5)Sterilized pipette tips
6)Silica membrane-based spin columns
7)Waste collection tubes
8)Buffer SP1 (with RNase A)
9)Buffer SP2
10)Buffer SP3
11)Wash Solution
12)Elution Buffer
13)Buffer DW1
14)Overnight bacterial culture
15)centrifuge tube racks

Method:
1)Verification:
Confirm RNase A has been added to Buffer SP1
Verify absolute ethanol has been added to Wash Solution
Check Buffers SP2 and SP3 for precipitation; warm to 37 °C if precipitated
2)Column equilibration:
Place spin column in a collection tube
Add 500 μL Buffer S to the column
Centrifuge at 12,000× g for 1 min
Discard flow-through and reassemble column-collection tube assembly
3)Bacterial Pellet Harvesting:
Transfer 1.5 mL bacterial culture to a microcentrifuge tube
Centrifuge at 8,000× g for 2 min
Discard supernatant completely
4)Cell Lysis and Neutralization
Resuspend pellet in 250 μL Buffer SP1 by vigorous pipetting
Add 250 μL Buffer SP2 and immediately mix by inverting tube 5–10 times
Incubate at room temperature for 2–4 min
Add 350 μL Buffer SP3 and immediately mix by inverting 5–10 times
Centrifuge at 12,000× g for 5–10 min.
5)DNA Binding and Washing
Transfer supernatant to the equilibrated spin column
Centrifuge at 8,000× g for 30 sec. Discard flow-through
Add 500 μL Buffer DW1, centrifuge at 9,000× g for 30 sec. Discard flow-through
Add 500 μL Wash Solution, centrifuge at 9,000× g for 30 sec. Discard flow-through
Repeat Wash Solution step
Centrifuge empty column at 9,000× g for 1 min to remove resting washing solution
6) DNA Elution
Transfer spin column to a clean 1.5 mL microcentrifuge tube
Add 50 μL Elution Buffer to the center of the membrane
Incubate at room temperature for 1 min
Centrifuge at 9,000× g for 1 min
Repeat elution buffer step
Conserve the DNA solution in tube

Obtaining target gene fragments by PCR
Experimental objective：
To amplify the target gene fragments (Nb-human, UrB-Nb6, UreB and Nb-SCFV ) for later recombination materials.

Equipment list:
1) 2×PrimeSTAR Max Premix
2) Sumo-Nb-human, Sumo-UrB-Nb6, UreB and Sumo-Nb-SCFV gene template
3) double distilled water
4) Primer-R
5) Primer-F
6) Centrifuge tube
7) PCR thermal cycler

Method:
1)Add 25 µL 2×PrimeSTAR Max Premix, pNb-human template 1µL, primer-R 1µL, and primer-F 1 µL to centrifuge tube. Add double distilled water to the centrifuge tube until reaching 50 µL. If bubbles are observed in the mixture, centrifuge the mixture for a few seconds to remove the bubbles.
2) Insert mixture into a PCR thermal cycler. Denature the fragments at 95 ˚C for 3 min
3) Perform 30 cycles of 30 sec 95 ˚C denaturing, 30 sec 55 ˚C annealing, and 90 sec extension 72 ˚C extension.
4) Extend the fragments for 1 min at 72 ˚C
5) If not use immediately, preserve the fragments at 4 ˚C
6) Repeat step 1-5 for Somo-UreB-Nb6, Sumo-UreB and Sumo-Nb-SCFV.

Vector linearization
Experimental objective：
To linearize the vector via double restriction enzyme digestion, generating sticky ends for downstream ligation.

Equipment list：
1)Ice bucket
2)Dry bath incubator
3)Adjustable micropipettes (0.5-10 μL, 10-100 μL)
4)centrifuge tubes
5)Waste collection tubes
6)Restriction enzyme: HindIII
7)Restriction enzyme: NheI
8)Compatible 10× reaction buffer
9)Plasmid extract (from previous step)
10)Double distilled water

Method:
1)Pre-cooling:
Thaw HindIII and NheI enzymes on ice
Chill 1.5 mL microcentrifuge tubes in ice bucket
2)Add 3 μL double distilled water into the centrifuge tube
3)Add 5 μL 10× Reaction Buffer into the centrifuge tube
4)Add 1.5 μg Plasmid extract into the centrifuge tube
5)Add 1 μL HindIII into the centrifuge tube
6)Add 1 μL NheI into the centrifuge tube
7)Incubate at 37 ˚C for 1 h in a calibrated dry bath incubator

Digestion of the target gene fragments
Experimental objective：
To digest the target genes by double restriction digestion, yielding compatible sticky ends for downstream ligation.

Equipment list：
1)Ice bucket
2)Dry bath incubator
3)Adjustable micropipettes (0.5-10 μL, 10-100 μL)
4)centrifuge tubes
5)Waste collection tubes
6)Restriction enzyme: HindIII
7)Restriction enzyme: NheI
8)Compatible 10× reaction buffer
9)PCR product (from previous step)
10)Double distilled water

Method:
1)Pre-cooling:
Thaw HindIII and NheI enzymes on ice
Chill 1.5 mL centrifuge tubes in ice bucket
2)Add 40.5 μL double distilled water into the centrifuge tube
3)Add 5 μL 10× Reaction Buffer into the centrifuge tube
4)Add 1.5 μg amplication of target genes extract into the centrifuge tube
5)Add 1 μL HindIII into the centrifuge tube
6)Add 1 μL NheI into the centrifuge tube
7)Incubate at 37 ˚C for 1 h in a calibrated dry bath incubator

Agarose gel preparation
Experimental objective:
To produce a functional and well-shaped gel for electrophoresis of DNA

Equipment list:
1)Casting Tray
2)Weighing scale
3)Measuring cylinder
4)Well comb
5)Microwave
6)1x TAE
7)Agarose powder
8)1000x Nucleic acid gel stain
9)Conical flask

Method:
1)Weigh out 0.8g Agarose powder by weighing scale
2)Transfer the Agarose powder into 250 mL conical flask
3)Add TAE (Running buffer) up to total volume of 100 mL by 100 mL measuring cylinder
4)Heat the material with microwave
5)Melt the mixture at 30 sec intervals
6)See whether bubbles are forming, if did successfully mix
7)Add 100 μL nucleic acid gel stain (1000x)
8)Let it cool for 5~10 min
9)Pour the agarose mixture into the casting tray
10)Place the appropriate well comb to create the wells
11)Wait to solidify at least 30 min
12)Remove the comb and place the gel in the gel box

Agarose gel electrophoresis
Experimental objective:
To verify the restriction digestion of the target gene fragments and vector, followed by gel purification of the digested products.

Equipment list：
1)sample
2)Marker (DNA ladder)
3)Prepared gel (from previous step)
4)10x Loading buffer (dye)
5)Horizontal gel electrophoresis system
6)Adjustable micropipettes

Method:
1)Extract 50 µL of every sample, add 5 µL of loading buffer (10x) to each
2)Place the gel in the horizontal electrophoresis system
3)Extract 50 µL of the mixed solution containing the sample, mix them till their colors are almost uniform
4)Add the marker to the first well and load 50 µL of each sample into the wells
5)The procedure runs at 120 V for 25 min
6)Afterwards, take out the gel from the device and proceed to following steps

Gel Extraction and Purification of DNA
Experimental objective:
To verify the DNA bands of digested target genes fragments and plasmids and thus preparing for the next step of gel extraction to extract the bands that are of the correct length.

Equipment list:
7)UV transilluminator
8)Clean scalpel
9)Weighing scale
10)Water bath (50 °C calibrated)
11)Microcentrifuge tube rack
12)Silica-membrane spin columns
13)Collection tubes (2 mL)
14)Nuclease-free 1.5 mL microcentrifuge tubes
15)Buffer B2 (Gel solubilization buffer)
16)Wash Solution
17)Elution Buffer
18)Agarose gel
19)pipettes and sterilized pipette heads
20)centrifuge

Method:
1)Pre-Extraction Preparation
Check Buffer B2 for precipitation
Preheat water bath to 50 °C
2)Excise target band under UV light, Minimize UV exposure (<30 sec)
3)Weigh gel slice and record the mass
4)Add Buffer B2 (3× gel weight)
5)Incubate at 50 °C for 5 min to melt the gel
6)Transfer lysate to spin column, centrifuge at 8,000× g for 30 sec
7)Discard flow-through
8)Add 500 μL Wash Solution centrifuge at 9,000× g for 30 sec
9)Repeat wash step centrifuge at 9,000× g for 30 sec
10)Dry column (empty) centrifuge at 9,000 ×g for 1 min
11)Transfer column to clean 1.5 mL tube
12)Add 20 μL Elution Buffer to membrane center, place at room temp for 1 min
13)Centrifuge to collect DNA at 9,000 ×g for 1 min
14)Store eluate

Ligation of the target gene and vector for plasmid construction
Experimental objective:
Ligate the plasmid fragment after enzymatic digestion with the target gene fragment, constructing the target gene-containing plasmid for subsequent protein expression and purification.

Equipment list:
1)T4 DNA ligase
2)T4 DNA ligase buffer
3)PCR products of target genes
4)vector（plasmid）
5)nanodrop
6)Adjustable micropipettes

Method:
1)Test the concentration of vector and target genes using Nanodrop
2)Prepare the system (20 μL)
Add 1 μL T4 DNA ligase
Add 2 μL T4 DNA ligase buffer
vector 2 μL
target DNA fragment 50 ng
Place the system at in a 16°C metal bath for 2 hours to react.

Transformation of competent cells and plating via heatshock
Experimental objective:
To transfer the target gene-carrying plasmid into competent cells for plasmid amplification (for subsequent verification) or recombinant protein expression.

Equipment list:
1)Ligated samples
2)LB Agar plates and liquid solution
3)Laminar flow cabinet
4)Inoculation loop
5)Shaking incubator
6)Antibiotic (kanamycin)
7)Component cells E.coli DH5α, E.coli BL21(DE3)

Method:
1)Ligated samples are added to component cells
2)Placed on ice for 30 min
3)Heat at 42 °C for 45 sec, then immediately put on ice for 2 min
4)Add 900 μL LB solution, incubate for 30 min
5)Shake at 220 rpm for 50 min at 37 °C using shaking incubator
6)Apply end product to agar plates with antibiotic (kanamycin), incubate for 12-16 h at 37 °C

Positive clone verification
Experimental objective:
To confirm the successful transformation of the target gene into competent cells, PCR amplification followed by gel electrophoresis was performed on bacterial colonies.

Equipment list:
1)Adjustable micropipettes
2)LB Agar plates with bacterial colonies
3)Centrifuge
4)Measuring cylinder
5)Centrifuge tube
6)2×PrimeSTAR Max Premix
7)Double distilled water
8)Primer-R
9)Primer-F
10)PCR thermal cycler
11)Marker (DNA ladder)
12)Prepared gel (from previous step)
13)10x Loading buffer (dye)
14)Horizontal gel electrophoresis system

Method:
1)Preparation of a 20 μL system
Add 10 µL 2×PrimeSTAR Max Premix, primer-R 1.0 µL, and primer-F 1.0 µL to centrifuge tube.
Add double distilled water to the centrifuge tube until reaching 20 µL.
If bubbles are observed in the mixture, centrifuge the mixture for a few seconds to remove the bubbles
2)Within sterilized fume hood workstation, remove a colony of preprepared bacteria from the agar plate and add to 1.5mL centrifuge tube
(Note: choose larger colonies but also be aware that colonies near the sides of the petri dish are most likely to be contaminated or are undesired bacteria and are less ideal)
3)Insert mixture into a PCR thermal cycler. Denature the fragments at 95 ˚C for 3 min
4)Perform 30 cycles of 30 sec 95 ˚C denaturing, 30 sec 55 ˚C annealing, and 90 sec extension 72 ˚C extension.
5)Extend the fragments for 1 min at 72 ˚C
6)Extract 50 µL of every sample
7)Place the gel in the horizontal electrophoresis system
8)Extract 50 µL of the mixed solution containing the sample, mix them till their colors are almost uniform
9)Add the marker to the first well and load 10 µL of each sample into the wells
10)The procedure runs at 120 V for 25 min
11)Afterwards, take out the gel from the device and observe the position of the bands using a gel imager

2.2 Construction of pGEX4T-1-Nb-human
The construction of the pGEX4T-1-Nb-human is same as the construction of pET28a-Sumo-Nb-human, pET28a-Sumo-Nb-SCFV, pET28a-Sumo-UreB-Nb6, pET28a-UreB. And there were some difference.
1. First, we used Nb-human as the target gene instead of all three target genes for expression and purification when the plasmid was constructed.
2. The vector was replaced by pGEX4T-1.
3. We transferred the constructed plasmid into Escherichia coli Nissle 1917 for expression and purification, and we got the desired vector carrying the target gene.

3.1 Protein expression of pET28a-Sumo-Nb-human, pET28a-Sumo-Nb-SCFV, pET28a-Sumo-UreB-Nb6, pET28a-UreB

Protein induction and expression in E.coli BL21
Experimental objective:
To scale up the cultivation of genetically verified bacterial clones for subsequent target protein induction, expression, and functional validation.

Equipment list：
1)Adjustable micropipettes
2)Culture tube
3)Spectrophotometer
4)Shaking incubator
5)Antibiotic (kanamycin)
6)Laminar flow cabinet
7)Conical flask
8)LB liquid solution
9)Colonies samples
10)IPTG (isopropyl β-D-1-thiogalactopyranoside)

Method:
1）Add 4 mL of LB liquid solution into a sterile culture tube
2）Based on the agarose gel electrophoresis results (from the previous step), select and transfer colonies confirmed to carry the target genes into the culture tube
3）Incubate the culture tube in a shaking incubator at 37 °C with 220 rpm shaking for 4 h
4）Add 100 mL of LB liquid medium with 50 μg/mL kanamycin into a sterile conical flask
5）Transfer the entire bacterial suspension from the culture tube into the conical flask
6）Place the flask in a shaking incubator and culture at 37 °C, 220rpm.
7）Measure the optical density (OD₆₀₀) using a spectrophotometer until the reading reaches 0.4–0.6
8）Add 100 µL of IPTG (isopropyl β-D-1-thiogalactopyranoside) to induce protein expression at 25°C or 16 °C, 20h

Protein extraction and purification
Experimental objective：
To lyse bacterial cells and extract proteins for subsequent purification and concentration measurement

Equipment list：
1)Centrifuge
2)LB solution (with sample)
3)50 mL centrifuge tubes
4)1.5 mL centrifuge tubes
5)Adjustable-volume micropipettes
6)Ultrasonic cell disruptor with microtip probe
7)Ice bath
8)Column stands with clamps
9)4 °C cold room or refrigerated cabinet
10)-20 °C freezer (for enzyme stock storage)
11)100 mg/mL Lysozyme
12)Non-denaturing lysis buffer with protease inhibitor cocktail
13)50% BeyoGold™ His-tag Purification Resin
14)Lateral shaker
15)Affinity chromatography column
16)Wash buffer
17)Elution buffer

Method：
1)Transfer the LB solution (with sample) into 50 mL centrifuge tubes
2)Centrifuge induced culture at 12,000 rpm for 4 min, discard supernatant completely
3)Add 4 mL non-denaturing lysis buffer with protease inhibitors per gram wet pellet, fully resuspend the bacterial pellet
4)Add lysozyme from 100 mg/mL stock to final 1 mg/mL, incubate on ice for 30 min
5)Sonicate on ice: 3 sec ON / 3 sec OFF, at 40% power for 15 min
6)Centrifuge to obtain the bacterial lysate supernatant
7)Transfer 1 mL of a well-mixed 50% slurry of BeyoGold™ His-tag Purification Resin into a microcentrifuge tube, centrifuge at 4 °C and discard the supernatant.
8)Add 0.5 mL of non-denaturing lysis buffer to the resin pellet, mix thoroughly to equilibrate the resin.
9)Add 4 ml of bacterial lysate supernatant to it and gently shake it at 4 ºC for 60 min by lateral shaker
10)Transfer the mixture into an empty affinity chromatography column
Remove the bottom cap of the purification column and allow the liquid to flow through by gravity. Collect 20 µL of the flow-through for subsequent analysis
11)Repeat the flow-through step a total of 2 times
12)Apply 0.5 mL of non-denaturing wash buffer, each time collecting 20 µL of the wash-through for subsequent analytical detection
Proceed with column washing and the subsequent elution step.
13)Repeat the washing step a total of 3 times
14)Apply 0.5 mL of non-denaturing elution buffer to elute the bound proteins, collecting each fraction in separate microcentrifuge tubes.
15)Repeat the elution step a total of 4 times. The collected eluates constitute the purified His-tagged protein samples

Dialysis for salt removal
Experimental objective：
The main purpose of dialysis and desalination of the target protein is to to remove the excess NaCl and imidazole ions in the protein solution, ensuring the purity and stability of the protein

Equipment list：
1) Dialysis bag
2) 20 mM Tris-HCl（pH = 8.0）
3) Beaker
4) Magnetic stirrer
5) Clip

Method:
1) The purified recombinant protein solution was placed into a dialysis bag with a molecular weight cut-off of 10 kDa.
2) The two ends of the dialysis bag were clamped into a beaker containing 20 mM Tris-HCl (pH = 8.0) dialysate
3) Placed the dialysis bag at 4℃, and dialyzed overnight with continuous stirring by a magnetic stirrers.

Concentrate the protein after dialysis
Experimental objective：
It can increase the concentration and reduce the volume in an efficient and gentle way, laying the foundation for further research or application of proteins.

Equipment list：
1) 10 kDa ultrafiltration tube
2) Cone shaped bottle
3) High-speed centrifuge
4)50 mL centrifuge tubes
5) 1.5 mL centrifuge tubes

Method:
The protein after dialysis was concentrated by ultrafiltration with a 10 kDa ultrafiltration tube, packaged and stored at -80℃

SDS-PAGE
A. PAGE gel preparation
Experimental objective：
To produce a functional and well-shaped gel for electrophoresis of proteins

Equipment list:
1)Casting tray
2)15 mL centrifuge tubes
3)Adjustable-volume micropipettes
4)Well comb
5)Resolving gel solution
6)Resolving gel buffer
7)Stacking gel solution
8)Colored stacking gel buffer
9)Polymerization accelerator

Method:
1)Add 4 mL of resolving gel solution and 4 mL of resolving gel buffer and mix thoroughly
2)Add 1 mL of stacking gel solution and 1 mL of colored stacking gel buffer and mix thoroughly
3)Add 80 μL of polymerization accelerator to the resolving gel mixture and mix gently
4)Immediately pour the solution into the gel-casting assembly, ensuring the liquid level remains 0.5 cm below the top edge of the short glass plate
5)After resolving gel solidification, add 20 μL of polymerization accelerator to the stacking gel mixture and mix gently
6)Carefully layer the stacking gel solution on top of the polymerized resolving gel, insert the well-forming comb, avoiding bubble formation
7)Allow gel polymerization, remove the comb carefully and rinse wells with running buffer before electrophoresis.
8)Wait for 40minutes
The percentage of the gel is 15% based on the target protein size.

SDS-PAGE gel electrophoresis
Experimental objective:
To verify successful expression and purification of the target protein by SDS-PAGE analysis of induced lysates and elution fractions.

Equipment list：
1)Sample
2)Marker
3)Prepared gel (from previous step)
4)Loading buffer (dye)
5)Vertical gel electrophoresis system
7)Adjustable micropipettes

Method:
1)Extract 20 µL of every sample, add 5 µL of loading buffer (5x) to each
2)Place the gel in the vertical electrophoresis system
3)Add the marker to the first well and load 15 µL of each sample into the wells
1)The procedure runs at 180V for 40 minutes.
2)Afterwards, take out the gel from the device
3)Stain with Coomassie Brilliant Blue R-250 for 15min and proceed to following steps

Target protein concentration determination
Experimental objective：
To quantify the target protein concentration and evaluate its expression using the Bicinchoninic Acid (BCA) assay.

Equipment list:
1)Adjustable micropipettes
2)Protein standard
3)Protein standard diluent
4)BCA Reagent A
5)BCA Reagent B
6)96-well plate
7)Protein samples
8)Incubator
9)Microplate reader

Method:
1)Add 0.8 mL of protein standard diluent to one vial of protein standard (20 mg BSA). After thorough dissolution, prepare a 25 mg/mL protein standard solution, then dilute* to a final concentration of 0.5 mg/mL.
*The protein samples should be in the same diluent as that used for the standard preparation
2)Prepare 7.65 mL of BCA working solution by mixing 7.5 mL of BCA Reagent A with 0.15 mL of BCA Reagent B, followed by thorough vortexing
3)Add 0, 1, 2, 4, 8, 12, 16, and 20 μL of the standard solution to the designated wells of a 96-well plate, adjusting the final volume in each well to 20 μL with standard diluent. The resulting standard concentrations are 0, 0.025, 0.05, 0.1, 0.2, 0.3, 0.4, and 0.5 mg/mL, respectively
4)Add 20 μL of protein samples to the designated sample wells of the 96-well plate
5)Add 200 μL of BCA working solution to each well and incubate at 37°C for 20–30 minutes
6)Measure the absorbance at 562 nm using a microplate reader
7)Calculate the protein concentration of the samples based on the standard curve and the sample volume used.

Western Blot (WB)
A.SDS-PAGE gel electrophoresis and Membrane Transfer
The SDS-PAGE operation is consistent with the above.

Experimental objective:
To transfer the extracted proteins from the PAGE gel to a PVDF membrane for subsequent analysis

Equipment list：
1)Gel sample (from previous step)
2)Adjustable micropipettes
3)Ice bath
4)PVDF membrane
5)Anhydrous methanol
6)Transfer cassette
7)Filter paper
8)Filter pad
9)Transfer tank
10)Pre-chilled transfer buffer

Method:
1)Pre-activate the PVDF membrane by soaking in methanol.
2)Assemble the transfer sandwich in the following order (from anode to cathode): transfer cassette, filter pad, filter paper, gel, PVDF membrane, filter paper, filter pad, and transfer cassette
Ensure all components are fully saturated with transfer buffer
3)Secure the assembled cassette and place it into the transfer tank filled with pre-chilled transfer buffer
4)Perform electrophoretic transfer at 150mA for 2h under ice-cooled conditions to prevent overheating

B. Non-specific binding blockade
Experimental objective：
To block non-specific binding sites

Equipment list：
1)Lateral shaker
2)1x PBST buffer
3)5% BSA-PBST blocking buffer

Method：
1)Remove the PVDF membrane from the transfer tank
2)Wash the PVDF membrane 3–5 times with PBST buffer
3)Block the PVDF membrane with 50 mL of blocking solution(5% BSA-PBST) and incubate with gentle shaking on a lateral shaker for 30 min

C. Antibody Incubation
Experimental objective：
To specifically identify the protein of interest, the membrane was incubated with a primary antibody, then with an HRP-labeled secondary antibody, and finally subjected to TMB for visualization

Equipment list：
1)Adjustable micropipettes
2)Lateral shaker
3)1x PBST buffer
4)Anti-His tag primary antibody
5)Rabbit Anti-Mouse IgG H&L HRP secondary antibody

Method：
1)Remove the PVDF membrane from the blocking buffer
2)Wash the PVDF membrane 3–5 times with PBST buffer
3)Incubate the membrane with anti-His tag primary antibody for 2 h at 4 °C temperature under gentle shaking
4)Wash the PVDF membrane 3–5 times with PBST buffer
5)Incubate the membrane with Rabbit Anti-Mouse IgG H&L HRP secondary antibody for 1 h at room temperature under gentle shaking

D. Detection
Experimental objective：
To validate the specificity of the target protein, the membrane was developed with TMB substrate, resulting in visible colorimetric signals at the sites of antibody binding

Equipment list：
1)Adjustable micropipettes
2)1x PBST buffer
3)TMB color-developing solution

Method：
1)Wash the PVDF membrane 3–5 times with PBST buffer
2)Uniformly drop 100 µL of TMB color-developing solution onto the membrane, covering the area on the membrane
3)Incubate in the dark for 5 to 30 min until the desired result is achieved

3.2 Protein expression of pGEX4T-1-Nb-human
The protein expression of Nb-human on the pGEX4T-1 is similar to the above. The induction condition was set to induce at 25℃ for 20 hours under the action of 1mM IPTG.

4.1 Function test of pET28a-Sumo-Nb-human, pET28a-Sumo-Nb-SCFV, pET28a-Sumo-UreB-Nb6, pET28a-UreB

Immunodiffusion Assay
Experimental objective：
To qualitatively verify whether the expressed antibody protein effectively binds to its target antigen

Equipment list：
1)Double distilled water
2)Weighing scale
3)Conical flask
4)Agarose powder
5)Measuring cylinder
6)Microwave
7)Ureb sample
8)Antibody sample (Nb-human, Nb6 and SCFV)
9)Culture dish
10)Incubator
11)Oxford cups
12)Adjustable micropipettes

Method：
1)Weigh out 1.00 g Agarose powder by weighing scale
2)Transfer the Agarose powder into 250 mL conical flask
3)Add double distilled water up to total volume of 100 mL by 100 mL measuring cylinder
4)Heat the material with microwave, melt the mixture at 30 sec intervals
5)Add 1 mL UREB sample into the conical flask
6)Transfer the mixture into a culture dish
7)After solidification of the culture medium, wells were created using Oxford cups, with clear labeling of antibody names and control groups
8)Add 100 μL antibody sample into each well
9)Incubate at 37 °C for 24-36h.

Nanobody-Mediated Urease Inhibition Assay
Experimental objective：
To quantitatively measure the inhibitory activity of recombinant nanobodies against urease

Equipment list:
1)Adjustable micropipettes
2)Diluent
3)Ureb sample
4)Antibody sample (Nb-human, Nb6 and SCFV)
5)96-well plate
6)Incubator
7)Microplate reader
8)Urea

Method：
1)Dilute Ureb sample to achieve a final concentration of 100 μg/mL
2)Perform two-fold serial dilutions of each antibody (Nb-human, Nb6 and SCFV) in assay buffer to create concentrations of 256, 128, 64, 32, 16, 8, 4, 2, 1, 0.5, and 0 μg/mL
3)Aliquot 100 μL of UreB solution (100 μg/mL) into each well of microtiter plates
4)Transfer 50 μL of diluted antibody solutions to corresponding antigen-coated wells.
5)Incubate at 4 °C overnight
6)Preparation of 1 M Urea Solution
7)Aliquot 50 μL of 1 M Urea solution into each well of microtiter plates
8)Incubation at 25 °C for 6-10 h
8)Measure the absorbance at 550 nm using a microplate reader and calculate inhibition ratio.