A nanobody against Clostridium perfringens alpha toxin and its application

Abstract

The present invention provides a Clostridium perfringens alpha-toxin nanobody and its application, belonging to the field of bioengineering technology. The amino acid sequence of the Clostridium perfringens alpha-toxin nanobody is shown in SEQ ID No. 1. The Clostridium perfringens alpha-toxin nanobody provided by the present invention can specifically bind to the Clostridium perfringens alpha-toxin protein with a minimum sensitivity of 0.625 μg / ml to the protein; and can detect Clostridium perfringens alpha-toxin in chicken and alpaca feces by ELISA with a sensitivity of 2 μg / ml.

Description

Technical Field

[0001] The present invention belongs to the field of bioengineering technology, and in particular relates to a Clostridium perfringens alpha toxin nanobody and application thereof. Background Art

[0002] Single-domain antibodies (SDOs) are specific antibodies found in camelids (alpacas, camels) and cartilaginous fish that lack heavy chains but still possess biological activity. The antigen-binding site (VHH) of a SDO has independent antigen recognition capabilities, and independently expressed VHHs are also known as nanobodies. Compared with traditional tetramer antibodies, SDOs have the following key advantages: small molecular weight, simple structure, and stable physicochemical properties. These excellent properties offer advantages in several areas: They can penetrate certain protective barriers within the animal body, such as the blood-brain barrier and the blood-testis barrier, to reach the site of disease and exert their effects. They can bind to hidden epitopes, making them particularly suitable for difficult-to-access antibody targets, such as GPCRs, ion channels, and enzyme activity centers. Their simple structure reduces production costs, making them easy to express in vitro and less prone to the formation of inclusion bodies, simplifying the production process. Furthermore, their small molecular weight and simple structure make them more amenable to genetic modification and humanization.

[0003] Clostridium perfringens, also known as Clostridium welchii or Bacillus perfringens, has been identified as producing up to 20 toxins and enzymes. Among these, alpha toxin (CPA), beta toxin (CPB), epsilon toxin (ETX), and iota toxin (ITX) are the most prominent toxins produced by C. perfringens and are the primary toxins that induce disease. Alpha toxin is the earliest and most studied of the C. perfringens toxins. It is essentially a zinc-containing phospholipase C that has been modified to confer toxin activity. The toxin contains 370 amino acids and has a molecular weight of approximately 43 kDa. It exhibits lethal, hemolytic, and dermatonecrotic activities, as well as phospholipase C and sphingomyelinase activities, and is a major causative factor in the development of gas gangrene. The development of nanoantibodies against C. perfringens alpha toxin could serve as a tool for rapid detection of C. perfringens alpha toxin and as a treatment for diseases caused by C. perfringens alpha toxin. Summary of the Invention

[0004] In view of this, the object of the present invention is to provide a Clostridium perfringens alpha toxin nanobody and its application. The Clostridium perfringens alpha toxin nanobody provided by the present invention can specifically bind to the Clostridium perfringens alpha toxin protein and can detect Clostridium perfringens alpha toxin in animal feces.

[0005] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0006] The present invention provides a Clostridium perfringens alpha toxin nanobody, the amino acid sequence of the Clostridium perfringens alpha toxin nanobody is shown in SEQ ID No.1.

[0007] The present invention also provides the use of the Clostridium perfringens alpha toxin nanobody described in the above technical solution in the preparation of a reagent that specifically binds to the Clostridium perfringens alpha toxin protein.

[0008] The present invention also provides the use of the Clostridium perfringens alpha toxin nanobody described in the above technical solution in the preparation of a reagent for detecting Clostridium perfringens alpha toxin in animal feces.

[0009] Preferably, the animals include chickens and / or alpacas.

[0010] The beneficial effects of the present invention are:

[0011] The Clostridium perfringens alpha toxin nanoantibody provided by the present invention can specifically bind to the Clostridium perfringens alpha toxin protein, and the sensitivity to the protein is as low as 0.625 μg / ml; it can detect Clostridium perfringens alpha toxin in chicken and alpaca feces by ELISA method with a sensitivity of 2 μg / ml. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 This is the gel electrophoresis image of the first round of PCR-VHH;

[0013] Figure 2 is the electrophoresis diagram of the second round of PCR-VHH;

[0014] Figure 3 The results of the natural nano library capacity were detected on a plate;

[0015] Figure 4 This is the result of plate detection of the abundance of natural nanolibrary;

[0016] Figure 5 This is the result of PCR detection of the insertion rate of the natural nano library;

[0017] Figure 6 For amino acid sequence homology analysis in natural nanobody libraries;

[0018] Figure 7 for SDS-PAGE detection of purified Clostridium perfringens α-toxin nanobodies;

[0019] Figure 8 Western Blot was used to detect nanoantibodies against α-toxin of Clostridium perfringens. DETAILED DESCRIPTION

[0020] The present invention provides a Clostridium perfringens alpha toxin nanobody, wherein the Clostridium perfringens alpha toxin nanobody has the amino acid sequence shown in SEQ ID No.1.

[0021] In the present invention, the Nanobody has the amino acid sequence shown in SEQ ID No. 1, and the specific sequence is as follows:

[0022] ESGGGLVQPGGSLRLSCAASGFTFSRSRMYWVRQAPGKGLEWVSGITNAGISTGYADSVKGRFYVSRGNDESTLYLQMNSLKPEDTALYFCTRLGSGWEYDYWGQGTHVTVSS.

[0023] In the present invention, the method for screening Clostridium perfringens alpha toxin nanobodies preferably comprises the following steps:

[0024] 1) The natural nanobody library was subjected to the first round of panning to obtain α-toxin-VHH1;

[0025] The coating concentration of the Clostridium perfringens alpha toxin protein in the first round of panning was 18-22 μg / ml;

[0026] 2) performing the second, third, and fourth rounds of panning on the α-toxin-VHH1 obtained in step 1) to obtain a phage solution;

[0027] The coating concentration of the Clostridium perfringens alpha toxin protein in the second round of panning was 8-12 μg / ml;

[0028] The coating concentration of the Clostridium perfringens alpha toxin protein in the third round of panning is 3-8 μg / ml;

[0029] The coating concentration of the Clostridium perfringens alpha toxin protein in the fourth round of panning is 3 to 8 μg / ml;

[0030] 3) mixing the phage solution obtained in step 2) with the TG1 bacterial solution, infecting the mixture, and culturing the mixture to obtain a strain;

[0031] 4) mixing and infecting the strain obtained in step 3) with KM13 helper phage, subjecting the obtained infection to a first shaking culture followed by a first centrifugation, resuspending the obtained first precipitate in liquid culture medium, subjecting the obtained first precipitate to a second shaking culture, and subjecting the obtained second centrifugation to a second centrifugation, mixing the obtained second supernatant with a blocking solution, incubating the mixture, and conducting an indirect ELISA test to test the reactivity of the second supernatant with alpha toxin protein of Clostridium perfringens to confirm that the strain is reactive with the alpha toxin protein of Clostridium perfringens, and preserving the positive strains;

[0032] The temperature of the first oscillation is 35-42°C, and the temperature of the second oscillation is 28-32°C;

[0033] The centrifugal force of the first centrifugation is 7500-8500g, and the centrifugal force of the second centrifugation is 2000-2100g;

[0034] 5) extracting a plasmid from the strain reactive with the alpha toxin protein of Clostridium perfringens obtained in step 4), performing PCR amplification using the plasmid primer pair and the plasmid as a template to obtain a Nanobody VHH fragment, and connecting the Nanobody VHH fragment to an expression vector to obtain a recombinant plasmid;

[0035] The plasmid primers include a plasmid upstream primer and a plasmid downstream primer. The plasmid upstream primer has a nucleotide sequence shown in SEQ ID No. 2, and the specific sequence is as follows:

[0036] CGGGATCCGAGTCTGGAGGAGGCTTGGT, where GGATCC is the BamHI restriction site.

[0037] The plasmid downstream primer has the nucleotide sequence shown in SEQ ID No. 3;

[0038] CGGAATTCTGAGGAGACGGTGACGTGG, where GAATTC is the EcoRⅠ restriction site.

[0039] 6) The recombinant plasmid and pET28a obtained in step 5) were transformed into Escherichia coli to obtain a nanobody expression strain. After the nanobody expression strain was induced by IPTG, the protein of the induced nanobody expression strain was extracted, and the protein was identified by Western Blotting based on the protein molecular weight and His-tag tag. The protein with a molecular weight of 15 kDa was a Clostridium perfringens alpha toxin nanobody.

[0040] The present invention preferably performs a first round of panning on the natural nanobody library to obtain α-toxin-VHH1; the coating concentration of the Clostridium perfringens α-toxin protein in the first round of panning is 18-22 μg / ml.

[0041] The present invention has no particular limitation on the natural nanoantibody library, and a conventionally used natural nanoantibody library can be used. In an embodiment of the present invention, the method for constructing the natural nanoantibody library preferably includes the following steps:

[0042] A. extracting total lymphocyte RNA from the blood of 200 animals, and reverse transcribing the total lymphocyte RNA into cDNA;

[0043] B. Using the cDNA obtained in step A as a template, perform a first PCR amplification using the primer pair Call001-F and Call002-R to obtain a 700 bp amplification product;

[0044] The Call001-F primer has the nucleotide sequence shown in SEQ ID No. 4, and the specific sequence is as follows:

[0045] GTCCTGGCTGCTCTTCTACAAGG;

[0046] The Call002-R primer has the nucleotide sequence shown in SEQ ID No. 5, and the specific sequence is as follows:

[0047] GGTACGTGCTGTTGAACTGTTCC.

[0048] C. Using the 700 bp amplified product obtained in step B as a template, perform a second PCR amplification using the VHH2-F and VHH2-R primer pairs to obtain the VHH fragment;

[0049] The VHH2-F primer has the nucleotide sequence shown in SEQ ID No. 6, and the specific sequence is as follows:

[0050] TTTCTATTACTAGGCCCAGCCGGCCGAGTTCTGGAGGRRGCTTGGTGCA;

[0051] The VHH2-R primer has the nucleotide sequence shown in SEQ ID No. 7, and the specific sequence is as follows: AAACCGTTGGCCATAATGGCCTGAGGAGACGRTGACSTSGGTC.

[0052] D. The VHH fragment obtained in step C and pCANTAB5e were digested twice with SfiI and then ligated to obtain a ligation product;

[0053] E. The ligation product obtained in step D is mixed with TG1 electroporation competent cells and then electroporated to obtain a natural nanobody library.

[0054] The present invention preferably extracts total RNA from lymphocytes in animal blood and reverse transcribes the total RNA from lymphocytes into cDNA.

[0055] In the present invention, the animal is preferably an alpaca.

[0056] The present invention has no particular limitation on the method for extracting total RNA from lymphocytes in animal blood, and conventional RNA extraction methods can be used.

[0057] In the present invention, the reverse transcription preferably comprises: 5 μg total RNA, 1 μl Random6 Primer, 1 μl Oligo (dT) 18 Primer, 1 μl dNTP Mix, and 7 μl ddH2O were mixed and treated at 65°C for 5 min. The resulting treated product was allowed to stand on ice for 5 min. The treated product was mixed with 4 μl 5X PrimeScript II buffer, 0.5 μl RNase inhibitor, 1 μl Primer Scrip II Rtase, and 4.5 μl ddH2O, and then treated at 40°C for 40 min and 70°C for 15 min, respectively, to obtain cDNA.

[0058] The present invention uses cDNA as a template and performs a first PCR amplification using the primer pair Call001-F and Call002-R to obtain a 700 bp amplification product;

[0059] The Call001-F primer has the nucleotide sequence shown in SEQ ID No.4; the Call002-R primer has the nucleotide sequence shown in SEQ ID No.5.

[0060] In the present invention, the Call001-F primer has the nucleotide sequence shown in SEQ ID No. 4, and the specific sequence is as follows:

[0061] GTCCTGGCTGCTCTTCTACAAGG;

[0062] The Call002-R primer has the nucleotide sequence shown in SEQ ID No. 5, and the specific sequence is as follows:

[0063] GGTACGTGCTGTTGAACTGTTCC.

[0064] In the present invention, the Call001-F primer corresponds to the leader region of alpaca antibodies, and the Call002-R primer corresponds to the second constant region (CH2) of alpaca antibodies, and can amplify the leader region and CH2 region of conventional antibodies (900 bp) and heavy chain antibodies (700 bp), respectively. In the present invention, the amplified 700 bp amplification product is subjected to a second amplification.

[0065] In the present invention, the first PCR amplification system preferably includes: 2 μl cDNA, 1 μl Call001-F with a concentration of 10 umol / μl, 1 μl Call001-R with a concentration of 10 umol / ul, 25 μl Taq Green PCR Mix and 21 μl ddH2O per 50 μl.

[0066] In the present invention, the program of the first PCR amplification preferably includes: 95°C for 5 min; 95°C for 30 s, 53°C for 30 s, 72°C for 40 s, 30 cycles; 72°C for 5 min.

[0067] The present invention uses the 700bp amplification product as a template and performs a second PCR amplification using a VHH2-F and VHH2-R primer pair to obtain a VHH fragment; the VHH2-F primer has the nucleotide sequence shown in SEQ ID No. 7; the VHH2-R primer has the nucleotide sequence shown in SEQ ID No. 7.

[0068] In the present invention, the VHH2-F primer has the nucleotide sequence shown in SEQ ID No. 6, and the specific sequence is as follows:

[0069] TTTCTATTACTAGGCCCAGCCGGCCGAGTTCTGGAGGRRGCTTGGTGCA;

[0070] The VHH2-R primer has a nucleotide sequence shown in SEQ ID No. 7, and the specific sequence is as follows:

[0071] AAACCGTTGGCCATAATGGCCTGAGGAGACGRTGACSTSGGTC.

[0072] According to the sequencing results of the first round of PCR products, the second round of amplification primers VHH2-F and VHH2-R were designed in the constant region FR1 and FR4 regions at both ends of the alpaca single domain antibody (VHH domain) to amplify the VHH sequence.

[0073] In the present invention, the obtained VHH fragment and pCANTAB5e are preferably digested twice with SfiI and then ligated to obtain a ligation product.

[0074] The present invention has no particular limitation on the enzymatic digestion of the VHH fragment and pCANTAB5e, and conventional enzymatic digestion methods can be used. In the present invention, the empty loading rate can be eliminated after two enzymatic digestions.

[0075] In the present invention, the enzyme used for the ligation is preferably T4 ligase. In the present invention, the ligation system preferably includes, per 200 μl, the following: 1 μg of the VHH fragment, 3 μg of the pCANTAB5e vector, 10 μl of T4 DNA Ligase, 20 μl of 10× Buffer, and ddH2O to make up to 200 μl. The present invention does not particularly limit the ligation conditions; conventional ligation conditions can be used.

[0076] The present invention mixes the ligation product with TG1 electroporation competent cells and then performs electroporation to obtain a natural nanobody library.

[0077] In the present invention, the electroconversion preferably includes: the electroconversion voltage is preferably 1.5 to 2.0 kV, more preferably 1.8 kV; the electroconversion resistance is preferably 180 to 220 Ω, more preferably 200 Ω; the electroconversion capacitance is preferably 20 to 30 μF, more preferably 25 μF; the electroconversion time is preferably 4 to 6 ms, more preferably 5 ms.

[0078] After electroporation, the present invention further comprises: incubating the obtained electroporation product at 37° C. for 1 hour, centrifuging the obtained product at 5000 g for 5 minutes, and adding the obtained precipitate to SOC liquid culture medium to obtain a natural nanobody library.

[0079] In the present invention, the coating concentration of the Clostridium perfringens alpha toxin protein in the first round of panning is preferably 18-22 μg / ml, more preferably 10 μg / ml. The present invention has no particular limitation on the method of the first round of panning, and conventional panning methods in the art can be used.

[0080] The present invention preferably subjects the obtained α-toxin-VHH1 to second, third and fourth rounds of panning in sequence to obtain a phage solution; the coating concentration of the α-toxin protein of Clostridium perfringens in the second round of panning is preferably 8-12 μg / ml; the coating concentration of the α-toxin protein of Clostridium perfringens in the third round of panning is preferably 3-8 μg / ml; and the coating concentration of the α-toxin protein of Clostridium perfringens in the fourth round of panning is preferably 3-8 μg / ml.

[0081] In the present invention, the coating concentration of the α-toxin protein of Clostridium perfringens in the second round of panning is preferably 8-12 μg / ml, more preferably 10 μg / ml; the coating concentration of the α-toxin protein of Clostridium perfringens in the third round of panning is preferably 3-8 μg / ml, more preferably 5 μg / ml; and the coating concentration of the α-toxin protein of Clostridium perfringens in the fourth round of panning is preferably 3-8 μg / ml, more preferably 5 μg / ml. The present invention does not particularly limit the methods of the second, third, and fourth rounds of panning, and conventional panning methods in the art can be used.

[0082] In the present invention, the obtained phage solution is preferably mixed with the TG1 bacterial solution, and cultured after infection to obtain a strain.

[0083] In the present invention, the volume ratio of the phage solution to the TG1 bacterial solution is preferably 1:4. In the present invention, the OD600 value of the TG1 bacterial solution is preferably 0.4. In the present invention, the culture temperature is preferably 25-35°C, more preferably 30°C.

[0084] The present invention preferably mixes and infects the obtained strain with KM13 helper phage, performs a first shaking culture on the obtained infection, and then performs a first centrifugation, resuspends the obtained first precipitate in a liquid culture medium, performs a second shaking culture, and then performs a second centrifugation, mixes the obtained second supernatant with a blocking solution, incubates, and then performs an indirect ELISA test to detect the reactivity of the second supernatant with the α-toxin protein of Clostridium perfringens to determine that the strain has reactivity with the α-toxin protein of Clostridium perfringens, and at the same time, the reaction-positive strain is preserved; the temperature of the first shaking is 35-42°C, and the temperature of the second shaking is 28-32°C; the centrifugal force of the first centrifugation is 1700-1900g, and the centrifugal force of the second centrifugation is 2000-2100g.

[0085] In the present invention, the strain is preferably mixed with the KM13 helper phage and infected in a static manner, and the infection time is preferably 25 to 35 minutes, preferably 30 minutes.

[0086] In the present invention, the temperature of the first oscillation is preferably 35-42°C, more preferably 37°C; the temperature of the second oscillation is preferably 28-32°C, more preferably 30°C; the centrifugal force of the first centrifugation is preferably 1700-1900g, more preferably the centrifugal force of the second centrifugation is 1800g; the centrifugal force of the second centrifugation is preferably 2000-2100g, more preferably 2020g.

[0087] The present invention preferably extracts the plasmid from a strain reactive with the alpha toxin protein of Clostridium perfringens, uses the plasmid as a template, and performs PCR amplification with a plasmid primer pair to obtain a nanobody VHH fragment, and connects the nanobody VHH fragment to an expression vector to obtain a recombinant plasmid; the plasmid primers include a plasmid upstream primer and a plasmid downstream primer, and the plasmid upstream primer has the nucleotide sequence shown in SEQ ID No. 2; the plasmid downstream primer has the nucleotide sequence shown in SEQ ID No. 3.

[0088] The present invention does not specifically limit the extraction of the plasmid, and conventional plasmid extraction methods can be used. The present invention does not specifically limit the system and procedure used for the PCR amplification, and conventional systems and procedures can be used. In the present invention, the plasmid primers include a plasmid upstream primer and a plasmid downstream primer. The plasmid upstream primer preferably has the nucleotide sequence shown in SEQ ID No. 2, which is as follows:

[0089] CGGGATCCGAGTCTGGAGGAGGCTTGGT;

[0090] The plasmid downstream primer has the nucleotide sequence shown in SEQ ID No. 3, and the specific sequence is as follows:

[0091] CGGAATTCTGAGGAGACGGTGACGTGG.

[0092] The present invention has no particular limitation on the method for connecting the Nanobody VHH fragment to the expression vector, and conventional connection methods in the art can be used.

[0093] The obtained recombinant plasmid, pET28a, is transformed into Escherichia coli to obtain a nanobody expression strain. The nanobody expression strain is induced by IPTG, and the protein of the induced nanobody expression strain is extracted. The protein is identified by Western blotting based on the protein molecular weight (about 15 kDa) and the His-tag label. The protein with a molecular weight of 15 kDa is a Clostridium perfringens alpha toxin nanobody.

[0094] The present invention does not specifically limit the method for introducing the recombinant plasmid, pET28a, into Escherichia coli; conventional methods can be used. The present invention does not specifically limit the method for inducing the nanobody expression strain with IPTG; conventional induction methods can be used. The present invention does not specifically limit the method for extracting protein from the induced nanobody expression strain; conventional methods for extracting protein from microorganisms can be used. The present invention does not specifically limit the method for SDS-PAGE identification; conventional methods can be used.

[0095] The present invention also provides the use of the Clostridium perfringens alpha toxin nanobody described in the above technical solution in the preparation of a reagent that specifically binds to the Clostridium perfringens alpha toxin protein.

[0096] The present invention also provides the use of the Clostridium perfringens alpha toxin nanobody described in the above technical solution in the preparation of a reagent for detecting Clostridium perfringens alpha toxin in animal feces. Preferably, the animal includes chickens and / or alpacas.

[0097] The following is a further detailed introduction to the Clostridium perfringens alpha toxin nanobody and its application in conjunction with specific examples. The technical solutions of the present invention include but are not limited to the following examples.

[0098] Example 1

[0099] Extracting total lymphocyte RNA from the blood of 200 animals, and reverse-transcribing the total lymphocyte RNA into cDNA;

[0100] After RNA extraction according to the instructions of the RNA extraction kit, the first chain of VHH was synthesized according to the instructions of the long-chain cDNA reverse transcription kit. The reaction system is shown in Table 1:

[0101] Table 1 Reaction system

[0102]

[0103]

[0104] The reaction system was vortexed instantaneously, and reverse transcription was performed using a PCR instrument according to the reaction system of 40°C for 40 min; 70°C for 15 min; and storage at 12°C.

[0105] The first round of PCR amplification of VHH fragments: The natural nanobody library was constructed using the nested PCR method. The primers for the first round of PCR amplification were Call001-F and Call002-R, where Call001-F corresponds to the leader region of alpaca antibodies, and Call002-R corresponds to the second constant region (CH2) of alpaca antibodies. They can amplify the leader region and CH2 region of conventional antibodies (900 bp) and heavy chain antibodies (700 bp), respectively. The specific method is as follows:

[0106] The first round PCR primers are shown in Table 2:

[0107] Table 2 Primer sequences

[0108] Call001-F: SEQ ID No.4 GTCCTGGCTGCTCTTCTACAAGG Call002-R: SEQ ID No.5 GGTACGTGCTGTTGAACTGTTCC

[0109] PCR amplification was performed using the synthesized first-strand cDNA as a template. The reverse transcription product was divided into 30 reactions. Each PCR reaction system was 50 μl. The reagent used in the reaction was 2xTap enzyme from Cosmos. The reaction system is shown in Table 3:

[0110] Table 3 Reaction system

[0111] cDNA 2 μl Call001-F (10umol / ul) 1 μl Call002-R10umol / ul) 1 μl Taq Green PCR Mix 25 μl Water 21 μl Total volume 50 μl

[0112] The PCR amplification reaction program is shown in Table 4:

[0113] Table 4 Amplification procedure

[0114]

[0115] Agarose gel electrophoresis analysis of the first-round PCR reaction products showed that they mainly contained two amplification products of 900 bp and 700 bp in size. The 700 bp nucleic acid was recovered by gel excision under UV light. The 700 bp nucleic acid fragment was recovered and purified using a gel recovery kit (Kangwei Century). 1 μl of the gel recovery product was ligated to the pMD19-T simple vector. The reaction system is shown in Table 5:

[0116] Table 5 Reaction system

[0117]

[0118]

[0119] The reaction system was vortexed overnight at 4°C. The heat-shocked ligation product was mixed with DH5α competent cells and incubated on ice for 25 minutes. Heat-shocked for 90 seconds at 42°C was performed, followed by another 4-minute incubation on ice. 400 μl of LB medium was added and incubated at 37°C, 200 rpm, for 40 minutes. 50 μl of the transformed bacterial suspension was evenly spread onto the surface of LB solid medium containing ampicillin (AMP). The plate was inverted and incubated overnight at 37°C. The next day, 20 single colonies were inoculated into 5 ml of LB liquid medium containing AMP resistance and cultured overnight before their base sequence determination. Sequencing results were analyzed using Vector NTI software, and second-round PCR primers VHH2-F and VHH2-R were designed for library construction.

[0120] Second round of PCR amplification of VHH: Using the 700bp fragment recovered from the first round of PCR as a template, amplify the VHH fragment using the sequencing-designed primers VHH2-F and VHH2-R. Perform 24 PCR reactions in a 50μl reaction system. See Table 6 for the specific reaction system:

[0121] Table 6 Reaction system

[0122] Gel recovery product 80μg / μl 6 μl VHH2-F (10 μmol / μl) 2 μl VHH2-R (10 μmol / μl) 2 μl Taq Green PCR Mix 25 μl Water 15 μl Total volume 50 μl

[0123] PCR amplification reaction conditions are shown in Table 7:

[0124] Table 7 Amplification procedure

[0125]

[0126]

[0127] A 2-μl sample of the second-round PCR product was analyzed by agarose gel electrophoresis. The amplified product was a single band. The second-round VHH-PCR product can be directly purified using the nucleic acid purification kit instructions.

[0128] Enzyme digestion and purification of the VHH fragment and vector: E. coli containing the pCANTAB5e plasmid was isolated by shake-blowing. A 10-μl aliquot of the plasmid was sequenced to verify the plasmid quality and the presence of mutated bases. The VHH fragment and plasmid pCANTAB5e were digested twice using SfiI (Thermo). The enzyme digestion system is shown in Table 8:

[0129] Table 8 Enzyme Digestion System

[0130]

[0131] The VHH fragment and pCANTAB5e vector were digested twice to eliminate empty vectors. In the first round, both the VHH and vector were digested for 20 reactions. The digestion conditions were: 1 hour at 50°C in a water bath. The VHH system was purified using a PCR product purification kit; the vector digestion system was first purified on agarose gel, followed by a gel recovery kit. The purified products from the first round of digestion were digested again for 1 hour at 50°C in a water bath, and both products were purified again.

[0132] Ligation and purification of digestion products: The VHH fragments purified after two rounds of digestion were ligated with the vector pCANTAB5e using T4 ligase (Thermo). The reaction system is shown in Table 9:

[0133] Table 9 Connection system

[0134] VHH fragments 1 μg pCANTAB5e vector 3 μg T4 DNA Ligase 10 μl 10×Buffer 20 μl Water Up to 200μl Total volume 200 μl

[0135] The reaction system was vortexed briefly and allowed to react overnight at 4°C. The next day, the ligation product was purified according to the manufacturer's instructions.

[0136] Construction and characterization of natural nanoantibody library: The purified pCANTAB5e-VHH ligation product (total volume 100ul) was added to freshly prepared TG1 electroporation competent cells, pipetted to mix, and placed in an ice bath for 10min; after transformation in an electroporator at a voltage of 1.8kV, a resistance of 200Ω, a capacitance of 25μF, and a time of 5ms, the TG1 cells after electroporation were transferred to a 50ml sterile centrifuge tube and incubated at 37°C for 1h; the incubated bacterial solution was centrifuged at 5000g for 5min, the supernatant was discarded, and 8ml of fresh SOC medium was added to resuspend the precipitate; 100μl of the bacterial solution was diluted gradiently and applied to 16 2×YTAG The storage capacity was determined on a solid culture plate (90 mm), with two plates for each gradient, for a total of 8 gradient plates. The remaining bacterial liquid was spread on 30 2×YTAG solid culture plates (150 mm plates) and cultured in a static incubator at 25°C overnight. The next day, after the colonies on the culture plates had grown well, 5 ml of 2×YT liquid culture medium was added to each of the 39 150 mm plates to wash the colonies and collect the washing liquid. The culture medium was centrifuged at 5000 g and 4°C for 12 min, and 90 ml of 2×YT liquid culture medium containing 15% glycerol was added to resuspend the precipitate. The tubes were sealed to obtain the prepared primary library bacteria, named natural nanoantibody library. 100 μl of the primary library bacteria was used to determine the abundance of the library, and the rest was aliquoted and frozen at -70°C.

[0137] Determination of library capacity: 100 μl of electroporated bacterial solution was diluted in a gradient manner, with the dilution ranging from 10 -1 ~10 -8; Take 100 μl of each dilution and coat two 2×YTAG solid culture plates, and culture them at 25℃ overnight; the next day, count the colonies on the gradient plates and calculate the storage capacity.

[0138] Determination of library abundance: Take 100 μl of primary library solution and perform gradient dilution from 10 -4 ~10 -10 ; Take 100 μl of bacterial solution from each dilution and spread it on two 2×YTAG solid culture plates, and culture them at 25°C overnight; the next day, count the colonies on the gradient plates and calculate the library abundance.

[0139] Determination of VHH insertion rate and insertion diversity: After the library size is determined, 50 monoclonal colonies are randomly selected from the solid culture plate used for the determination and inoculated into 1 ml of 2×YTAG medium. Culture the cells at 37°C with shaking overnight. The next day, 50 clones are identified by PCR, and the remaining cells are sent to a sequencing company for sequencing. Analyze the PCR and sequencing results, and calculate the VHH insertion rate and insertion diversity.

[0140] Experimental results:

[0141] 1. Lymphocyte Isolation and VHH Fragment Amplification

[0142] The number of alpaca lymphocytes isolated by cell counting was 3.6×10 7 Lymphocytes were divided into 10 tubes, 1 ml of TRIzol was added to each tube, total RNA was extracted according to the steps, long fragment cDNA was synthesized by two-step reverse transcription, and the leader region to FR2 region of VHH was amplified by primers Call001 and Calloo2. The bands were detected by gel electrophoresis. Figure 1 It was found that one round of PCR primers could amplify VHH fragments of traditional antibodies (900 bp) and heavy chain antibodies (700 bp).

[0143] After the first-round PCR products were ligated to the pMD19T vector, 20 monoclonal plaques were selected for sequencing analysis. The sequencing results were analyzed and compared using Vector NTI software (Vector NTI 11.5.1). Second-round PCR primers VHH2-F and VHH2-R were designed for secondary in vitro amplification of the VHH fragment. The second-round PCR amplification system was the same as the previous round PCR amplification system. The electrophoresis pattern of the second-round PCR products was as follows: Figure 2 As shown: The second-round PCR product is about 400 bp in size, with a single band, and the size is consistent with expectations.

[0144] 3. Construction of natural nanoantibody library

[0145] Gradient dilution method to detect the natural nanoantibody library capacity: 10-8 There are 4 monoclonal colonies on the dilution plate, 4÷(100×10 -8 )×30×10 3 =1.2×10 11 indivual( Figure 3 ), the abundance of the library is 10÷(100×10 -8 )×395×10 3 =3.95×10 13 / ml( Figure 4 ). All 58 bacterial liquid PCR samples were consistent with the VHH fragment length ( Figure 5 ), of the 54 sequenced samples, only two clones failed sequencing, not VHH fragments. NCBI analysis of the sequence of the qualified samples found that they were all VHH sequences. Therefore, the insertion rate of VHH fragments in the library can be calculated to be 100%, and the sequence diversity is 93%. Software analysis of the amino acid sequences corresponding to the 54 sample sequences and their structure shows that the natural nanobody library is divided into distinct constant and variable regions. The analysis results are as follows Figure 6 .

[0146] All of the above indicate that the natural nanoantibody library is an immune library with excellent characteristics and is suitable for screening specific nanoantibodies.

[0147] Example 2

[0148] The natural nanobody library prepared in Example 1 was subjected to the first round of panning to obtain α-toxin-VHH1, which was aliquoted and frozen at -70°C.

[0149] During the panning, 50 mM sodium carbonate / sodium bicarbonate buffer was used as the coating buffer, the coating concentration was 20 μg / ml, the coating volume was 2 ml, and the immunotube was coated with the α-toxin protein of Clostridium perfringens.

[0150] The washing method is as follows:

[0151] (1) 500 μl of the natural nanobody library prepared in Example 1 was inoculated into 100 ml of 2×YTAG medium and cultured with shaking at 200 rpm at 37°C for 1 hour until the OD600 reached 0.4;

[0152] 2) Add KM13 helper phage: add 100 μl KM13 helper phage to 100 ml of bacterial solution, incubate at 37°C for 30 minutes, and then shake incubate for 30 minutes;

[0153] 3) Centrifuge at 4000 × g for 10 minutes, remove the supernatant, resuspend the pellet in 100 ml of 2× YTAK medium, and culture at 30°C with shaking at 200 rpm overnight;

[0154] 4) The next morning, centrifuge the overnight culture at 11,000 × g at 4°C for 10 minutes. Transfer the supernatant to a new centrifuge bottle and add 20 ml of PEG / NaCl solution. Mix well and incubate on ice for 70 minutes.

[0155] 5) Centrifuge at 11,000 × g, 4°C for 30 minutes, discard the supernatant, and then centrifuge again for 2 minutes to completely aspirate the supernatant;

[0156] 6) Resuspend the pellet in 2.6 ml of PBS buffer, divide the pellet into two 1.5 ml centrifuge tubes, and centrifuge at 11,600 × g for 10 minutes.

[0157] 7) Recover the supernatant, named ZJ-α-toxin-VHH1, take 100 μl for titer determination, mix the remainder with 1.6 ml of MPBS solution, and incubate at room temperature for 1 hour to obtain a mixed solution (α-toxin-VHH1 treated with MPBS solution) for later use.

[0158] Coating protein treatment:

[0159] (1) The day after protein coating, pour out the liquid in the immune tube and wash the tube three times with PBS buffer.

[0160] (2) Fill each tube with MPBS, block at room temperature for 2 hours, and then wash the tube three times with PBS buffer.

[0161] (3) Add 2 ml of the mixture obtained in step (7) above to the immunotube, incubate at room temperature for 2 h, and then wash the tube 10 times with PBST solution and then wash the tube 10 times with PBS buffer.

[0162] (4) Add 2 ml of 100 mM TEA solution to each tube, shake gently at room temperature for 15 min to elute the bound phage, and then add 2 ml of Tris-HCl solution to neutralize.

[0163] (5) Transfer the eluted phage (named XT-α toxin-VHH1) to a 50 ml centrifuge tube and add 16 ml of TG1 bacterial solution with an OD600 of 0.4. Incubate in a 37°C water bath for 30 minutes to allow the eluted phage to infect the TG1 bacterial solution. (Add 4 ml of TG1 bacterial solution with an OD600 of 0.4 to the immunotube for infection and combine them for a total volume of 24 ml.)

[0164] (6) Take 100 μl of the bacterial solution for titer determination and centrifuge the remaining bacterial solution at 4000 g for 10 min.

[0165] (7) Resuspend the bacterial pellet in 1 ml of 2×YT medium, spread the resuspended bacterial solution on five 2×YTAG solid culture plates (150 mm plates), and incubate at 30°C overnight.

[0166] (8) The next day, the colonies grown on the plates were collected using 2×YT medium, and 60% glycerol was added to a final concentration of 15%. This was the primary library bacteria, named α-toxin-VHH1, and was aliquoted and frozen at -70°C.

[0167] Determine the titer of rescued phage: ZJ-α toxin-VHH1 was serially diluted from 10 -7 ~10 -13 ; Take 10 μl of phage from each dilution to infect 190 μl of TG1 bacterial solution with an OD600 of 0.4; take 100 μl of bacterial solution from each dilution to coat 2×YTAG solid culture plates and place them in a 30°C incubator for overnight culture; count the colonies on the assay plate and calculate the ZJ-α toxin-VHH1 titer.

[0168] Determine the titer of eluted phage: dilute XT-α toxin-VHH1 in a gradient from 10 -1 ~10 -5 ; Take 100 μl of bacterial solution for each dilution and spread it on 2×YTAG solid culture plates, place them in a 30°C incubator and culture overnight; count the colonies on the assay plate and calculate the XT-α toxin-VHH1 titer; then calculate the input-output ratio I / O of the first round of panning.

[0169] On the basis of one round of panning, two to four rounds of panning were carried out in sequence: the coating concentrations of Clostridium perfringens α-toxin protein were 10 μg / ml, 5 μg / ml, and 5 μg / ml respectively; the dilutions for the titer determination of rescued phages were 10 -7 ~10 -12 , 10 -8 ~10 -11 , 10 -8 ~10 -11 The titer of eluted phage was determined by dilution of 10 -1 ~10 -6 , 10 -1 ~10 -6 , 10 -1 ~10 -6 After the eluted phage was neutralized with Tris-HCl solution (1M, pH 7.4), 200 μl of phage was taken to infect 800 μl of TG1 bacterial solution with an OD600 of 0.4 (100 μl was taken for gradient dilution and the rest was used for bacterial preservation), and then 10 -3 ~10 -6 There are 4 dilutions in total. Each dilution is coated on three 2×YTAG solid culture plates (150 mm plates), with 100 μl of bacterial solution on each plate, and cultured at 30°C overnight; count the colonies on the culture plates, calculate the titer, mark the culture plates as plates, and place them in a 4°C refrigerator for use.

[0170] Screening of specific nanoantibodies:

[0171] Preparation of monoclonal phage supernatant: 192 monoclonal strains were selected from each plate and inoculated into two 96-well deep-well plates, each containing 200 μl of 2×YTAG medium. The plates were labeled E-1 and E-2 and incubated at 30°C with shaking. After 8 hours, 20 μl of the bacterial suspension from each well was inoculated into 180 μl of 2×YTAG medium and incubated at 37°C with shaking. The remaining bacterial suspension from the original plate was supplemented with 60 μl of 60% glycerol to a final concentration of 15% and stored frozen at -80°C. After incubation on the transferred plates with shaking for 1 hour, 20 μl of KM13 helper phage (60 μl KM13 + 12 ml 2×YTAG) was added to each well. The infection was allowed to proceed at 37°C for 30 minutes, followed by incubation at 37°C with shaking for 40 minutes. Centrifuge the deep-well plate at 1800 × g for 10 minutes, discard the supernatant, and add 400 μl of 2× YTAK medium to each well to resuspend the pellet. Incubate with shaking at 30°C overnight. The next day, centrifuge at a maximum speed of 2020 × g for 20 minutes. Aspirate 250 μl of phage supernatant from each well and transfer it to a new deep-well plate. Add 250 μl of blocking solution (PBS buffer containing 3% BSA) to each well and incubate at room temperature for 1 hour before use in indirect ELISA.

[0172] Identification of specific monoclonal phage: Phage supernatants were tested for reactivity with Clostridium perfringens α-toxin protein by indirect ELISA. The following method was used: 100 μl of α-toxin protein was coated into a 96-well microtiter plate at a concentration of 2 μg / ml, and the plate was incubated at 4°C overnight. The next day, the coating solution was discarded, and 100 μl of blocking solution was added to each well and blocked at 37°C for 1 hour. The blocking solution was discarded, and 100 μl of phage supernatant obtained from four rounds of selection, treated with blocking solution, was added to each well as the primary antibody and incubated at 37°C for 1 hour. The plate was washed 12 times with PBST. 100 μl of secondary antibody (HRP-M13 Antibody, dilution 1:10,000) was added to each well and incubated at 37°C for 1 hour. The plate was washed 12 times with PBST. Add 100 μl of chromogenic substrate to each well and incubate in the dark for 5-15 minutes. Terminate the reaction by adding 50 μl of stop solution to each well. Place the 96-well plate on a plate reader and read the OD450 absorbance. Analyze the ELISA results and determine the positive well numbers.

[0173] Phage supernatants corresponding to 192 monoclonal clones were tested for reactivity with the α-toxin protein of Clostridium perfringens using an indirect ELISA method. Based on the results of the indirect ELISA test, 20 monoclonal clones were selected, all of which showed good reactivity with the α-toxin protein of Clostridium perfringens. Cultures of these 20 monoclonal clones were sent to a sequencing company for sequencing.

[0174] Activity and affinity of nanobodies against Clostridium perfringens alpha-toxin

[0175] Construction of a prokaryotic expression recombinant plasmid: 5 ml of 2×YTAG medium was inoculated with a glycerol stock of the clone identified by sequencing. The plasmid was extracted using a plasmid miniprep kit and used as a template for prokaryotic expression. Primers for prokaryotic expression were then designed, with BamHI and EcoRI restriction sites introduced at the 5' and 3' ends, respectively. The nanobody VHH sequence was amplified using the designed primers and ligated into the pET28a prokaryotic expression vector via the aforementioned restriction sites. This construct was used to construct a prokaryotic expression recombinant plasmid for specific characterization of the nanobody against Clostridium perfringens alpha toxin.

[0176] Primers for prokaryotic expression:

[0177] (SEQ ID No. 2) F: CGGGATCCGAGTCTGGAGGAGGCTTGGT;

[0178] (SEQ ID No. 3) R: CGGAATTCTGAGGAGACGGTGACGTGG.

[0179] The screening steps are as follows

[0180] The recombinant plasmid and pET28a were transformed into the BL21(DE3) strain to obtain the corresponding nanobody expression strain. The nanobody was then induced to express as follows:

[0181] Culture the transformed bacterial solution on the plate overnight, pick a single colony on the plate the next day and culture it overnight.

[0182] (Pipette 10 μl of glycerol bacteria into 5 ml of Kan-resistant LB medium and culture at 37°C with shaking overnight;)

[0183] On the second day, 50 μl of bacterial solution was inoculated into 5 ml of Kan-resistant LB medium, two tubes were inoculated each, and cultured at 37°C with shaking until the OD600 reached 0.6;

[0184] IPTG was added to one tube of bacterial solution for induction (final concentration 0.8 mM), and IPTG was not added to the other tube as an uninduced control. The cells were cultured at 15°C with shaking overnight.

[0185] At the same time, a BL21 (DE3) empty strain control was made, and the empty strain control was cultured using LB medium without resistance.

[0186] SDS-PAGE identification of nanobodies:

[0187] The expression of nanobodies will be identified by SDS-PAGE, the specific method is as follows:

[0188] Pipette 1 ml of bacterial solution into a 1.5 ml centrifuge tube and centrifuge at 13000 rpm for 2 min;

[0189] The supernatant was discarded and the bacterial pellet was washed twice with PBS buffer;

[0190] Resuspend the bacterial pellet in 20 μl of PBS buffer, then add 5 μl of 5× protein loading buffer and boil the sample in boiling water for 5 minutes. Electrophorese the sample on a 10% polyacrylamide gel. After electrophoresis, stain the gel with Coomassie Brilliant Blue for 1 hour and then destain with a destaining solution.

[0191] Screening for Nanobodies with Neutralizing Activity against Clostridium perfringens α-toxin: The corresponding glycerol strains of the screened Nanobodies Nb.1, Nb.2, Nb.3, Nb4, and Nb.5 were inoculated into 5 ml of Kan-resistant LB medium, cultured with shaking at 37°C for 10 h, and then transferred to 500 ml of Kan-resistant LB medium. The culture was shaken at 37°C until the OD600 reached 0.6, at which time IPTG (final concentration 0.8 mM) was added to induce expression. The culture was shaken at 15°C overnight. The next day, the five Nanobodies were purified.

[0192] Affinity of Clostridium perfringens alpha toxin nanoantibody: ELISA plate was coated with 5μg / ml Clostridium perfringens alpha toxin; after BSA blocking, the purified and diluted Clostridium perfringens alpha toxin nanoantibody was used as the primary antibody and gradiently diluted to 5μg / ml, 2.5μg / ml, 1.25μg / ml, 0.625μg / ml, and 0.3125μg / ml for ELISA identification.

[0193] Identification of the purified product: The nanoantibody with the best affinity to Clostridium perfringens α-toxin was screened out by ELISA, and the antibody was identified by his tag using Western Blotting: after SDS-PAGE electrophoresis, it was transferred to NC membrane and directly labeled with his secondary antibody, and the antibody was displayed by development.

[0194] Application of nanoantibodies: Different sheep and alpaca feces were coated by ELISA, and the purified nanoantibodies were used as primary antibodies, and his-tag antibodies were used as secondary antibodies for labeling and color development to show the alpha toxin of Clostridium perfringens in different feces.

[0195] result:

[0196] ELISA screening results

[0197] Phage supernatants corresponding to 192 monoclonal clones were tested for reactivity with the α-toxin protein of Clostridium perfringens using an indirect ELISA method. Based on the results of the indirect ELISA test, 20 monoclonal clones were selected. These monoclonal clones all showed strong reactivity with the α-toxin protein of Clostridium perfringens and weak reactivity with BSA protein (Table 11). Cultures of these 20 monoclonal clones were sent to a sequencing company for sequencing.

[0198] Table 11 Clostridium perfringens alpha toxin monoclonal ELISA screening results

[0199]

[0200]

[0201] After ELISA screening, the 20 most positive clones were selected for sequencing. After sequencing and screening, 5 single clones met the requirements. The predicted amino acid sequences of these 5 single clones were:

[0202] Nb.1 (α-toxin-VHH1) amino acid sequence (SEQ ID No. 1):

[0203] ESGGGLVQPGGSLRLSCAASGFTFSRSRMYWVRQAPGKGLEWVSGITNAGISTGYADSVKGRFYVSRGNDESTLYLQMNSLKPEDTALYFCTRLGSGWEYDYWGQGTHVTVSS.

[0204] Nb.2 (α-toxin-VHH2) amino acid sequence (SEQ ID No. 8):

[0205] ESGGGLVQPGGSLRLSCVVSGSIFSINSLGWYRQTPGKKRELVAAVFASGSIQYTDSVKGRFTVTGDIEKKTVDLQMNSLKPEDTGVYFCNTDVDDDGSGFWRPRWGQGTQVTVSS.

[0206] Nb.3 (α-toxin-VHH4) amino acid sequence (SEQ ID No. 9):

[0207] ESGGGLVQSGGAVKLSCAASGFTVHTSAVGWFRQAPGREREGIACIDKTGRETNYADSVKGRFTISKDKDMNTVSLQMNSLKPEDTGVYYCAIEALRGRRITADQAKCVSRDEYDYWGQGTEVIVSS.

[0208] Nb.4 (α-toxin-VHH5) amino acid sequence (SEQ ID No. 10):

[0209] ESGGGLVQSGESLTLSCKAHGFAMDWYTIGWFRQASGKEREALSCIDNRNGNTHYADSAKGRFAISKDSADDTVYLKMSDLKPEDTATYICSAREWVDCGDTYYSVPISQFSFWGQGTHVIVSS.

[0210] Nb.5 (α-toxin-VHH6) amino acid sequence (SEQ ID No. 11):

[0211] ESGGGLVQPGGSLRLSCAASGSILRINTMGWYRQAPGKQRELVAGITSGGSTNYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCNADQWARRYWGQGTQVTVSS.

[0212] Affinity detection of nanoantibodies against Clostridium perfringens α-toxin:

[0213] After expressing the five clones, affinity testing revealed that the following affinity tests revealed that the sensitivity of the Clostridium perfringens α-toxin nanobody, Nb.1, to the α-toxin protein was 0.625 μg / ml; the sensitivity of the Clostridium perfringens α-toxin nanobodies, Nb.2 and Nb.3, to the α-toxin protein was 1.25 μg / ml; and the sensitivity of the Clostridium perfringens α-toxin nanobodies, Nb.4 and Nb.5, to the α-toxin protein was weaker, at only 5 μg / ml. Western blotting of the purified antibodies revealed a His-positive band with a molecular weight of approximately 15 kDa, consistent with the size of the nanobody.

[0214] ELISA test results for chicken and alpaca feces:

[0215] Through ELISA testing, it was found that among the 30 chicken feces to be tested, 23 were positive; among the 18 alpaca feces to be tested, 17 were positive; the above results show that the purified Clostridium perfringens α-toxin nanoantibody can be used for fecal detection, with a detection sensitivity of 2 μg / ml (see Table 12).

[0216] Table 12 Detection results of Clostridium perfringens α-toxin in chicken and alpaca feces

[0217]

[0218]

[0219] From the above examples, it can be seen that the Clostridium perfringens alpha toxin nanoantibody provided by the present invention can specifically bind to the Clostridium perfringens alpha toxin protein, with a sensitivity to the protein of 0.625 μg / ml; and can detect 2 μg / ml of α in chicken and alpaca feces by ELISA.

[0220] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. A Clostridium perfringens alpha toxin nanobody, characterized in that The amino acid sequence of the Clostridium perfringens alpha toxin nanobody is shown in SEQ ID No.

1.

2. Use of the Clostridium perfringens alpha-toxin nanobody according to claim 1 in the preparation of a reagent for detecting Clostridium perfringens alpha-toxin in animal feces.

3. The use according to claim 2, characterized in that The animals are chickens and / or alpacas.

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