TWI759750B - Preparation of antivenom using recombinant toxins thereof - Google Patents

Abstract

The present invention relates to recombinant DsbC-neurotoxin chimera proteins and recombinant snake alpha-neurotoxins, preparation methods and application thereof. Further, the present invention relates to preparation of antivenom using recombinant toxins and thereof.

Description

A kind of method for making antivenom with recombinant toxin

The present invention relates to the preparation of antivenoms and methods thereof using recombinant toxins.

Snake envenomation has been a neglected public health problem, causing more than 110,000 bites in Southeast Asia every year. Among these bites, poisoning by cobra (Naja sp.) is one of the deadliest, accounting for about 17%, 12%, 34%, 16% and 17%. The Zhoushan cobra (Naja atra), also known as the Chinese cobra, mainly lives in China, Vietnam and Taiwan, and the Bengal cobra (Naja kaouthia) is mainly native to Bangladesh, Thailand, Vietnam and Malaysia, among other Species such as: Siamese cobra (Naja sumatrana) in Vietnam and Thailand, Sumatran spitting cobra (Naja sumatrana) in Malaysia and Sumatra, also known as Equatorial spitting cobra, and Malay spitting cobra in Java and southern Indonesia Cobra (Naja sputatrix), also known as Java spitting cobra (Javen spitting cobra).

In the past few decades, through the results of venom proteomics, the components of the snake venom have been analyzed and the most clinically relevant toxins have been identified as the three-fingered toxin family, the third of which is the three-fingered toxin family. Type I and Type II alpha-neurotoxins are antagonists of muscle/neuron acetylcholine receptors (AchRs) and are therefore known as It is the deadliest toxin of cobra venom; in addition, it is conceivable that the entire toxicity of cobra venom can be attributed to any one alpha-neurotoxin. For example: the neurotoxicity of N.atra and N.sputatrix venoms is thought to be the effect of short-chain neurotoxins, while Thai monocled cobra (N.kaouthia) and Indian common cobra (N.naja) is caused by the action of long-chain neurotoxins. In the literature, the neutralizing ability of snake antivenoms can be judged by the ability of antibodies to bind to any neurotoxin (such as: N.kaouthia, N.naja and N.atra), so consistent with the above point of view, the Neurotoxins are the most important cause of death.

Due to lack of government attention and support, most countries in Southeast Asia are unable to produce regional antivenoms, so only polyvalent antivenoms from the closest related species, heterologous snake venom, are used. Although effective in general, the paraspecificity of antivenom is greatly hindered by the weak immunogenicity and intra-species diversity of the venom, which compromises effectiveness. Therefore, many efforts have been made to optimize immunogen mixtures or to develop recombinant monoclonal antibodies/antivenoms, aiming to design a broad-spectrum, pan-Asian polyvalent antivenom (broad-spectrum, pan-Asian polyvalent antivenom). antivenom), which is one of the strategies proposed by the Global Snakebite Initiative to treat venomous snakebites.

Recently, a pan-specific antiserum capable of neutralizing 16 medically important cobra families (elapids) in Asia was successfully generated by using multiple venom and toxin fractions as immunogens. The proof-of-concept inclusion of multiple major toxins in an immunogenic cocktail is a simple strategy to overcome the poor paraspecificity of cobra antivenom, but the collection of venom is rather difficult.

To date, E. coli remains the most popular "biofactory" for many proteins expressing drug targets. Escherichia coli B is an ideal host for the production of genetically engineered proteins, Due to its features such as lack of proteolytic enzymes, low acetate production at high glucose concentrations, and enhanced permeability, BL21(DE3) also carries a recombinant phage with T7 RNA polymerase gene and DE3 , therefore, under the control of the T7 promoter, the genes of the vector can be expressed in large quantities.

By co-expressing a thio-disulfide isomerase, a corrected folded and biologically active disulfide-rich protein (eg, three-finger toxin) can be expressed in cells, The protein is a protein that is difficult to express in the cytoplasm of E. coli, thus making the method for preparing natural toxins scalable and cost-effective. Therefore, many of the major toxins found in snake venom of the Cobra family have been successfully recombinantly expressed and purified in E. coli systems, which means that E. coli-derived recombinant toxins have potential as alternative sources of immune antigens.

Due to the diversity of snake venom, there are 60 sequenced α-neurotoxins (α-NTX) in the Cobra genus stored in the UniProt database so far, including: 29 short-chain NTX (SNTX), 16 Long chain NTX (LNTX) and 15 weak NTX. Since α-NTXs have been tailored to maintain a retained and fixed three-fingered scaffolds through four or five pairs of disulfide bonds throughout evolution, many of their functions or structures have been preserved. Stability-related retained epitopes; accordingly, cross-neutralization of α-NTX has been reported through heterologous antivenoms belonging to the same subfamily (eg, long-chain neurotoxins) or with The same α-NTX produced antivenom for cross identification. Nonetheless, it is speculated that antibodies in currently used antivenoms rarely cross-recognize these two types of α-NTX. Considering that short-chain α-NTX and long-chain α-NTX play a crucial role in integrating the toxicity of the whole snake venom, therefore, having an effective pan-antivenom to neutralize these two types of α-NTX is important.

In one aspect, the present invention relates to a recombinant DsbC-NTX chimeric protein, wherein the recombinant DsbC-NTX chimeric protein is composed of a DsbC-Linker-NTX sequence, wherein: DsbC is a DsbC protein sequence; Linker is a linker protein sequence , wherein the connexin sequence comprises a 6xHis repeat and a TEV proteolytic site sequence is attached after the 6xHis repeat; and NTX is a recombinant snake alpha-neurotoxin sequence, wherein the recombinant snake alpha-neurotoxin sequence is selected from From a SEQ ID NO.1 or a SEQ ID NO.6.

In another aspect, the present invention relates to a method for preparing a recombinant snake α-neurotoxin, comprising the following steps: (a) expressing the recombinant DsbC-NTX chimeric DsbC-NTX as described in item 1 of the scope of application in a bacterial protein expression system (b) purifying the recombinant DsbC-NTX chimeric protein with a metal affinity chromatography; (c) cleaving the purified recombinant DsbC-NTX chimeric protein with a TEV protease; and (d) using a cationic The recombinant snake alpha-neurotoxin free of DsbC sequence and connexin sequence was repurified by exchange chromatography.

In another aspect, the present invention relates to a recombinant snake alpha-neurotoxin for the production of an immunogen for the production of a cobra effective antivenom, wherein the recombinant snake alpha-neurotoxin is represented by SEQ ID NO.1 or SEQ ID NO.6 composed of sequences

Figure 1. Multiple sequence alignment of major (A) long-chain NTX and (B) short-chain NTX in Naja genus. (a: accession number in UniProt database, b: percentage (%) of relative content in corresponding venoms reported in literature, c: same as P01391 (for LNTXs) or The percentage of identical or similar amino acid sequences at the same position compared to the sequences of P60770 (for SNTXs). Identical amino acids in all sequences of the same subfamily are shown in red with a bright yellow background; while similar amino acids are shown in black with a blue or green background.

Figure 2. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) of recombinant short-chain neurotoxin and long-chain neurotoxin. Protein samples of (A) recombinant short-chain neurotoxin and (B) long-chain neurotoxin were separated by 4-12% reducing gradient gel and stained with Coomassie blue R-250 (left panel) Or transferred to PVDF membrane for analysis by immunoblotting with BAV anti-venom and NPAV anti-venom antibodies (right panel). Lane M: pre-stained protein marker (kDa), lanes 1 and 5: total lysate after induction, lanes 2 and 6: total lysate before induction, lanes 3 and 7: soluble fraction, lanes 4 and 8: Purified recombinant neurotoxin.

Figure 3. CD spectra of native, recombinant and reduced forms of neurotoxins. The spectra of the native toxin are shown as black lines, the spectra of recombinant toxins are shown as red lines, and the spectra of reduced recombinant toxins pretreated with 30 mM dithiothreitol are shown as dashed lines, and the results are shown as the average of five scans , where the scanned wavelength range is from 190 to 260 nm. SNTX: native short-chain NTX, rSNTX: recombinant short-chain NTX, RSNTX: reduced short-chain NTX.

Figure 4. MS ² mass spectrum of dimethyl-labeled and disulfide-linked peptides of long-chain alpha-neurotoxins. An ion with a mass-to-charge ratio (m/z) of 616.2637 (2+) was observed in the digests of (A) rLNTX and (B) native LNTX representing the trypsin fragment of TWC ₂₆ DAFC ₃₀ SIR with C ₂₆ C ₃₀ are connected by disulfide bonds. The only enhanced a1 ion, 106.1212, indicates that the fragment consists of a single chain in which threonine (T) is the N-terminal amino acid. The ions observed from digests of rLNTX (C) and native LNTX (D) with a mass-to-charge ratio (m/z) of 846.9937 (7+) represent C ₃ FITPDITSK-DC ₁₇ PGHVC ₂₄ YTK-VDLGC ₄₁ AATC ₄₄ PTVK-TGVDIQC ₅₄ C ₅₅ STDNC ₆₀ Trypsin fragment of PFPTR in which all eight cysteines are oxidized. The four enhanced a1 ions 106.0628, 120.0963, 104.1446 and 106.1249 illustrate that the peptide consists of four chains, of which cysteine (C), aspartic acid (D), valine (V) and threonine (T) is the first N-terminal amino acid.

Figure 5. MS ² mass spectra of dimethyl-labeled and disulfide-linked peptides of short-chain alpha-neurotoxins. Ions with mass-to-charge ratios 899.94 (6+) and 909.39 (6+) were observed from digests of (A) _rSNTX and (B) native _SNTX representing _{GLEC4HNQQSSQTPTTTGC18SGGETNC25Y - GC42GC44PSVK-} NGIEINC ₅₅ C ₅₆ TTDRGC-SG ₆₁ CKCC-CNCC-C ₂₄ CC ₆₀ NN in which all eight cysteines are oxidized. The four enhanced a1 ions 118.1596, 62.1038, 119.1195 and 106.0682 indicate that the peptide consists of four chains, including leucine (L), glycine (G), asparagine (N) and half Cystine (C) is a fragment of the first N-terminal amino acid. The lack of a1 ion (118.1534) in the above figure is due to the fact that the first amino acid of recombinant SNTX is glycine rather than leucine.

Figure 6. (A) NMR spectra of rSNTX (blue) and native SNTX (red); (B) rLNTX (blue) and native LNTX (red).

Figure 7. Toxin-specific antibody responses and survival curves of immunized mice. (A) Serum antibody responses to short-chain and long-chain neurotoxins in immunized mice, results are quantified and presented as mean ± SD (n=12); (B) at 3 x _LD50 N.Atra venom (LD ₅₀ = 0.67 μg/g) after challenge, the survival curve of mice immunized with double recombinant toxin (●), double venom (▲) or PBS (■); (C) Bengal cobra (N) with 3 times the _LD50 dose. .siamensis) venom (LD ₅₀ =0.15 μg/g) challenged mice survival curve; (D) after challenge with 3 times LD ₅₀ dose of Thai cobra (N. siamensis) venom (LD ₅₀ =0.19 μg/g) Survival curves of mice.

The present invention relates to a recombinant DsbC-NTX chimeric protein. The recombinant DsbC-NTX chimeric protein is composed of a DsbC-Linker-NTX sequence, wherein: DsbC is a DsbC protein sequence; Linker is a linker protein sequence, wherein The connexin sequence comprises a 6xHis repeat and a TEV proteolytic site sequence followed by the 6xHis repeat; and NTX is a recombinant snake alpha-neurotoxin sequence, wherein the recombinant snake alpha-neurotoxin sequence is selected from a SEQ ID NO.1 or a SEQ ID NO.6; wherein in the sequence of the SEQ ID NO.1, X ₉ =I or V; X ₁₂ =K, T or Q; X ₁₃ =D or I; X ₁₅ =P or A; _X28 =A, G or N; _X31 =S or A; _X32 =I or S; _X35 =K or R; _X38 =D or E; _X49 =K or R; _X50 = X ₅₅ =Q or K; X ₆₉ =K, P or N; and X ₇₀ - ₇₁ =RP or none; and wherein X ₁ =L or M in the sequence of SEQ ID NO. 6; X ₂ X ₄ =H or Y; X ₅ =N, D or K; X ₉ =S or L; X ₁₁ =T, A, P or F; X ₁₃ =T or I; X _15-16 =TG, KT or KV; _X18 =S or P; _X20 =G or none; _X22 =T or K; _X28 =R, W, S or Q; _X30 =R or S; _X31 =D or G; _X35-37 =YRT, TII or _YRI ; X45=S or K; _X48 =N, P or K; _X50-52 =IEI, VNL or VKI; _X56 and _X57 =T or R ; X ₅₉ =R or K; X ₆₂ =N or R.

In one aspect of the invention, the connexin sequence is a 6xHis repeat followed by a TEV proteolytic site sequence, the connexin sequence is located after the DsbC protein sequence and before the recombinant snake alpha-neurotoxin sequence .

In another aspect of the present invention, the recombinant snake α-neurotoxin sequence consists of a sequence selected from SEQ ID NO.1, wherein the SEQ ID NO.1 and SEQ ID NO.2 have at least 80% of the sequence Identity and 90% sequence similarity.

In one embodiment, the above-mentioned recombinant snake α-neurotoxin sequence is selected from SEQ ID Nos. 2-5. In a preferred embodiment, the recombinant snake α-neurotoxin is selected from SEQ ID NO.2.

In another aspect of the present invention, a recombinant snake alpha-neurotoxin sequence consisting of a sequence selected from SEQ ID NO. 1, wherein the SEQ ID NO. 1 and SEQ ID NO. 6 have at least 69% sequence identity and 75% sequence similarity.

In one embodiment, the above-mentioned recombinant snake α-neurotoxin sequence is selected from SEQ ID Nos. 7-16. In a preferred embodiment, the recombinant snake α-neurotoxin is selected from SEQ ID NO.7.

In one aspect, the present invention relates to a method for preparing recombinant snake α-neurotoxin, which comprises the following steps: (a) expressing the recombinant DsbC-NTX chimera described in item 1 of the application scope in a bacterial protein expression system protein; (b) purifying the recombinant DsbC-NTX chimeric protein with a metal affinity chromatography; (c) cleaving the purified recombinant DsbC-NTX chimeric protein with a TEV protease; and (d) with a cation exchange The recombinant snake alpha-neurotoxin free of DsbC sequence and connexin sequence was repurified by chromatography.

In one embodiment, wherein the bacterial protein expression system uses an E. coli BL21(DE3) strain to produce the recombinant DsbC-NTX chimeric protein.

In one embodiment, the metal affinity chromatography method is a Ni-NTA affinity chromatography method. In another embodiment, wherein the cation exchange chromatography is a sulfopropyl strong cation exchange chromatography.

In the above method, the method further comprises passing the purified protein through a Q Steps to remove endotoxins on Sepharose Fast Flow columns.

In another aspect, the present invention relates to a recombinant snake alpha-neurotoxin for the production of an immunogen for the production of a cobra effective antivenom, wherein the recombinant snake alpha-neurotoxin is represented by SEQ ID NO.1 or SEQ ID NO.6 In the sequence of SEQ ID NO.1, X ₉ =I or V; X ₁₂ =K, T or Q; X ₁₃ =D or I; X ₁₅ =P or A; X ₂₈ =A, G or N; _X31 =S or A; _X32 =I or S; _X35 =K or R; _X38 =D or E; _X49 =K or R; _X50 =T or P; _X55 =Q or K; _X69 =K, P or N; and _X70-71 =RP or none; and wherein in the sequence of SEQ ID NO. _{6 ,} X1 ₌ L or M; X2=E or I; _X4 =H or Y; X ₅ =N, D or K; X ₉ =S or L; X ₁₁ =T, A, P or F; X ₁₃ =T or I; X _15-16 =TG, KT or KV; X ₁₈ =S or P; _X20 =G or none; _X22 =T or K; _X28 =R, W, S or Q; _X30 =R or S; _{X31=D or G; X35-37 =} YRT , TII or _YRI ; X45=S or K; _X48 =N, P or K; _X50-52 =IEI, VNL or VKI; _X56 and _X57 =T or R; _X59 =R or K; X ₆₂ = N or R.

In one aspect, the above-mentioned recombinant snake α-neurotoxin, wherein the SEQ ID NO. 1 and SEQ ID NO. 2 have at least 80% sequence identity and 90% sequence similarity. In one embodiment, the recombinant snake alpha-neurotoxin sequence is selected from SEQ ID NOs. 2-5. In another aspect, wherein the recombinant snake alpha-neurotoxin sequence is selected from SEQ ID NO.2.

In one embodiment, the recombinant snake alpha-neurotoxin sequence is selected from SEQ ID NOs. 7-16. In a preferred embodiment, the recombinant snake α-neurotoxin sequence is selected from SEQ ID NO.7.

Example

The practical application range of the present invention can be further proved by the following specific examples around. It is only a preferred embodiment of the present invention, and does not limit the scope of the present invention. Therefore, any simple changes and modifications made in accordance with the scope of the present invention and the contents of the description of the present invention are still covered by the scope of the present invention.

Considering both the relative medical importance and relative content of snake venoms, one type I α -neurotoxin and one type I α-neurotoxin were selected from a total of 13 major α-neurotoxins (4 LNTX and 9 SNTX) in Asian cobra venom Type II alpha-neurotoxins were soluble as designed fusion DsbC proteins (Tables 1 and 2).

Table 1. Sequence listing of long alpha-neurotoxins (LNTXs) identified in Asian cobra venom.

Table 2. Sequence listing of long alpha-neurotoxins (LNTX) identified in Asian cobra venom

The results of structural and functional analysis can prove that recombinant NTX is a correctly folded structure and has a biologically active form, which can mimic natural toxins. Antisera collected from two rabbits immunized with recombinant NTXs also exhibited strong to moderate cross-neutralization ability against the venoms of N.atra, N.kaouthia, and N.siamensis. In summary, the present invention discloses a cost-effective, scalable and robust production platform that improves formulations for the production of anti-Cobra antivenoms by using recombinant toxins as immunogens.

Example 1. Design, bacterial expression and purification of alpha-neurotoxins.

Materials and Method

Expression of rDsbC-NTX chimera in Escherichia coli

DsbC (Disulfide bond C) is a prokaryotic disulfide bond isomerase. The formation of native disulfide bonds plays an important role in the correct folding of proteins and in stabilizing the tertiary structure of proteins. DsbC is one of the six proteins of the Dsb family in prokaryotes. DsbC interacts with improperly folded proteins to correct non-native disulfide bonds. Tobacco etch virus (TEV) protease is a highly specific cysteine protease that recognizes the amino acid sequence Glu-Asn-Leu-Tyr-Phe-Gln-(Gly/Ser) and Cleavage between Gln and Gly/Ser residues. In addition, the TEV protease recognition site is tolerant to all other residues at its C-terminus (except proline), confirming that the removal of the tag does not require the addition of any non-native residues at the N-terminus of the recombinant peptide that might affect activity molecular. Therefore, TEV protease can be used to remove marker molecules such as polyhistidine in fusion proteins for affinity purification. To express the rDsbC-NTX chimeric protein, the optimized dsbC gene sequence (651 bp) and the gene encoding cobra alpha-neurotoxin (cobrotoxin, 186 bp, P60770 or alpha-elapitoxin-Nk2a, 213 bp) base, P01391) were synthesized and linked together by a 39-nucleotide linker sequence (5'-catcatcaccaccaccacgagaacctgtactttcagggc-3') that would translate a histidine marker molecule (6x histidine) and The TEV proteolytic site (ENLYFQG) was then translated. However, the last amino acid of the TEV proteolytic site can be replaced with any other amino acid other than proline. The resulting gene was treated with NdeI and BamHI restriction enzymes and cloned into pET-9a plastids under the control of the T7 promoter.

The resulting plastids named pDsbC-His-SNTX and pDsbC-His-LNTX were transformed into BL21(DE3) or Rosetta ^(TM) (DE3) cells and cultured in 2L standard flasks containing 34mg/L Conmycin Heterologous expression was performed with 800 mL of LB growth medium and the flask was placed at 37°C with shaking at 180 rpm. Protein expression was induced by adding 1 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) when bacterial growth entered exponential phase (OD600=0.5-0.6). Three hours after induction, cells were pelleted and redissolved in PBS and disrupted with an ultra-high pressure homogenizer (French press) at 27 Kpsi (Constant Systems, Daventry, UK). The clarified supernatant containing the rDsbC-NTX chimeric protein and bacterial endogenous cytoplasmic proteins was collected by ultracentrifugation (32,000 rpm, 40 min, 4° C.), followed by filtration and downstream purification.

Purification of recombinant alpha-neurotoxin

First, the rDsbC-NTX chimeric protein was isolated from soluble cell lysates by passing through an immobilized metal affinity chromatography (IMAC) at 4°C. The lysate was placed into an open chromatography column with Ni-NTA (Ni-nitrilotriacetic acid) agar colloid (QIAGEN) and washed with 10 column volumes (CV) of wash buffer (PBS, 40 mM imidazole, pH = 7.4) The column was washed and then eluted by gravity with 3 CV of elution buffer (PBS, 500 mM imidazole, pH = 7.4). The flushed eluate was dialyzed against PBS and lysed with TEV (1:3, w/w) in a solution containing 0.1 mM DTT at 16°C overnight. The next day, the finished mixture was dialyzed 3 times against 20 mM sodium phosphate buffer (pH=5.8). The dialyzed mixture was added to a chromatographic column of Ni-NTA resin equilibrated with 20 mM sodium phosphate buffer to adsorb TEV with polyhistidine, DsbC protein fragments and pro-chimeras that were not cleaved by TEV enzymes. protein.

In contrast, the recombinant alpha-neurotoxin was mainly present in the effluent and wash fractions, and the two fractions were collected and pooled together and added to another chromatography tube containing sulfopropyl strong cation exchange resin Column (GE Healthcare). with 3CV of wash buffer (25mM After washing with ammonium bicarbonate, ABC), the recombinant alpha-neurotoxin was eluted with 3 CV of elution buffer (25 mM ABC buffer, 500 mM NaCl) and dialyzed against 25 mM ABC buffer overnight at 4°C. The dialyzed eluate was added to Q Sepharose Fast Flow resin (GE Healthcare) pre-equilibrated with 25 mM ABC buffer to remove endotoxins. The effluent was collected and lyophilized and stored at -20°C until use. The endotoxin residue rSNTX in the final recombinant toxin was 0.03-0.30Eu/mg and rLNTX was 0.35-3.50 Eu/mg.

result

First, data mining was performed on recently published articles describing the main snake venom data and relative content of Cobra genus and its identity, in which the results of the protein sequence alignment are summarized in Figure 1. As shown in Figure 1, there are mainly 9 different forms of type I α -neurotoxin and 10 different forms of type II α-neurotoxin in Asian cobra venom. For example: the venoms of N. kaouthia and N. siamemsis contain very high levels of α-elaptoxin-Nk2a (33.3% and 22.4%, respectively, accession number: P01391), the most abundant LNTX identified to date. More than 80% identity and 90% similarity to the minor content LNTX found in N. sumatrana, N. naja and N. melanoleuca venoms. On the other hand, the venom of N.atra contains a high content of cobrotoxin (~12%, accession number: P60770), which is the most abundant SNTX and is 75% similar to the remaining SNTX of other cobra species. Therefore, the genes for the two most abundant neurotoxins were synthesized separately and fused to the dsbC gene separated by a His-tag and a TEV proteolytic site (ENLYFQG) and cloned into pET-9a plastids. Further, in E. coli expressed in.

After cell rupture, the recombinant fusion protein was collected from the clarified supernatant and fixed using Metal affinity chromatography (IMAC) was used for separation. After TEV cleavage, rSNTX and rLNTX were confirmed to carry an additional glycine residue at the N-terminus by N-terminal sequencing data. The first three amino acid sequences of rSNTX are GLE, and the first three amino acid sequences of rLNTX are GIR. In addition, rSNTX and rLNTX were successfully separated from TEV, DsbC and the original chimeric protein again by IMAC and further purified by ion exchange resin to remove impurities and lipopolysaccharide from E. coli. The purity and immunorecognition properties of the SDS-PAGE separated reduced form of the recombinant toxin were detected by Western blotting with Coomassie blue staining and equivalent antivenom (Figure 2).

Meanwhile, the purity and hydrophobicity of the protein were analyzed by reversed-phase chromatography, and it was observed that the natural toxin and the corresponding recombinant toxin had similar retention times. In summary, no protein refolding is required, and high-purity recombinant anti-short-chain/long-chain neurotoxins recognized by antivenom antibodies are obtained at the end of the purification, where the yield per liter of bacterial culture is 10 mg/L ( rSNTX) and 2.5 mg/L (rLNTX).

Example 2. Toxicity and structural characterization of neurotoxins.

Materials and Method

circular dichroism spectrum

Spectra were read in the wavelength range of 190 to 260 nm at 20°C in a 0.5 mm path length quartz tube using a circular dichroism spectrometer (JASCO J-815). All protein samples were prepared at protein concentrations of 7-90 μM in distilled water. After subtracting the value of the blank group, calculate the ellipticity from millidegrees (mdeg) according to the following formula (Ellipticity, [θ], deg x cm2 x dmol-1) = (millidegrees x average residual weight)/(path length (mm) ) × protein concentration (mg/ml)), Wherein average residue weight=(molecular weight)/(residue number-1). The content of α-helices and β-sheets was estimated by the web server K2D3 (Louis-Jeune C, et al. Proteins 2012, 80, 374-381, 10.1002/prot.23188).

result

The neurotoxicity of recombinant short/long chain neurotoxins can be directly assessed by measuring their lethal activity in mice by intraperitoneal injection. Various doses of the toxin were diluted in PBS, and then injected intraperitoneally into different groups of mice (19-22 g, body weight, n=6), and the number of survival was observed 48 hours after injection, and the results of the survival rate were calculated and calculated. shown in Table 3. The lethal dose (LD50) mean values of rSNTX and rLNTX were 0.25 μg/g (0.19-0.33, 95% confidence interval, C.I) and 0.21 μg/g (0.18-0.24, 95% confidence, respectively, according to the survival rate at the corresponding treatment doses) interval, C.I), both were slightly higher than the LD50 of the corresponding natural toxins (0.23 μg/g for SNTX and 0.15 μg/g for LNTX). Acute toxicity showed that both E. coli-derived short-chain/long-chain neurotoxins could fold correctly and cause sudden death in mice after antagonizing AChRs.

Table 3. Survival of mice challenged with native or recombinant NTXs.

As has been demonstrated, neutralizing monoclonal antibodies recognize mainly conformational rather than linear cobrotoxin epitopes. The major toxins responsible for cardiac arrest and cytotoxicity in snake venoms, where E. coli-derived short/long chain NTXs were used as immunogens, were identified Their structure obviously matters. First, the circular dichroism (CD) spectra of the recombinant toxin and its native toxin were compared, revealing two CD spectra of short-chain NTX and long-chain NTX, the former showing two positive ellipticities at 228.5 and 199.5 nm , while the latter is characterized by a strong ellipticity at 211.5 nm. Compared with the corresponding native toxins, both E. coli-derived NTXs showed similar CD spectra, indicating their identity in secondary structures. More specifically, native and recombinant sNTX predicted 54.5% and 53.4% anti-parallel β-strands, and native and recombinant LNTX predicted 9.8% and 9.2% α-helix and 33.5% and 34.7% antiparallel beta strands (Figure 3). In addition, it is speculated that the cysteine of both recombinant toxins may exist in the oxidized form because the structure is completely destroyed after reduction.

In conclusion, the two most abundant NTXs identified in cobra venom, including: α-elapitoxin-Nk2a and cobrotoxin, were recombinantly produced in biologically active forms and exhibited typical secondary structures. Since the finger-shaped structures of neurotoxins are connected through disulfide bonds (DB), and the integrity of the connections is not only important for their toxicity but also necessary for the induction of structure-dependent antibodies, it is worth investing more in energy to study whether recombinant toxins have similar natural disulfide bonds.

Example 3. Structural analysis of recombinant neurotoxins.

Materials and Method

Enzymatic digestion and dimethyl labeling of disulfide-linked peptides

The procedure to determine the disulfide bonds of neurotoxins was performed according to the protocol of Huang et al. previously described (Anal Chem 2014, 86, 8742-8750, 10.1021/ac501931t). Briefly, 40 μg neurotoxin was dissolved in 35.6 μl of 50 mM sodium acetate (pH=6.0) containing 0.2% (w/v) RapiGest ^™ SF surfactant (Waters, MA, USA). Afterwards, the protein was treated with 10 mM N-ethylmaleimide (NEM) for 30 minutes at room temperature to block any free cysteine. Digestion was performed overnight at 37°C with a trypsin/protein ratio of 1:50 (w/w). The next day, protein digests were diluted twice with 100 mM sodium acetate (pH=5.0) and then dimethyl-labeled by adding formaldehyde D2 to 4% (w/v) and sodium cyanoborohydride to 20 mM. After 30 minutes at room temperature, an equal volume of 1 M HCl was added to the digested mixture, followed by incubation at 37°C for 30 minutes and centrifugation at 14,000 rpm for 5 minutes to remove RapiGest ^™ SF. The clear supernatant containing the labeled peptide mixture was carefully collected and analyzed by ESI-Q-TOF mass spectrometer coupled to a UPLC system (Waters, MA, USA).

LC-MS/MS analysis of dimethyl-labeled and disulfide-linked peptides

An ESI-Q-TOF mass spectrometer (Synapt HDMS) coupled to a nanoACQUITY UPLC system (Waters, MA, USA) was used for disulfide-linked peptide analysis. LC separation was performed on a reverse phase C18 column (75 μm x 100 mm, 1.7 μm, Waters, MA, USA) and a trap column (180 μm x 20 mm, 5 μm, Waters, MA, USA) ) on. Distilled water with 0.1% formic acid (FA) and acetonitrile with 0.1% FA (JT Baker, Phillipsburg, NJ, USA) were used as mobile phases A and B, respectively, with gradient settings as follows: 30 min over a total separation time of 60 min 1% B to 50% B in 30-40 minutes, 50% B to 65% B in 30-40 minutes, 65% B in 5 minutes. The full scan was set to the m/z range of 400-1600, and six channels were used for simultaneous MS/MS detection. The raw data was processed into a peak list file using Proteome Discoverer 1.3 prior to RADAR searches using RADAR 3.0 ( http://www.mass-solutions.com.tw/ ). Searches were performed using neurotoxin sequences (P60770 and P01391) with the following parameters. In addition to KP and RP, enzymatic cleavage at the C-terminus of lysine and arginine allows up to two missed cleavages. All amines are deuterium labeled. Mass tolerances were set to ±0.01 Da and ±0.2 Da for a1 and total mass, respectively. The signal strength cutoff was set at 10% and the maximum number of chains was defined as 4.

Immunoreactivity of antisera to toxins and venoms

Antiserum reactivity to toxin and venom was tested by indirect enzyme-linked immunosorbent assay (ELISA) following previously described procedures. Briefly, serum serially diluted in PBS buffer containing 1% bovine serum albumin was added to 96-well microtiter plates (Corning®, USA) coated with 0.5 μg of toxin or venom. After 2 hours at room temperature, they were washed four times with PBS buffer containing 0.05% (v/v) Tween 20 (PBST) and mixed with goat anti-mouse or horseradish peroxidase conjugated Goat anti-rabbit IgG was incubated for 10 minutes, followed by development for 15 minutes using SureBlue Reserve ^™ TMB (3,3',5,5'-tetramethylbenzidine) microwell peroxidase substrate (KPL, Gaithersburg, MD, USA). The reaction was stopped by adding 50 μl of sulfuric acid (2N H ₂ SO ₄ ), and the absorbance at 450 nm (OD 450 ) was measured by a microplate analyzer (SUNRISE, TECAN). ELISA intra-point titers were obtained from titration curves by interpolation and defined as the reciprocal of the serum dilution that maintained an OD450 value of 0.3. All measurements were repeated in triplicate and results are presented as mean ± standard deviation (SD).

result

Producing properly folded proteins in E. coli cells has always been a difficult task, especially for DB-rich proteins, which tend to aggregate and precipitate in inclusion bodies due to mismatches or confusion of disulfide bridges ). Interestingly, with the help of DsbC and expression in specific bacterial strains, both recombinant NTXs containing four to five pairs of DB could be partially produced in soluble form.

A method utilizing dimethyl labeling combined with LC-MS/MS and the RADAR (Rapid Assignment of Disulfide Bonds by a1 Ion Recognition) algorithm, a previously established method for the analysis of whole cysteine linkages of DB-rich proteins , _{representing 616.2392 disulfide - linked peptides} ( ₂₊ , _m / _z ) and _987.9949 ( 6+,m/z) was observed in both native LNTX and recombinant LNTX, indicating that both have the correct DB pattern. However, by detecting 670.3887(2+,m/z), a minor mismatch was found in _rLNTX , which is due to the connection between C41 and C45, and this chaotic structure may partly explain _rLNTX were less toxic (Figure 4).

On the other hand, both rSNTX and its corresponding natural toxin were found to have C ₃ C ₁₇ C by detecting the corresponding disulfide-linked peptides 909.39 (6+, m/z) and 899.94 (6+, m/z) ₂₄ - _C41C43 - _C54C55 - _C60 arrangement ₍ Figure ₅ ).

In addition, the two-dimensional ^1H - ^1H NMR spectra of recombinant and native NTX clearly showed nearly overlapping cross-peaks in the amide regions, indicating that all residues were sterically located in similar orientations. From these results, we can conclude that both long-chain and short-chain recombinant NTX share similar DB bond linkage patterns and spatial structures, and represent C ₃ FITPDITSK-DC ₁₇ PNGHVC ₂₄ YTK-VDLGC ₄₁ AATC ₄₄ PTVK- Trypsin fragment of TGVDIQC ₅₄ C ₅₅ STDNC ₆₀ PFPTR in which all eight cysteines are oxidized. The four enhanced a1 ions (106.0628, 120.0963, 104.1446 and 106.1249) indicate that the peptide consists of four chains, among which cysteine (C), aspartate (D), valine (V) and threonine (T) as the first N-terminal amino acid (Figure 6).

Example 4. Protection of immunized mice from lethal challenge

Materials and Method

Animal experiment

ICR (CD-1, body weight 19-21 g) mice purchased from BioLASCO Taiwan Co., Ltd. were housed in the National Institutes of Health (NHRI) Animal Center according to the protocol (NHRI-IACUC-105108A). Approved by the NHRI Institutional Animal Care and Use Committee. New Zealand White rabbits (NZW) were housed in GeneTex's animal facility according to approved protocols. Animals had ad libitum access to food and water.

Determination of lethality of venom

The assay procedure for venom lethality was performed according to the previously described protocol. Briefly, 200 μL of serially diluted venom was injected intraperitoneally (i.p) into ICR mice (19-21 g, n=6). The survival rate of mice was recorded 48 hours after the injection of venom, and the toxicity was expressed as LD50 (μg/g, the amount of toxin/mouse body weight), calculated by the Trimmed Spearman-Karber method, in the amount corresponding to the death of half of the mice in the experiment Get the fatality rate.

result

To investigate whether the E. coli-derived toxin is immunogenic and sufficient to elicit lethal neutralization against multiple cobra venoms, each group of mice (n =12) Immunization. Specific antibody production was assessed by indirect ELISA using LNTX or SNTX as the surface-coated antigen. Serological analysis showed that after three immunizations, a robust antibody response with an average antibody titer of over 31,622 (pre-immunity titer: 11.7) was generated (Figure 7A), implying the most important epitopes associated with antibody production Retained in recombinant toxin. In addition, comparable survival percentages (83-100%) were observed in groups of mice immunized with either mixed venom or dual forms of recombinant neurotoxin following lethal dose venom challenge, suggesting that the immunogen is composed of only short-chain and Long-chain NTX can be used as a general antivenom for development potential candidates for alpha-nuclein (Fig. 7B-D).

Example 5. Neutralizing ability of rabbit hyperimmune serum to cobra venom

Materials and Method

In vivo neutralization assay

Rabbit serum was passed through a HiTrap® Protein A column (GE Healthcare) to purify immunoglobulin G (IgG). After extensive washing with buffer, bound IgG was eluted with 0.1M glycine (pH=2.7) and rapidly neutralized with 1.0M Tris-HCl pH 8.9. IgG from two rabbits from the same group was pooled, dialyzed, concentrated, and stored at -20°C until use. To evaluate the neutralizing potency of IgG derived from immunized rabbit plasma on venom lethality, groups of six ICR mice were challenged with mixtures containing defined doses of venom and various dilutions of IgG. The mixture was preconditioned at 37°C for 30 minutes and then centrifuged at 4,000 g for 10 minutes to remove the precipitate. Then, 400 μL of the supernatant was injected intraperitoneally into the mice and their 48-hour survival was recorded. The mean effective dose (ED50) of antivenom was calculated by the Trimmed Spearman-Karber method. where defined as the dose at which half of the mice survive venom challenge, where the dose is shown as the weight ratio of venom (mg) to antivenom (g). The effectiveness of antivenoms is also expressed in terms of neutralizing power (P). Since the protein concentration in the product of antivenoms varies, the potency was further normalized and expressed as milligrams of venom neutralized per gram of antivenom protein.

Immunization

For mouse experiments, 50 μg of venom or toxin detoxified with glutaraldehyde (0.25% (v/v)) was formulated in Freund's adjuvant prior to intramuscular injection (i.m). Two consecutive booster immunizations with the same amount of antigen in incomplete Freund's adjuvant were administered at two-week intervals, and 2 weeks after the final immunization, the Blood was collected via tail vein bleeding.

NZW rabbits were immunized 5 times with 1 mg of mixed venom (NAV and NKV, 250 μg each) or 285 μg of recombinant toxin (225 μg rL and 60 μg rS) following the detoxification procedure described above. The serum obtained was decomplemented (30 minutes at 56°C) and then stored at -20°C until use.

Statistical Analysis

Differences in survival were compared by One-way analysis of variance followed by Fisher's least-significant difference test. Statistical analysis of antivenin neutralization efficacy was performed by One-way analysis of variance followed by Bonferroni's multiple comparisons test. An unpaired two-tailed t-test was performed to compare differences in antibody titers. Results with p-value < 0.05 were considered significant. All statistical analyses were performed using GraphPad Prism software.

result

One of the aims of this study was to demonstrate the concept of an anti-cobra snake venom produced using recombinant toxins and which has a more extended neutralizing effect, which has the advantage of not requiring venom acquisition and snake farming. To this end, rabbit populations were immunized with mixed venom or recombinant toxins to obtain antivenoms, which were then further evaluated for their ability to neutralize the venom of three medically important venomous cobras in Asia. Again, quantification was performed by indirect ELISA based on response to antibody valence using either a single toxin or the entire venom as antigen covering the surface (Table 4).

Table 4. Antibody titers in rabbit serum

The NZW rabbit group (n=2) was immunized 5 times with mixed venom or recombinant toxin. Immunoreactivity of antisera against crude snake venom (NAV and NAK) or single toxins (a: Cobrotoxin, b: Alpha-elapitoxin-Nk2a) was assessed by indirect ELISA and expressed as the mean of titers±SD. In each column, the top two with the highest power prices are shown in bold. Serum before immunization was used as a negative control, and its titer was defined as a non-specific signal, and all experiments were repeated three times.

As expected, the bivalent antiserum excelled at neutralizing the lethality of the two homologous venoms with valences of 8.3 and 4.7, respectively (Table 5); in addition, it showed moderate neutralization ability against N. siamensis venom, and there is currently no antivenom available for proper treatment of N. siamensis bites. Compared with the bivalent antiserum, the antiserum produced by the recombinant toxin not only had comparable neutralizing ability against the venoms of N. Good neutralizing ability (P=13.4), which means that the antiserum produced by the recombinant toxin has broad-spectrum properties and has the potential to develop into a broad-neutralizing spectrum.

Table 5. Neutralizing ability of rabbit IgG to neutralize the lethal effects of venoms of N. atra, N. kaouthia and N. siamensis.

Antibodies from two rabbits immunized with the same immunogen were purified and pooled, collected, and A known challenge dose of each snake venom was pre-acted at 37°C for 30 minutes and then injected into mice. The mean effective dose (ED50) was estimated by the Trimmed Spearman-Karber method, and the neutralizing potency (P) was calculated according to the method of Morais et al., (J. Venom.Anim.Toxins incl.Trop.Dis.V.16,n.2 , p.191-193, 2010.)

<110> National Institutes of Health

<120> A method for producing antivenom with recombinant toxin

<150> 62854980

<151> 2019-05-31

<160> 21

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<213> Bengal cobra (Naja kaouthia)

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<221> MISC_FEATURE

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<221> MISC_FEATURE

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<221> MISC_FEATURE

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<221> MISC_FEATURE

<222> (38)..(38)

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<221> MISC_FEATURE

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<221> MISC_FEATURE

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<221> MISC_FEATURE

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<221> MISC_FEATURE

<222> (69)..(69)

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<221> MISC_FEATURE

<222> (70)..(71)

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<221> MISC_FEATURE

<222> (1)..(1)

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<221> MISC_FEATURE

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<221> MISC_FEATURE

<222> (4)..(4)

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<221> MISC_FEATURE

<222> (5)..(5)

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<221> MISC_FEATURE

<222> (9)..(9)

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<221> MISC_FEATURE

<222> (11)..(11)

<223> X11 is Thr, Ala, Pro or Phe

<220>

<221> MISC_FEATURE

<222> (13)..(13)

<223> X13 is Thr or Ile

<220>

<221> MISC_FEATURE

<222> (15)..(16)

<223> X15-16 is ThrGly, LysThr or LysVal

<220>

<221> MISC_FEATURE

<222> (18)..(18)

<223> X18 is Ser or Pro

<220>

<221> MISC_FEATURE

<222> (20)..(20)

<223> X20 is Gly or none

<220>

<221> MISC_FEATURE

<222> (22)..(22)

<223> X22 is Thr or Lys

<220>

<221> MISC_FEATURE

<222> (28)..(28)

<223> X28 is Arg, Trp, Ser or Gln

<220>

<221> MISC_FEATURE

<222> (30)..(30)

<223> X30 is Arg or Ser

<220>

<221> MISC_FEATURE

<222> (31)..(31)

<223> X31 is Asp or Gly

<220>

<221> MISC_FEATURE

<222> (35)..(37)

<223> X35-37 is TyrArgThr, ThrIleIle or TyrArgIle

<220>

<221> MISC_FEATURE

<222> (45)..(45)

<223> X45 is Ser or Lys

<220>

<221> MISC_FEATURE

<222> (48)..(48)

<223> X48 is Asn, Pro or Lys

<220>

<221> MISC_FEATURE

<222> (50)..(52)

<223> X50-52 is IleGluIle, ValAsnLeu or ValLysIle

<220>

<221> MISC_FEATURE

<222> (56)..(56)

<223> X56 is Thr or Arg

<220>

<221> MISC_FEATURE

<222> (57)..(57)

<223> X57 is Thr or Arg

<220>

<221> MISC_FEATURE

<222> (59)..(59)

<223> X59 is Arg or Lys

<220>

<221> MISC_FEATURE

<222> (62)..(62)

<223> X62 is Asn or Arg

<400> 6

<210> 7

<211> 62

<212> PRT

<213> Chinese Cobra (Naja atra)

<400> 7

<210> 8

<211> 62

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<400> 8

<210> 9

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<212> PRT

<213> Bengal cobra (Naja kaouthia)

<400> 9

<210> 10

<211> 61

<212> PRT

<213> Central Asian cobra (Naja oxiana)

<400> 10

<210> 11

<211> 61

<212> PRT

<213> Bengal cobra (Naja kaouthia)

<400> 11

<210> 12

<211> 61

<212> PRT

<213> Philippine cobra (Naja philippinensis)

<400> 12

<210> 13

<211> 62

<212> PRT

<213> Naja sputatrix

<400> 13

<210> 14

<211> 62

<212> PRT

<213> Naja sputatrix

<400> 14

<210> 15

<211> 61

<212> PRT

<213> Egyptian Cobra (Naja haje)

<400> 15

<210> 16

<211> 61

<212> PRT

<213> Forest cobra (Naja melanoleuca)

<400> 16

<210> 17

<211> 236

<212> PRT

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<210> 18

<211> 7

<212> PRT

<213> Synthetic sequences

<220>

<223> TEV proteolytic site sequence

<220>

<221> misc_feature

<222> (7)..(7)

<223> X7 is not Pro

<400> 18

<210> 19

<211> 39

<212> DNA

<213> Synthetic sequences

<220>

<223> Connexin sequence

<400> 19

<210> 20

<211> 72

<212> PRT

<213> Synthetic sequences

<400> 20

<210> 21

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<212> PRT

<213> Synthetic sequences

<400> 21

Claims (4)

A recombinant snake alpha-neurotoxin sequence, wherein the recombinant snake alpha-neurotoxin sequence comprises SEQ ID NO.20. A composition comprising two recombinant snake alpha-neurotoxin sequences, wherein the two recombinant snake alpha-neurotoxin sequences are SEQ ID NO. 20 and SEQ ID NO. 21, respectively. Use of a composition for preparing an anti-cobra venom serum, wherein the composition comprises two recombinant snake alpha-neurotoxin sequences, wherein the two recombinant snake alpha-neurotoxin sequences are SEQ ID NO.20 and SEQ ID respectively NO.21. The use according to claim 3, wherein the anticobra venom is used against Bengal cobra venom, Chinese cobra venom and Thai cobra venom.

Images