Introduction

Hemiscorpius lepturus (H. lepturus), belonging to the Hemiscorpiidae family, is one of the deadliest scorpion species recurrently reported in Iran1,2. Envenomation by H. lepturus is clinically a severe problem in the southwestern area of the country, especially in Khuzestan Province3. H. lepturus is responsible for only about 15% of scorpion stings throughout Iran; however, owing to the nature of its venom, it accounts for about 95% of all mortalities caused by such injuries4.

Due to the painless nature of H. lepturus venom and the slow development of the symptoms in the victims, the stung patients often fail to attend health centers for medical treatment5. The most rigorous toxicity and lethality have mainly been observed in children6. Several complicated symptoms include nephrotic syndrome, dermonecrotic lesions, erythema, renal failure, and immediate death7,8. Other clinical symptoms entail vascular leakage, persistent inflammation, cardiovascular diseases, severe hemolysis, platelet aggregation, and hematuria, ultimately leading to renal failure3,9. Hence, Identifying the biochemical properties of venom-derived components seems necessary. The H. lepturus venom contains various proteins, compounds (salts, nucleotides, and biogenic amines), and enzymes (hyaluronidase, L-amino acid oxidase, metalloproteinase, serine protease, mucoproteins, and phospholipases)10,11. It also comprises varying characterized peptides and enzymes, e.g., hemicalcin, hemitoxin, hemilipin, and heminecrolysin (HNc), which trigger erythrocyte lysis, antimicrobial peptides (AMPs), and phospholipases such as A1, A2, B, C, and D2,12,13,14.

This study represents the first comprehensive biochemical identification and characterization of phospholipase A2 (PLA2), a new enzyme isolated from H. lepturus and expressed in E. coli, to obtain complete information on its activity and biological role. Such investigations are crucial as they provide fundamental insights into the venom’s composition, which could pave the way for developing new therapeutic approaches against scorpion envenomation15. The identification of PLA2 in H. lepturus venom is particularly significant given its potential role in modulating inflammatory responses and its involvement in various physiological and pathophysiological processes16. Recent studies have highlighted the pharmacological activity of PLA2, demonstrating its involvement in inflammatory responses and its potential as a therapeutic target. For instance, Oliveira et al. (2008) showed that PLA2 activity could be modulated by BTL-2, an isolectin isolated from the red alga Bryothamnion triquetrum, indicating its significant role in disease modulation17. Furthermore, Zhang et al. (2024) demonstrated that serum levels of lipoprotein-associated phospholipase A2 (Lp-PLA2) are significantly associated with coronary atherosclerotic plaque progression in diabetic and non-diabetic patients, underscoring its role in cardiovascular diseases18. Additionally, Sun et al. (2009) reviewed the role of PLA2 in inflammatory responses in the central nervous system, highlighting its involvement in receptor signaling and transcriptional pathways that link oxidative events to inflammatory responses underlying many neurodegenerative diseases19. In Iranian H. lepturus venom, PLA2 isoforms have been identified as major components, contributing to the venom’s overall toxicity and pharmacological effects. The variation in PLA2 levels among different geographical populations of H. lepturus can lead to significant differences in venom potency and composition, impacting the efficacy of antivenoms. Studies have shown that commercial antivenoms may have variable effectiveness in neutralizing venoms from different regions, underscoring the need for region-specific formulations to ensure optimal treatment outcomes20. Moreover, the exploration of specific proteins and peptides found in the venom of the Iranian scorpion, H. lepturus, has been linked to the control of certain conditions, including cancer21,22,23, and may also significantly contribute to the enhancement of genetic reservoirs, advancing antiserum production, and the development of novel therapeutic candidates.

Results

Molecular cloning, purification, and western blotting of PLA2

The full-length sequence of the PLA2 gene was cloned into the pET-26b(+) vector using NdeI and XhoI restriction sites. The expression of PLA2 in E. coli BL21(DE3) pLysS yielded a protein with an approximate molecular weight of 14 kDa (Fig. 1a). The protein was purified using a Ni-NTA affinity column, and its purity was confirmed by SDS-PAGE, which showed a single band corresponding to the expected molecular weight. Western blotting using anti-His antibodies confirmed the identity of the purified PLA2 protein (Fig. 1b).

Fig. 1
figure 1

(A) Expressed and Purified PLA2 on SDS-PAGE: 14 kDa band. Lane 1: Negative control (before protein induction); Lane 2: Protein marker; Lanes 3: Expressed PLA2 (after protein induction); Lane 4: Purified PLA2 elution. (B) Detection of Expressed PLA2 by Western Blot. Lane 1: Marker; Lane 2: Purified PLA2.

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Structure of recombinant protein by CD spectroscopy

Circular Dichroism (CD) spectroscopy in the wavelength range of 190–250 nm was used to investigate the secondary structure of the recombinant protein. The CD spectrum acquired at 25 °C, as depicted in Fig. 2, clearly shows the presence of different kinds of secondary structural elements like alpha-helices, beta-sheets, and random coils. In the CD spectrum analysis, shown in Table 1, the secondary structural components have been quantified as a percentage composition of the protein’s secondary structure.

Fig. 2
figure 2

PLA2 CD Spectra in the Far-UV Region (190–250 nm).

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Table 1 The percentage of protein structure.
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Enzymatic activity assay for PLA2

Phospholipase activity is measured by hydrolysis of lecithin. As indicated by Fig. 3, lecithin could be hydrolyzed by PLA2. At the same concentrations, PLA2 exhibited greater phospholipase activity than crude venom. PLA2 reached its maximum activity at 6.25 µg/mL.

Fig. 3
figure 3

Activity Analysis of PLA2: PLA2 and crude venom at different concentrations were incubated with a reaction mixture containing 3.5 mM lecithin. The PLA2 activity percentage was derived from the variations in phenol red absorbance due to fatty acid release. PBS was the negative control. PLA2 showed peak activity at 6.25 µg/mL (N = 3).

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Enzymatic activity assay for PLA2 at different temperatures

PLA2 peaked in activity at 6.25 µg/mL, as Fig. 5 displays. At this point, various temperatures were used to measure the percentage of phospholipase activity. The findings indicated that the highest level of phospholipase activity was observed at 25 °C, with over 70%; however, at 37 °C, this amount decreased to approximately 62% (Fig. 4).

Fig. 4
figure 4

Phospholipase Activity Temperature Analysis: The highest activity was observed at 25 °C (> 70%), which decreased to about 62% at 37 °C (N = 3). PLA2 activity was unmeasurable due to the protein turning yellow in acidic and red in alkaline pH.

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Fig. 5
figure 5

The cross-reactivity of H. lepturus PLA2 against anti-PLA2 polyclonal antibodies in rabbits’ serum was evaluated by ELISA. The result shows the highest anti-PLA2 polyclonal antibody titer level in rabbits’ serum after the fifth injection.

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ELISA assay

The cross-reactivity of H. lepturus PLA2 against anti-PLA2 polyclonal antibodies in rabbit serum was evaluated by ELISA. This evaluation was conducted over a series of five injections, administered at intervals of 10–12 days. The absorbance was measured at 450 nm, and the test was conducted in triplicate using different diluents to ascertain the reliability of the findings. The results showed that after every injection, the amount of anti-PLA2 antibodies in the rabbit serum increased gradually. This increase was consistently observed across the different dilutions tested. Notably, the antibody levels peaked after the fifth injection, indicating a robust immune response elicited by the repeated exposure to the antigen (Fig. 5). These data highlight H. lepturus PLA2’s immunogenic potential and provide critical new insights into its antigenic characteristics.

MIC and MBC

The results of our study indicated that the Minimum Inhibitory Concentration (MIC) of the recombinant PLA2 protein was determined to be 31.25 µg/ml. At this concentration, the PLA2 protein effectively inhibited the growth of all bacteria in the test culture, preventing their proliferation. This demonstrates the strong antibacterial properties of the PLA2 protein, which can halt bacterial growth at relatively low concentrations.

Furthermore, the Minimum Bactericidal Concentration (MBC) was found to be 0.5 mg/ml. At this concentration, the recombinant PLA2 protein was able to kill 100% of the bacteria in the culture, indicating its potent bactericidal effect. The ability of PLA2 to achieve complete bacterial eradication at this concentration highlights its potential as a powerful antimicrobial agent.

Discussion

The present study aimed to analyze the enzymatic activity and structural features of PLA2 from H. lepturus venom. The identification and characterization of PLA2 from various sources, including scorpion venom, provide valuable insights into the enzyme’s biochemical properties and potential applications. PLA2-specific antibodies are essential due to the high content of PLA2 in the total proteome of H. lepturus venom. These antibodies can help neutralize the toxic effects of PLA2 and are critical for the development of effective antivenoms. Studies on the venom proteome composition of H. lepturus and related species have shown that PLA2 is a major component, highlighting its importance in venom toxicity and its potential as a therapeutic target24,25. Consistent with prior research, our investigations revealed that this PLA2 isomer possesses acceptable phospholipase activity26,27,28. Utilizing the pET expression vectors leveraging the His-tag marker facilitated efficient purification and detection of the recombinant protein29. Shahbazzadeh’s venom research group effectively expressed several proteins extracted from scorpion venom using this expression vector, following our methodology2. In this study, the PLA2 gene was cloned into the pET-26b (+) vector and transformed to the E. coli BL21(DE3) pLysS strain, which has been modified for maximum protein expression. The optimization of expression conditions revealed that the best expression was obtained six hours post-induction with IPTG. The SDS-PAGE and Western blot results confirmed robust expression of PLA2 in this strain. In order to preserve the protein’s original form, the purification procedures in the following step were initially carried out using the imidazole buffer purification approach. This procedure was also utilized by Kapoor et al. to isolate PLA2 in their research30. Subsequent analysis, however, showed that no protein was detected in the elution solution using this method, indicating that the solubility of the protein in its natural form is very low. To address this, the protein was denatured using purification buffers containing urea, facilitating PLA2 expression and purification and allowing it to be refolded into its natural form for structural, biochemical, and antibacterial activity investigations31,32. As evidenced by Kazemi et al., proteins are frequently denatured by urea during chromatography-based purification. Therefore, PLA2 was renaturated following purification by gradually eliminating urea and then concentrating the protein for further processing33,34. Further investigation into the phospholipase activity of the PLA2 protein indicates that at varying concentrations, the protein exhibits around 60% of phospholipase activity. This activity level is comparable to that of the crude extract at these concentrations, highlighting the significant role of the purified PLA2 in the whole venom activity. These results are also consistent with those obtained in the research projects carried out at the Pasteur Institute of Iran for other isomers of PLA2 protein35. Additionally, other PLA2 isomers have been reported to exhibit 70% phospholipase activity in other studies36,37. In line with the findings of the pH analysis of phospholipase activity, this PLA2 protein is yellow in acidic pH and turns red in alkaline pH. As a result, this method could not be used to assess this activity reliably. Using the same approach, Toufani et al. additionally glanced at the PLA2 protein’s activity in Iranian snake venom. They discovered that even minimal pH variations can affect color, which may lead to errors in wavelength measurements and other calculations38. These results are in accordance with our current findings. The temperature-dependent activity profile of PLA2 observed in this study revealed that the enzyme exhibits its highest phospholipase activity at 25 °C. While between 37 °C and about 57 °C, this activity diminishes, and between 70 °C and 100 °C, no phospholipase activity is seen. These discoveries are consistent with the results of some other researchers, who also observed comparable temperature-dependent PLA2 activity profiles39,40. The observed decline in activity at higher temperatures could be attributed to the thermal denaturation of the enzyme, which disrupts its tertiary structure and catalytic efficiency. Additionally, research on a particular PLA2 isoform from the venom of Heterometrus fulvipes scorpions supports our findings with similar activity patterns41. All things considered, the comprehensive biochemical analysis of PLA2, encompassing its activity that is reliant on pH and temperature, improves our comprehension of its functional characteristics. These findings are essential for developing the creation of PLA2-based drugs and opening up new insights for venom research. The dependence of PLA2 activity on temperature and the observed challenges with pH assessment underscore the need to consider these factors when developing therapeutic applications. Future research should focus on refining pH measurement techniques to obtain more precise data and conducting further studies to explore the full potential of PLA2 in therapeutic contexts. The CD spectrum, recorded in the wavelength range of 190–250 nm at 25 °C, reveals distinct features corresponding to alpha-helices, beta-sheets, and random coils. Notably, the quantification of secondary structural elements shows that beta-sheets constitute 45.1% and random coils 36.6%, which are significantly higher than the alpha-helix and turn content. In order to evaluate the protein’s functional integrity under the used conditions, this structural knowledge is crucial. The quantitative analysis of the CD spectrum highlights the importance of secondary structure composition in determining the protein’s stability and functionality. These findings are also crucial for understanding the protein’s folding and stability. The characteristic negative peaks around 208 nm and 222 nm, indicative of alpha-helical content, and the positive peak near 195 nm, suggesting the presence of beta-sheets, align with the observed structural composition42,43. The higher percentage of beta-sheets and random coils in PLA2 suggests a unique conformational state that may influence its enzymatic activity and interaction with other molecules44. Comparing these findings with known structures or literature values can offer insights into the protein’s behavior. Maintaining the CD experimental conditions, especially at 25 °C, is crucial for preserving the protein’s structural integrity, as variations in temperature or other environmental factors can alter the CD spectrum and affect the interpretation of the protein’s secondary structure45,46.

Phospholipases have been identified in both vertebrates and invertebrates, including mammals, snakes, scorpions, and bees47. H. lepturus anti-scorpion serum from horses was initially used to treat scorpion stings. However, individuals treated with antiserum, especially those native to scorpion-prone areas, are at risk of repeated stings. This necessitates the administration of higher doses of antiserum, which may cause anaphylactic shock and possibly lead to death47,48. One potential solution is to use a specific part of the venom compound with minimal toxic capabilities. This method preserves the person stung by the scorpion while facilitating the development and production of antibodies. On the other hand, for decades, antiserum production facilities have relied on the breeding and euthanizing of scorpions and snakes to harvest their whole venom. This traditional method presents significant ethical and practical challenges, including the need for large-scale hunting, breeding, and the associated costs. Additionally, it risks the depletion of snake and scorpion populations in nature. The identification and characterization of specific venom components, such as PLA2, can serve as alternatives to whole venom extraction, reducing the dependency on these traditional practices49,50,51. Antivenom production centers typically rely on carefully breeding and maintaining snakes, spiders, and scorpions in captivity to extract venom without euthanizing the animals. This practice ensures a sustainable supply of venom while preserving animal populations52. However, using venoms as a limiting factor for antivenom production poses significant challenges due to the complexity and variability of venom composition. Additionally, the production of antisera using rabbits or other animals can face challenges related to ethical considerations and resource constraints. To address these challenges, we propose a combined approach that leverages recombinant DNA technology to produce specific venom components, such as PLA2, in bacterial or mammalian expression systems. This method can reduce the dependency on animal-sourced venoms and provide a more consistent and scalable source of antigens for antivenom production. Additionally, we emphasize the potential of next-generation antivenoms that target specific venom components, offering improved efficacy and safety53. By utilizing recombinant DNA technology, we can ensure a sustainable and ethical production process while maintaining the efficacy of antivenoms. This approach not only addresses the limitations associated with using whole venoms but also enhances the precision and specificity of antivenom therapies. We hope these clarifications address the concerns raised and provide a more balanced perspective on the modernization of antivenom production20.

Recent studies have also shown promising results in PLA2 anticancer activities. Oh et al. (2023) revealed that the lipoprotein-associated phospholipase A2 (Lp-PLA2) inhibitor Darapladib sensitizes cancer cells to ferroptosis by remodeling lipid metabolism, suggesting a potential therapeutic strategy54. Furthermore, Khunsap et al. (2016) investigated the anticancer properties of phospholipase A2 from Daboia siamensis venom, showing its cytotoxic effects on human skin melanoma cells55. Future research endeavors ought to concentrate on utilizing the recombinant proteins examined in the current research, other PLA2 protein isomers, and even recombinant phospholipase D derived from earlier studies3. Additionally, it is recommended that these proteins’ immunogenicity be investigated using an animal model, as this method could potentially replace the current approach of immunizing animals with whole scorpion venom.

Materials and methods

Chemicals, plasmid, bacteria, and mature PLA2

Complete and Incomplete Freund’s Adjuvants were acquired from Sigma Aldrich (St Louis, MO, USA). All chemicals and other reagents were procured from Merck Whitehouse Station, NJ, USA). E. coli as competent cells were obtained from the Pasteur Institute of Iran (Tehran, Iran), and the mature chain was provided from a previous study21. E. Coli BL21 (DE3) pLysS has DE3 on the chromosome, which includes the T7 RNA polymerase gene under the control of the Lac promoter. PlysS is a 4886 bp plasmid with a low-copy number in which the T7 lysozyme gene is located in its BamHI cleavage region. This gene suppresses the basal expression of the T7 promoter by expressing the T7 lysozyme protein, which is considered a natural inhibitor for T7 RNA polymerase. This is while the expression hosts without PlysS plasmid have a primary expression of the recombinant protein in the absence of an inducer (Isopropyl-ß-D-thiogalactopyranoside (IPTG), as a result of which proteins are incompletely synthesized at a low scale, and the purity percentage of complete proteins decreases56.

Plasmid preparation, expression, and purification of PLA2

Codon optimization was applied to the DNA sequence correlated with the target protein in order to facilitate its production in E. coli cells. The protein sequence containing an N-terminal His-tag flanked by NdeI and XhoI was constructed by an external source (Biomatik Co., Kitchener, ON, Canada), which was subsequently cloned into pET-26b(+) vector (Thermo Fisher, Invitrogen™, Waltham, MA, USA). The recombinant vector was introduced into E. coli BL21 (DE3) pLysS cells for expression. The culture was then supplemented with 0.5 mM IPTG (Sigma Aldrich, St Louis, MO, USA) as soon as the cell density at 600 nm reached 0.6. An LB broth medium was used to cultivate bacterial cultures in a shaking incubator overnight. The cells were incubated at 37 °C for 3 h. The suspension was subjected to a centrifugation process (10,000 g, 10 min, 4 °C) and sonication (Hielscher Co. Teltow, Germany) using short pulses (30 pulses of 30 s with a 1-min interval in between, 80% amplitude). The supernatant was separated after the debris was eliminated by centrifugation (10,000 g, 10 min, 4 °C). The presence of the target protein was confirmed by the sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and western blotting. Subsequently, a large-scale cell culture was conducted to produce more protein. Once more, bacterial cultures were grown in a shaking incubator in an LB broth medium overnight. The suspension was centrifuged and sonicated with short pulses, the same as in the previous steps. The debris was removed by centrifugation (10,000 g, 10 min, 4 °C), and the supernatant containing soluble protein was subjected to affinity chromatography using a Ni-NTA agarose column (Qiagen Co., Hilden, Germany). A washing buffer (50 mM sodium phosphate buffer, 300 mM NaCl, 20 mM imidazole, 8 M urea at pH 8.0) was used to wash and equilibrate the Ni-NTA agarose column. As previously indicated, the sample was transferred into the column and washed with washing buffer to remove the contaminants. The His-tagged protein was eluted using elution buffer (50 mM sodium phosphate buffer, 300 mM NaCl, 250 mM imidazole, and 8 M urea at pH 8.0). Following purification, the protein was dialyzed using PBS 1X (pH 7.4) at 4 °C and assessed with SDS-PAGE.

Recovery of the natural form of protein by dialysis

Once purified, the protein was poured into a dialysis bag with a specific pore size (12 kDa) to restore the protein structure. After tightening both sides, the bag was placed in a completely clean 1000-ml protein-free beaker and immersed in dialysis buffer. The beaker containing the magnet was placed on a stirrer in a cold room at 4 °C for 16 h. Urea will be gradually eliminated from the protein samples over the four days of dialysis by altering the body’s buffer according to the following protocol: (First day: one liter of dialysis buffer containing 6 M urea; Second day: one liter of dialysis buffer containing 4 M urea; Third day: one liter of dialysis buffer containing 2 M urea and Fourth day: one liter of dialysis buffer without the presence of urea).

Analysis of PLA2 by SDS-PAGE

We adopted the previously mentioned SDS-PAGE method to assess PLA257. In a nutshell, for the protein sample preparation, PLA2 was mixed with a sample buffer, which was then heated at 95 °C for 5 min. The sample (15 µl) was pipetted into one of the wells in the 15% gel. The protein molecular weight marker (Thermo Fisher, Invitrogen™, Waltham, MA, USA) was also loaded into its own well in the gel. Then, a 15-mA electrophoretic flow was applied to the gel for an initial 30 min, followed by 25-mA for 2 h, to migrate negatively charged protein through the gel in the direction of the anode pole. Finally, the gel was brought out of the glass plates and placed in the Coomassie Brilliant Blue R-250 (Thermo Fisher, Invitrogen™, Waltham, MA, USA) solution for staining at room temperature for 12 h, followed by immersing gel into the destaining solution until the background of the gel was fully destained and protein bands became visible.

Western blotting analysis

In reducing circumstances, the recombinant protein was analyzed on 12% SDS-PAGE. Afterward, a semi-dry blotting apparatus (Bio-Rad Laboratories, CA, USA) was implemented to transfer the protein to the nitrocellulose membrane. After blocking the nitrocellulose membrane with 5% skim milk at room temperature for 1 h, mice monoclonal anti-poly histidine antibody was introduced, and the membrane was incubated for a further 2 h at room temperature. Three washing cycles were then conducted using phosphate-buffered saline with 0.05% Tween (PBST). A secondary antibody (1:10000, Sigma Aldrich, St Louis, MO, USA) conjugated to anti-mouse IgG was incubated on nitrocellulose membranes for 1 h at room temperature. After additional washing steps, the membrane was developed using 3, 3′-diaminobenzidine (DAB).

Determination of protein concentration

The protein concentration in the samples was determined by the colorimetric technique of Bradford’s protein assay, using bovine serum albumin (BSA) at a concentration of 1 mg/mL as the standard58. To ascertain the protein quantity in the unidentified samples, their absorbance was measured and compared to a standard curve generated from known quantities of BSA.

Biochemical identification and characterization of PLA2

Circular dichroism (CD) spectroscopy

To determine the characteristics of the secondary structures of the PLA2, the far UV CD spectrum (190–250 nm) was analyzed. Parts of the protein in the alpha-helical configuration, the beta-sheet structure, and other possible combinations can be recognized with CD spectroscopy. Both the PLA2 and standard samples were subjected to this procedure. The buffer of the PLA2 sample was prepared using an Amicon filter with a cutoff of 3 kDa, which was replaced with injected deionized water and concentrated. The sample (PLA2) was dialyzed against dH2O at 4 °C to obtain a final 0.2 mg/ml concentration. CD spectra recorded using a 1 mm cuvette were measured on a Jasco J-715 polarimeter spectrometer (Jasco, Tokyo, Japan). The samples (400 µl) were poured into the Jasco J-810 device, and reading was done in the specified range. The results of the second structure of proteins were obtained as molar ellipticity. The percentage of each secondary structure was calculated using Jasco Secondary Structure Estimation Software. All the experiments were performed at a 100 nm/min speed using 1 s response time and 1 nm bandwidth. The cuvettes’ temperature was maintained at 25 °C throughout the experiment.

Phospholipase activity of purified PLA2

A colorimetric method based on earlier studies was used to evaluate PLA2 phospholipase activity59. The protein was produced at serial doses ranging from 3.125 to 150 µg/mL at a microplate. Each well was then filled with the substrate solution and incubated for 15 min at 37 °C. The H. lepturus scorpion’s crude venom served as the positive control and PBS 1X as the negative control. In a microplate Epoch spectrophotometer (PowerWave HT, BioTek Instruments, USA), the optical density (OD) was measured at 550 nm. The PLA2 phospholipase activity was assessed at various pH values (3–8) and temperatures (4–100 °C).

Determination of MIC and MBC

The MIC for PLA2 was determined by broth microdilution assays according to the Clinical and Laboratory Standards Institute (CLSI) recommendations59. Briefly, bacterial cells were grown in Muller Hilton Broth Overnight. The number of bacteria was adjusted to 0.5 McFarland standard by spectrophotometry at 625 nm. Phospholipase serial dilutions were combined with 100 µL of each bacterial strain, and the mixture was incubated for 2 h at 37 °C. The tests were performed in duplicate. The lowest concentration of phospholipase that inhibited visible bacterial growth was recorded for the MIC. For lethality assessment, 10 µL of the inhibitory concentrations and above were plated on agar plates and incubated at 37 °C for 24 h. The MIC and MBC were calculated based on the colony counts, providing an extensive evaluation of the antibacterial activity of the phospholipase. The MBC was defined as the lowest amount of sample required to kill 100% of the bacteria. Staphylococcus aureus ATCC 25923 and Escherichia coli ATCC 25922 were used as the quality control strains60.

Immunization trials in rabbits

Male New Zealand White rabbits weighing 2–3 kg (N = 3) were used in the present study. The animals were obtained from the Pasteur Institute of Iran (Karaj, Iran), housed in standard laboratory conditions with controlled temperature (22 ± 2 °C), humidity (50 ± 10%), and a 12-hour light/12-hour dark cycle. The rabbits were also housed in individual cages to monitor their health and behavior. The bedding was provided and changed regularly to maintain hygiene. The animals were acclimatized to the laboratory conditions for one week prior to the start of the experiments. All experiments were performed in full compliance with the relevant laws and institutional guidelines for animal welfare rules and in accordance with the Council Directive of the European Communities of November 24, 1986 (86/609/EEC). The study was conducted in accordance with the ARRIVE guidelines for reporting experiments involving animals.

Production of rabbit anti-PLA2 serum

The rabbits were immunized with 10 µg/ml of PLA2 over five injections at 10-12-day intervals. The initial immunization was conducted using Freund’s complete adjuvant, with subsequent immunizations using Freund’s incomplete adjuvant. Each immunogen was emulsified with an equal volume of the respective adjuvant.

Detection of specific rabbit’s anti-PLA2 polyclonal antibody

An ELISA assay was used to detect the presence of specific anti-PLA2 polyclonal antibodies in rabbit serum. In general, H. lepturus PLA2 (1µg/well) was immobilized and coated triplicate on 96-well flat bottom polystyrene microtitration plates (Nunc GmbH, Wiesbaden, Germany) at 4°C. PBST was used to wash the plates. After being blocked with PBS containing bovine serum albumin 1% (BSA) as blocking buffer for 1 h at 37°C, plates were washed with washing buffer. Then 100 µl of rabbit’s anti-PLA2 serum in PBS containing 1% bovine serum albumin was added twofold serial diluent to the appropriate well and incubated for 1 h at 37°C. Plates were washed with PBST and detected with peroxidase-conjugated goat anti-rabbit IgG diluted at 1/5000 in blocking buffer were added to each well. After a one-hour incubation period at 37°C, the plates were washed with PBST. 100 µl of 3, 3, 5, 5’-Tetramethylbenzidine (TMB) was added to each well, and the plates were incubated for 10 min at room temperature. 100 µl of H2SO4 (2 N) was added to stop the enzymatic reaction, and the OD was measured at 450 nm.

Randomization and blinding

Rabbits were randomly assigned to treatment groups. Blinding was not applicable due to the nature of the study design.

Sample size

Three rabbits were chosen based on previous studies that demonstrated sufficient statistical power for detecting immunological responses.

Method of euthanasia and anesthesia

The rabbits used in this study were humanely euthanized at the end of the experiment. Euthanasia was performed using an overdose of sodium pentobarbital administered intravenously. Prior to euthanasia, the rabbits were anesthetized using a combination of ketamine (35 mg/kg) and xylazine (5 mg/kg) administered intramuscularly to ensure they were fully unconscious and did not experience any pain or distress.

Statistical analysis

Statistical analyses were performed using GraphPad Prism 8 software (San Diego, CA, USA). All experiments were conducted in triplicate, and data are expressed as mean ± standard deviation (SD). The differences between the treated and control groups were analyzed by one-way ANOVA, and p-values ≤ 0.05 were considered statistically significant.

Conclusion

This study successfully achieved the characterization of the biochemical properties and immunogenic potential of the PLA2 protein isolated from H. lepturus venom and expressed in E. coli. The findings demonstrate that the recombinant PLA2 exhibits considerable phospholipase activity, making it a promising candidate for further biochemical and structural studies. The potential application of recombinant PLA2 in antivenom research is indicated by its immunogenicity, as evidenced by the existence of polyclonal antibodies in vivo. Future clinical research should focus on investigating the therapeutic applications of this enzyme protein, such as its potential to serve as a pharmaceutical or an effective vaccine candidate against H. lepturus-induced scorpion envenomation. Lowering the requirement for venom extraction from wild populations not only provides a novel technique for treating scorpion stings but also helps conserve scorpion species. The capacity to produce and purify recombinant venom proteins provides novel opportunities for investigation and treatment advancement, ultimately augmenting our comprehension and management of scorpion envenomation.

In summary, this research represents a significant advancement in our understanding of scorpion venom components and their potential therapeutic applications. By successfully expressing and characterizing the PLA2 enzyme from H. lepturus, we have paved the way for innovative approaches in antivenom development and venom research. The generation of specific polyclonal antibodies against PLA2 highlights its immunogenic potential and provides a foundation for the creation of targeted therapies. This study also underscores the importance of using recombinant technologies to produce venom proteins, which can lead to more sustainable and ethical practices in venom research and antivenom production. Overall, the insights gained from this work will contribute to the development of more effective treatments for scorpion stings and enhance our ability to manage envenomation cases more efficiently.