Based on the results of homology analysis using protein-protein basic local alignment search tool (BLASTP), the best ORF with highest identity was selected. The candidate ORF and its main chain were determined as chains with lengths of 324 and 286 amino acids respectively (Figure 1). Hl-PLD1 demonstrated the highest sequence similarity with the isoforms of PLD from L. intermedia (identity 46%–48%). Other PLDs from Loxoscelidae were highly similar to Hl-PLD1 (identity 45%–49%).

Molecular weight, isoelectric point (pI) and Grand Average of hydropathicity (GRAVY) of the main chain were calculated as 32,720.20 Da, 7.77 and -0.58 respectively.

ClustalW analyses showed that almost all of the Loxoscelidae PLDs (especially L. intermedia) were similar to Hl-PLD1 (Figure 2). A conserved motif at N-terminal region, “RPIWXXGH”, in L. intermedia PLDs was a suitable key point to use for prediction of the Hl-PLD1 main chain. According to homology comparison between the mature chain of Hl-PLD1 and L. intermedia PLDs, the “CXCXXXC” motif and two histidine residues (H12 and H48) were determined as a conserved region and active sites respectively. The catalytic site of Hl-PLD1 was determined based on the previous studies examining Loxosceles PLDs (Figure 2).

The results indicate that the predicted confirmation contains several central parallel beta sheets and a number of peripheral alpha helices (Figure 3). The predicted structure was relatively similar to a phospholipase D isoform from L. intermedia, LiSicTox-alphalA1bii [28]. According to this significant similarity, Hl-PLD1 was classified as having a “TIM beta/alpha-barrel” structure. Beta sheets, alpha helices and coils were calculated by AntheProt 3D software as making up 17.1%, 35.3% and 47.6% respectively of the predicted structure.

UCSF Chimera software performed a structural alignment of the predicted form of Hl-PLD1 with one of the previously determined PLD from L. intermedia, isoform LiSicTox-alphaIA1bii. The results clearly showed a significant structural homology between these isoforms (Figure 4). The structure of LiSicTox-alphaIA1bii was classified as “TIM beta/alpha-barrel” according to Coronado et al. in 2015 [28].

The accuracy of the main chain sequence was confirmed by RT-PCR and DNA sequencing. A PCR product of approximately 250 bp confirmed correct amplification of the internal fragment corresponding to the Hl-PLD1 sequence (Figure 5). Further verification was achieved by sequencing of RT-PCR products.

The recombinant toxin herein was designated as Hl-RecPLD1. Figure 6a demonstrates the successful expression of H1-RecPLD1. Sodium Dodecyl Polyacrylamide Gel Electrophoresis (SDS-PAGE) showed a single purified protein at the expected weight of Hl-RecPLD1 (~32 KDa) (Figure 6b).

A horse was immunized with H. lepturus crude venom at intervals of 14 days over the course of 70 days. The highest titer of horse antibody was obtained in the 5th injection. Western blot analysis was performed for recombinant toxin using horse antisera. As shown in Figure 7, the recombinant phospholipase D, Hl-RecPLD1, was recognized by horse antibodies.

The results showed that Hl-RecPLD1 at the amounts of 5, 10 and 15 µg was able to hydrolyze 26, 30 and 39 nmol of trinitrophenyl-aminolauryl-sphingomyelin (TNPAL-SM, Sigma Chemical Co., St Louis, MO, USA) respectively. At these same amounts, crude venom hydrolyzed 20, 21 and 24 nmol of the substrate respectively (Figure 8). Sphingomyelinase activity of Hl-RecPLD1 was 1.4-fold higher than the crude venom (positive control) at all examined amounts. Increased amounts of recombinant toxin led to increased sphingomyelinase activity.

To determine the dermonecrotic activity of Hl-RecPLD1, 1 µg of recombinant toxin was intradermally injected into rabbit skin. The lesion area was checked and measured. The necrosis area reached approximately 0.7 cm² after 72 h (Figure 9a,b).

The mortality induced by recombinant toxin in the mice was documented for 4 days after intraperitoneal injections. The LD₅₀ and lethal dose 100 (LD₁₀₀) of the recombinant toxin were determined as 3.1 and 3.7 µg respectively for each mouse.

No hemolytic activity was detected with injections of up to 50 µg with or without human serum.

Hemiscorpius lepturus is usually less than 9 cm in length and can be easily distinguished by its unique bead-shaped jointed tail, dark brown chelicerae and yellow–brown body (Figure 10). One hundred H. lepturus samples were collected manually from the suburban area of Ahvaz city in the Khuzestan province of Iran (31°00′ N 49°00′ E), with the specimens then being transferred to the laboratory (Figure 11).

The venom of H. lepturus scorpions were collected by the milking method [38] and two days later, the venom glands were cut and kept frozen in liquid nitrogen. The glands were cut into small pieces and grounded in liquid nitrogen to obtain a fine powder. The powder was re-suspended in Trizol (Invitrogen Co., Carlsbad, CA, USA) and centrifuged at 12,470 g. The quality of the extracted RNA was checked by electrophoresis on 1.5% agarose gel at 70 V for 40 min using Tris Acetate EDTA (TAE, 1x) buffer. The extracted RNA was transferred to an external facility (Source BioScience Co., Nottingham, UK).

The gene (GenBank accession number: KY287766) was codon–optimized for the E. coli strain K12. The gene designated as Hl-RecPLD1, with a 6x His-Tag at the C-terminus flanked by NdeI and XhoI, was synthesized by Generay Biotech Co. (Hong Kong, China) and inserted into pET-22b expression vector.

The recombinant construct was transformed into E. coli BL21 (DE3) cells [48]. E. coli BL21 (DE3) cells were cultured in Luria-Bertani broth (LB) overnight. The cells were harvested at 3615 g for 5 min at room temperature and re-suspended in 100 µL cold CaCl₂ (0.1 M) before being incubated on ice for 20 min. The cells were centrifuged and the pellet was washed two times in 100 µL cold CaCl₂ (0.1 M) by centrifugation at 3615 g for 5 min. Subsequently, the pellet was re-suspended in 100 µL cold CaCl₂ (0.1 M). The pET-22b expression vector (1 µg) was then added to the suspension and heated at 42 °C for 90 s followed by 2 min incubation on ice. Finally, 1 mL of LB broth was added to the suspension and incubated at 37 °C for one hour. The cells were centrifuged again at 3615 g for 5 min and re-suspended in 100 µL fresh LB broth. Fifty microliters of the transformants were plated on LB agar by ampicillin (100 µg/mL) overnight.

A single colony of the transformants was inoculated into LB medium containing ampicillin (100 µg/mL) overnight. Fifty microliters were then sub-cultured in fresh LB broth containing 100 µg/mL ampicillin (5 mL) to reach an optical density at 600 nm wavelength (OD₆₀₀) of 0.6. Induction was performed using 0.1 mM isopropyl β-d-thiogalactoside (IPTG, Thermo Fisher Scientific Co., Waltham, MA, USA) and the gene was expressed for 3.5 h at 30 °C in a shaker incubator at 170 RPM. The cells were harvested by centrifugation (10,397 g, 10 min, 4 °C) (Sigma 3-18K, Osterode am Harz, Germany), and frozen at −20 °C.

Host cells were suspended in 15 mL of lysis buffer (50 mM sodium phosphate buffer, 300 mM NaCl and 10 mM Imidazole with a pH of 8.0) and disrupted through sonication (Hielscher Co., Teltow, Germany) at 30% amplitude and cycled 30 s for 4.5 min. The cell lysate was centrifuged at 10,397 g for 10 min at 4 °C before being filtered through a membrane with 0.22 µm porosity.

The Ni-NTA agarose (Qiagen Co., Hilden, Germany) column was washed and equilibrated with washing buffer (50 mM sodium phosphate buffer, 300 mM NaCl and 20 mM imidazole at a pH of 8.0). The suspension was loaded onto the column and was washed by washing buffer as mentioned above to remove the impurities. The recombinant proteins were eluted with 10 ml of elution buffer (50 mM sodium phosphate buffer, 300 mM NaCl and 250 mM imidazole at a pH of 8.0). The eluted fraction was collected and dialyzed against PBS (1x). To check the purity of the eluted Hl-RecPLD1, the collected fraction was subjected to SDS-PAGE, which was performed according to the Laemmli method [49] with some modification. The purified recombinant toxin was loaded onto a 15% polyacrylamide gel and electrophoresed at initially 15 mA for 30 min, followed by 25 mA for 2 h. Protein concentrations were determined using the BCA Protein Assay Kit according to manufacturer instructions (iNtRON Biotechnology Co., Seoul, Korea).

To perform this step with specific antibodies, monovalent antisera were prepared as outlined below.

Sphingomyelinase activity of the purified recombinant protein was assessed by measuring the enzyme-catalyzed hydrolysis of a sphingomyelin analog, TNPAL-SM, according to Borchani et al. in 2011 [27]. The increasing amounts of recombinant toxin and crude venom, including 5, 10 and 15 µg, were incubated with an incubation buffer (190 µL) containing 250 mM Tris–HCl, 20 mM MgCl₂, 0.1% Triton X-100, and 60 nmol of TNPAL-SM at a pH of 7.4. The reaction mixtures were gently shaken for 2 h at 37 °C. Subsequently, the reactions were stopped by adding 375 µL of isopropanol/heptane/H2SO₄ (40:10:1 v/v). 200 µL of both heptane and water were then added per sample. The tubes were centrifuged at 664 g for 5 min to separate the resulting two phases. The upper phase was transferred to a 96-well microplate and OD was read at 410 nm.

The H. lepturus crude venom and PBS (1x) were used as positive and negative controls respectively. The sphingomyelin hydrolysis was expressed as the quantity of TNPAL-SM (as nmol) hydrolyzed per mg of toxin (1 nmol of hydrolyzed TNPAL-SM corresponds to 0.023 absorbance units) [27].

Adult male BALB/c mice (25 g) and female New Zealand Albino rabbits (2–3 kg) were purchased from the Pasteur Institute of Iran. Upon arrival, the animals were allowed to adapt for a week before taking part in the experiment. The dark and light duration cycled every 12 h. The room temperature was 22 ± 1 °C and the relative humidity adjusted at 50% ± 5%. The animals were given a standard pellet diet and fresh clean tap water. All experiments involving animals were performed in accordance with the ethical committee on research animal care agreement in Pasteur Institute of Iran (approval number: IR.PII.REC.1394.86). All the tests were done in triplicate.

This assay was performed as described in literature [52], with some modifications. Fresh human blood was obtained from a healthy donor and washed 3 times with PBS (1x). Serial dilutions of recombinant protein (from 50 to 0.39 µg) were prepared in 100 µL PBS (1x) and 100 µL of washed RBCs suspension (2%) was added to each well of a 96-well microplate (Nunc, Sigma Co., St. Louis, MO, USA).

To evaluate the effect of serum on hemolysis, in a separate series of assays, serial dilutions of recombinant protein (from 50 to 0.39 µg) were prepared in 100 µL human serum. The washed RBCs (2%) were added to each well as detailed above. The microplate was incubated at 37 °C for 2 h and centrifuged at 664 g for 10 min. The supernatant was transferred to a new plate and OD was read at 540 nm in a microplate spectrophotometer (EPOCH, BioTek Co., Winooski, VT, USA). Triton X-100 (2%) and PBS buffer (1x) were used as positive and negative controls respectively. The degree of hemolysis was determined using the below formula.

OD   sample   −   OD   negative   control OD   positive   control   −   OD   negative   control × 100