2.1. Genomic DNA Isolation, Polymerase Chain Reaction (PCR) Amplification, and Sequencing

Genomic DNA from honeybee was extracted by CTAB extraction method. Briefly, 500 ul of lysis buffer (2% w/v CTAB, 100 mM Tris (pH 8), 20mM EDTA, 1.4 M NaCl), and 20 ul of 20 mg/ml proteinase-K were added to the homogenized sample and incubated at 65°C for 1 h. The sample was centrifuged at 13,000 rpm for 1 min and the supernatant was transferred to a fresh 1.5 ml tube. The DNA was purified using phenol: chloroform: isoamyl alcohol (25:24:1), precipitated using isoamyl alcohol, washed with 70% ethanol, air dried, and dissolved in 50 ul nuclease free water. Melittin gene was amplified using forward- 5’GAATTAACAGCATTAACACA-3’ and reverse- 5’-GATTTGATGAATGAAAAATTTAT-3’ primers using Apis cerana cerana melt gene as reference (AJ786346.1) and the PCR amplicon was purified and sequenced.

2.2. Antigenicity Prediction

The antigenicity of melittin was performed using Kolaskar and Tongaonkar antigenicity prediction method. This program predicts those segments from within a melittin protein from A. cerana indica sequence which is likely to be antigenic by eliciting an antibody response and the potent antigenic regions were highlighted with its residues [21].

2.3. Protein Secondary Structure Prediction

The secondary structure of melittin was predicted by GOR Secondary Structure Prediction method on Job GOR4 (ID: 940d2603e1f1) running on NPS@ server. The GOR method analyses sequences to predict alpha helices, beta sheets, turn, or random coil secondary structure at each position based on 17 amino acid sequence windows [22].

2.4. Physicochemical Composition Analysis

ProtParam analysis tool available on Expasy server was used to analyze the physicochemical properties such as molecular weight, instability index, amino acid composition, theoretical pI, and Grand average of hydrophobicity of melittin [23].

2.5. Location in Solvent Accessible Region

ProtScale analysis tool in Expasy server was used to compute the hydrophobic and hydrophilic characteristics of amino acids for any given protein and represent the profile as an amino acid scale. Kyte and Doolittle [24], Hopp and Woods [25], Manavalan and Ponnuswamy [26], Eisenberg et al. [27], Fauchere [28], Janin [29], and Rao and Argos [30] amino acid scale scales were generated for the amino acids in the melittin protein. Determining the position in the protein’s solvent-accessible sections is essential predicting membrane-spanning domains, possible antigenic sites, and areas that are probably exposed on the protein surface.

2.6. Plasmid Construction for Transient Expression

The DNA fragment of the melittin intron was PCR amplified with the melittin PCR amplicon as the template using 2 primer sets; forward-1 (5’-ATATGCTAGCCTGAACCAGAACCG-3’) with Nhe-1, reverse-1 (5’-ATATCTTAAGAATCCTGTGGTTAATACTTT-3’) with Afl-2, forward-2 (5’-TATATGAATTCCTGAACCAGAACCGG-3’) with EcoR-1, and reverse-2 (5’-ATATTCTAGAAATCCTGTGGTTAATACTT-3’) Xba-1 restriction site. Construction of vector for evaluating the transgene expression is based on pVAX-1 plasmid. pVAX-1 is a non-viral 3 KB plasmid with Human cytomegalovirus immediate-early (CMV) promoter, Bovine growth hormone (BGH) polyadenylation (Poly-A) signal sequence, and Kanamycin resistance gene for selection. DNA sequence for coding EGFP was inserted between Hind-III and BamH-1 site to generate pVAX-1/E and used as a control vector. The plasmid pVAX-1/MEL-CIE was constructed by inserting Mel-intron upstream to the expression cassette between CMV promotor and the EGFP using the first set of primers. Similarly, pVAX-1/MEL-CEI was constructed by inserting Mel-intron downstream to the expression cassette between EGFP and BGH-Poly-A tail using the second set of primers. A schematic representation of Melittin-intron cloned in the pVAX-1/EGFP plasmid is shown in Figure 1.

Figure 1: Construction of pVAX-1/Mel-CIE and pVAX-1/Mel-CEI plasmid. (a) Vector map of pVAX-1 plasmid. (b) A schematic representation of intron-less and intron cloned plasmid. pVAX-1/EGFP was constructed by inserting EGFP sequence between Hind-III and BamH-1 site, pVAX-1/Mel-CIE was constructed by cloning melittin-intron between CMV promotor and the EGFP, and pVAX-1/Mel-CEI was constructed cloning melittin-intron EGFP and BGH-Poly-A tail. [Click here to view]

2.7. Cell Culture and Transfection

HEK-293 and CHO-K1 were purchased from NCCS Pune. HEK-293 and CHO-K1 cells were grown in DMEM and RPMI media supplemented with 10% FBS and 1X Pen-Step antibiotics in a humidified incubator with 5% CO₂ at 37°C, respectively. Transient transfection of all the plasmids was performed in 6-well plates using Lipofectamine 2000 (ThermoFisher Scientific) according to the manufacturer’s instruction. 1-day before transfection, 2 ml of exponentially growing cells with cell density 0.3 × 10⁶ cells/ml was seeded in the complete media. At the day of transfection, the complete media was removed and cells were washed with ×1 PBS. Both cell lines were transfected with 2 mg of plasmids in incomplete media for 5 h. After incubation, incomplete media was replaced with complete media and incubated to 24 h. All the transfection experiments were performed in triplicates.

2.8. Fluorescent Microscopy and Real-Time PCR (RT-PCR)

EGFP expression in HEK-293 and CHO-K1 cells was qualitatively determined by visualizing the EGFP expression using Inverted Fluorescent Microscope (Olympus CKX53) and quantitatively measured using RTq-PCR. Total RNA was isolated using TRIZOL, quantified, and converted to cDNA using cDNA conversion kit (PrimeScript™ RT Master Mix, Takara). RTq-PCR was performed using real time Sybrgreen PCR mastermix without ROX (TB Green® Premix Ex Taq™ II (Tli RNase H Plus), Takara) in a RT-PCR (Insta Q48 m, Himedia) according to manufacturer’s instruction and the fold change was calculated by 2-^DDCt method. EGFP expression was relatively quantified at the mRNA level using forward: 5’-AAGCTGACCCTGAAGTTCATCTGC-3’ and reverse: 5’- CTTGTAGTTGCCGTCGTCCTTGAA-3’ primers. GAPDH (Glyceraldehyde phosphate dehydrogenase) gene was used as internal control and primers used for HEK-293 cell line were forward: 5’-GTCTCCTCTGACTTCAACAGCG-3’, Hu-GAPDH-reverse: 5’- ACCACCCTGTTGCTGTAGCCAA-3’, and CHO-K1 cell line were forward: 5’- GAAAGCTGTGGCGTGATGG-3’, and reverse: 5’- CATACTTGGCAGGTTTCTCCAG-3’.

2.9. Statistical Analysis

One-way ANOVA analysis with 95%CI was carried out for statistical comparison between the EGFP expression among the intron and intron-less groups, and P < 0.05 was considered to be statistically significant. Statistical analysis was performed using GraphPaD Prism V 9.0.

3.1. Antigenicity

The partial melittin gene sequence was submitted to the GenBank and received the accession number (MZ356198). The 26 amino acid sequence “GIGAVLKVLTTGLPALISWIKRKRQQ” of matured melittin was considered for all of the in-silico analysis. The antigenic peptide of melittin was predicted using Kolaskar and Tongaonkar antigenicity prediction method. The potential antigenic region had eight residues “AVLKVLTT” showing highest peak starting from position 4–11 and the predicted residues scores of melittin are tabulated in Table 1 with an average antigenic propensity of 1.067. The antigenic plot is shown in Figure 2, where x-axis shows sequence number and y-axis shows average antigenic propensity.

Table 1: Kolaskar and Tongaonkar antigenicity predicted residue score.

Figure 2: Kolaskar and Tongaonkar antigenicity plot of Melittin. The potential antigenic region starts from position 4 to 11 with 8 residues and antigenic propensity was calculated to be 1.067. [Click here to view]

3.2. Protein Secondary Structure

The GOR method generates the code that links the amino acid sequence to the secondary structure of proteins using information theory. To determine the likelihood of each amino acid being present at each position, three scoring matrices are created to retrieve results a data mining approach called cluster analysis is applied. The predicted secondary structure of melittin showed 26.92% of alpha helix, 30.77% of extended strand, 42.31% of random coil, and no beta turn [Table 2]. The result from Kolaskar and Tongaonkar antigenicity prediction method further implies that the eight amino acid residues in the beta sheet is more antigenic when compared to the helical region of the melittin peptide.

Table 2: Secondary structures of Melittin.

3.3. Physicochemical Composition

Utilizing the ProtParam analysis tool on the Expasy server, the physicochemical makeup of melittin was examined and the properties are tabulated in Table 3. The instability index II for the melittin was computed to be 44.73 and the protein was classified as unstable. With respect to the theoretical pI, and the number of charged amino acid residues, the melittin was found to be basic and cationic in nature. Even though the native protein is unstable, the antigenic peptide region could play an essential role in designing peptide and subunit therapeutics.

Table 3: Physicochemical parameters of Melittin.

3.4. Solvent Accessible Region

The solvent accessible region scales are widely used scales for delineating hydrophobic and hydrophilic character of a protein. Because the N- and C-terminal region of a protein are often unstructured, solvent accessible, and the antibodies against these areas are more likely to recognize the native protein, predicting solvent accessibility is crucial to identifying the antigenic location of a specific protein. Since melittin is highly antigenic in nature, we considered Kyte and Doolittle, Hopp and Woods, Manavalan and Ponnuswamy, Eisenberg et al., Fauchere and Pliska, Janin, and Rao and Argos amino acid scale scales. The hydrophobic and hydrophilic indexes showed that the N- terminal region of the melittin protein is hydrophobic and the C-terminal region is hydrophilic in nature and the predicted antigenic peptide was in the N- terminal region [Figure 3].

Figure 3: Hydrophobicity scale for Melittin protein. (a) Kyte-Doolittle hydrophobicity scale for Melittin protein, (b) Hopp-Woods hydrophobicity scale for Melittin protein, (c) Manavalan hydrophobicity scale for Melittin protein, (d) Eisenberg hydrophobicity scale for Melittin protein, (e) Fauchere hydrophobicity scale for Melittin protein, (f) Janin hydrophobicity scale for Melittin protein, and (g) Rao and Argos hydrophobicity scale for Melittin protein. [Click here to view]

3.5. Influence of Melittin Intron in EGFP Expression

To enhance or improve gene expression in mammalian cells, several expression systems use components of the expression module, such as promoters, upstream enhancers and terminator, and Poly-A signals [31]. Non-coding enhancer DNA sequences including introns can enhance exogenous gene expression by positively influencing at the transcriptional and translational level. This phenomenon is known as intron-mediated enhancement (IME) and it is greatly influenced by physiological conditions of the cell, and cell type [32-34]. To test the influence of Melittin-Intron in transgene expression in mammalian cell lines based on the presence and special arrangements of the intron in the expression cassette, we constructed two sets of plasmids containing the intron in two sites, one: between the CMV promoter and EGFP sequence: upstream in the expression cassette (pVAX-1/Mel-CIE), and another between EGFP sequence and BHG Poly A-tail (pVAX-1/Mel-CEI): downstream in the expression cassette. Plasmid containing only EGFP sequence was considered as intron-less control (pVAX-1/EGFP). Results showed that CHO-K1 cells transfected with pVAX-1/Mel-CIE and pVAX-1/Mel-CEI showed no significant changes in the EGFP expression when compared to CHO-K1 transfected with the intron-less control plasmid. Similarly, HEK-293 cells transfected pVAX-1/Mel-CIE showed no significant changes in the EGFP expression when compared to the cells transfected with intron-less control plasmid. However, significant reduction in EGFP expression was observed in HEK-293 cells transfected with pVAX-1/Mel-CEI when compared to the HEK-293 cells transfected with the intron-less plasmid and pVAX-1/Mel-CIE. IME is a network of several known and unknown mechanism that affects gene expression in all levels of gene expression in different cell types and tissue types differently.

Various studies have shown that the introns positioned between the promoter and the gene of interest might increase gene expression, whereas introns that are in the 3’ UTR may have the opposite effect and even lower gene expression [35]. In our present investigation, we found no appreciable increase in the EGFP mRNA transcript in both HEK-293 and CHO-K1 cells when the intron was upstream on the expression cassette and found no appreciable IME. However, EGFP mRNA was severely decreased in HEK-293 when transfected with plasmid containing melittin intron downstream in the expression cassette. This reduction in exogenous gene expression might be due to the increased distance between the U1 snRNA recognition sites in the intron to the transcription start site and associated inefficient transcription re-initiation by the U1 snRNA, transcription factor II human, and RNA polymerase II complex [36-38]. Another possible reason for the reduction in the transgene expression could be due to the nonsense-mediated decay (NMD) of the mRNA. The mRNA molecule might have gotten degraded by proteins involved in the NMD process as the termination codon might have been detected as premature [39-42] [Figures 4 and 5]. Furthermore, melittin intron did not influence exogenous gene expression in CHO-K1 regardless of its position in the expression cassette.

Figure 4: Expression of EGFP in HEK-293 and CHO-K1 cells transfected with pVAX-1/E or pVAX-1/Mel-CIE or pVAX-1/Mel-CEI plasmid. Cells were seeded into 6-well plates and transfected with pVAX-1/E or melittin intron cloned pVAX-1 plasmid. The transfection efficiency was evaluated 24 h after transfection. [Click here to view]

Figure 5: Relative quantification of EGFP expression in HEK-293 (a) and CHO-K1 (b) cells transfected with pVAX-1/E or pVAX-1/Mel-CIE or pVAX-1/Mel-CEI plasmid. The cells were treated with 2 μg of plasmids and Lipofectamin 2000 in 1:2 ratio for 5 h in incomplete media. After incubation the media was replaced with complete media. One way ANOVA analysis with 95% CI was carried out for statistical comparison of the EGFP expression among the intron and intron-less groups, and P < 0.05 was considered to be statistically significant. [Click here to view]