Antibodies targeting the European lobster (Palinurus elephas) vitellogenin developed by mRNA isolation and in-silico-designed antigenic peptides

Abstract

Vitellogenin is an essential protein involved in ovary maturation in many animals. Detection of this protein correlated with reproductive capacity may be important if carried out on marine organisms such as the red spiny lobster Palinurus elephas, a crustacean that is an economically important crop from wild fish catches. Moreover, in recent years, vitellogenin has assumed an important role as a possible biomarker of marine environmental pollution, as its expression levels can be influenced by the presence of similar estrogen pollutants and can affect the reproductive sphere of marine organisms such as crustaceans. The P. elephas vitellogenin protein and its coding gene have never been isolated, so there is little information about its presence in this lobster. The aim of the present study was to develop a molecular strategy to create, for the first time, an antibody for the detection and quantization of vitellogenin in P. elephas.

Keywords: Palinurus elephas; Antibody anti-vitellogenin; CODEHOP; ELISA; Vitellogenin; mRNA.

Fig. 1.

(A) Satellite view, obtained through Google Earth, of the Mediterranean Sea, where Sardinia occupies a central position. (B) Sardinia island with the position of FPAs Su Pallosu (central western coast) and Buggerru (southwestern coast) shown with yellow points. (C) Number of ovigerous P. elephas females (eggs stage 2) captured inside (IN) and outside (OUT) FPAs, as well as their size range and egg diameter. The presence of external eggs on the pleopods and carapace length (CL) were used as indicators of maturity.

Fig. 2.

(A) Nucleotides and, below, deduced amino acid sequences of VTG from P. elephas. The underlined regions are predicted to be antigenic regions. (B) Information related to synthetic peptides. An extra ‘C’ (highlighted in green) is added to the C terminus (or N terminus) to facilitate conjugation. The antigenic positively charged residues (K, R) and negatively charged residues (E) are in blue and in red, respectively. The synthesis and epitope predictions were made using GenScript. Residues of VTG enzyme and synthetic peptides are reported with the one-letter code.

Fig. 3.

SDS-PAGE of P. elephas anti-VTG1 and anti-VTG-2 antibodies. The electrophoretic profile obtained under non-reducing conditions indicates the presence of a single band of about 130 kDa for anti-VTG1 (Ab1) and anti-VTG2 (Ab2). Bands of about 25 and 50 kDa (lanes Ab1 red and Ab2 red) were obtained for each antibody after treatment with β-mercaptoethanol. The arrows (←) indicate the anti-bodies anti-VTG bands. The size of the bands (kDa) of the protein ladder (M) is reported on the left.

Fig. 4.

(A) Dispersion plot illustrating the distribution of VTG concentrations obtained with the ELISA using anti-VTG1and (B) anti VTG2. The red dots refer to VTG determinations carried out on the samples of eggs of females captured outside the FPAs, while the green dots refer to determinations carried out on the egg fields of females captured inside the FPAs. The standard curve (A) was obtained using antigenic peptide 1 (R²=0.9757) in the presence of the anti-VTG1 antibody. The standard curve (B) was obtained using antigenic peptide 2 (R²=0.9757) in the presence of the anti-VTG2 antibody. For both antigenic peptides concentrations used were 250, 125, 62.5, 31.25, 15.65, 7.81, 3.9, 1.95 and 0.97 ng/ml. Samples were read at 460 nm. (B) The number of ovigerous females and the estimated VTG concentration range inside and outside FPAs (A,B). The VTG concentration's relationship with both the inside and outside FPAs did not indicate any statistical differences. Fisher's exact test statistic=1; P>0.05.

Fig. 5.

Results of SDS-PAGE (A) and western blot analysis using anti-VTG1 antibody (B). An aliquot of all homogenate samples with a concentration of VTG120-180 ng/ml were pooled into one sample (S1). Similarly, an aliquot of all samples with a concentration of VTG 200-260 ng/ml (S2) was pooled. The same S1 and S2 samples were digested with the subtilisin protease (S1D and S2D samples). The SDS-PAGE (A) and western blot profiles (B) did not show any particular differences between the samples treated and untreated with the protease. The different concentrations of VTG detected by ELISA were also not reflected in the number of immunoreactive bands to the anti-VTG1 antibody. The arrows indicate the presence of an immunoreactive band of about 120 kDa and two very close bands between 80–75 kDa. Similar results were obtained using anti-VTG2 antibody (Fig. S3).

Fig. 6.

An overview of steps involved in the development of P. elephas anti-VTG antibodies. (A) Total RNA was extracted from P. elephas eggs. (B) To obtain cDNAs, P. elephas RNAs were reverse transcribed; to detect the unknown nucleotide sequences of the Vtg gene, CODEHOP PCR was used, starting from aligning the multiple sequences of VTG proteins. (C) The PCR products obtained were used for the preparation of the sequencing samples. (D) The nucleotide sequence acquired experimentally was translated into the amino acid sequence and aligned with protein sequences deposited in NCBI databases. (E) The potential presence of two epitopes in the virtual VTG peptide allowed the biotechnological synthesis of two peptides (peptide 1 and peptide 2), which were used to create two different antibodies for ELISA and western blot experiments (F) to detect the VTG in the females of P. elephas in the second stage of egg maturation.