Sub-cloning and expression of BuPAG-8: A potential antigen for buffalo pregnancy diagnosis

Introduction

Accurate and early pregnancy detection in buffaloes (Bubalus bubalis) is essential for efficient herd management, improved reproductive performance, and sustained milk production (Abdullah et al. 2017; Rajamanickam et al. 2022) [1,14] . Delayed or inaccurate diagnosis leads to prolonged calving intervals, economic loss, and suboptimal reproductive efficiency (Batra et al. 2019; Van Hanh et al. 2020) [3,20] . Conventional methods such as rectal palpation and ultrasonography, although widely used, have practical limitations in early gestation. Biochemical markers offer a more sensitive and non-invasive alternative, and pregnancy-associated glycoproteins (PAGs) have emerged as promising candidates for this role (Szenci 2021; Barbato et al. 2022; Ferraz et al. 2024) [18,2,4] . PAGs are secreted by binucleate trophoblast cells of the placenta and are released into maternal circulation shortly after implantation. Their presence in serum and milk reflects ongoing pregnancy, making them attractive biomarkers for diagnostic assays. In cattle, PAGs have been extensively studied and successfully applied in radioimmunoassay and ELISA-based tests (Holton et al. 2022; Kline et al. 2024) [5,9] . However, in buffalo, despite their recognized importance, the diagnostic application of PAGs is complicated by the large number of isoforms and the limited understanding of their temporal expression patterns (Lotfan et al. 2018, 2019) [11,12] . Recent studies have demonstrated that PAG concentrations in serum and milk can be used not only for early pregnancy detection but also for predicting embryonic mortality in buffaloes (Barbato et al. 2022) [2] . These findings highlight the potential of PAGs in improving reproductive efficiency. Still, a major limitation lies in the lack of purified isoform-specific antigens, which are essential for developing standardized immunoassays. Among the identified isoforms, BuPAG-8 has been recognized as a prominent candidate. The molecular identity of BuPAG-8 has been established through sequence characterization, but its recombinant expression has not yet been reported. [15,6] . For PAGs, such an approach is particularly valuable as it would enable the production of large amounts of antigen for antibody generation and immunoassay development. were prepared from overnight cultures and stored at-80 °C for long-term preservation. Plasmid DNA was extracted from overnight-grown cultures using the Wizard Plus SV Miniprep DNA purification system (Promega, USA). Briefly, bacterial pellets were lysed, neutralized, and clarified, and the resulting supernatant was passed through silica spin columns. After washing, plasmid DNA was eluted in nuclease-free water and stored at-20 °C until further use.

PCR confirmation, restriction digestion, and ligation

The BuPAG-8 gene (1105 bp) was amplified using genespecific primers carrying NdeI and NotI sites. Each 25 µl PCR reaction contained 1× DreamTaq buffer, 0.2 mM dNTPs, 0.5 µM of each primer, 1 µl template DNA, and 1 U DreamTaq polymerase. Amplification conditions were: initial denaturation at 95 °C for 30 s; 30 cycles of denaturation (95 °C, 10 s), annealing (69 °C, 40 s), and extension (72 °C, 1 min); and a final extension at 72 °C for 5 min. The PCR-amplified BuPAG-8 fragment and pET 2 2b(+) vector were digested with NdeI and NotI in Tango buffer at 37 °C for 4 h. Digested fragments were purified from agarose gel and quantified. Ligation was carried out using T 4 DNA ligase (Thermo Scientific, USA) with a 3:1 insert-to-vector molar ratio in 1× ligation buffer.

Reactions were incubated at room temperature for 6-8 h, and the ligation mix was used directly for transformation.

Bacterial transformation

Competent E. coli TOP10 cells were prepared using the CaCl₂ method. Briefly, exponentially growing cultures (OD₆₀₀ 0.25-0.3) were chilled on ice, harvested by centrifugation, and resuspended in ice-cold 0.1 M CaCl₂ followed by storage in 0.1 M CaCl₂ with 15% glycerol at-70 °C. For transformation, 5 µl of ligation mixture was added to 50 µl of competent cells, incubated on ice for 30 min, and subjected to heat shock at 42 °C for 90 s. Cells were immediately chilled, recovered in SOC medium for 45 min at 37 °C, and plated on LB agar containing ampicillin (100 µg/ml). Plates were incubated overnight at 37 °C, and transformants were further propagated in LB broth with ampicillin for plasmid isolation and verification.

Induction and verification of recombinant protein expression

Recombinant E. coli BL21 (DE3) Lemo21 harbouring pET 2 2b(+)-BuPAG-8 was grown overnight in LB broth with ampicillin (100 µg/ml). A 1% inoculum was sub-cultured and grown at 37 °C until OD₆₀₀ reached 0.5-1.0. Expression was induced with 1.0 mM IPTG, while parallel uninduced cultures served as controls. Cultures were incubated for 4-5 h at 37 °C with shaking. Following induction, cells were harvested, resuspended in PBS, and lysed. Soluble and insoluble fractions were separated by centrifugation. Samples from uninduced and induced cultures, as well as soluble and pellet fractions, were mixed with SDS sample buffer, boiled for 3-5 min, and analysed by 12% SDS-PAGE. Gels were stained with Coomassie Brilliant Blue to confirm BuPAG-8 expression and assess its solubility.

Results

Primer design and cloning of BuPAG-8

Specific primers containing NdeI and NotI restriction sites were designed to amplify the BuPAG-8 open reading frame (Table 1). PCR amplification yielded a single product of 1105 bp, which matched the expected size of the target gene (Fig. 1A). The fragment was ligated into the pET 2 2b(+) vector under the control of the T 7 promoter and in-frame with the vector-encoded C-terminal His₆ tag, ensuring compatibility for downstream expression and purification (Fig. 1B). Recombinant plasmids were validated by restriction digestion, which released two distinct fragments corresponding to the 5375 bp pET 2 2b(+) backbone and the 1105 bp BuPAG-8 insert (Fig. 1C). Colony PCR screening of randomly selected clones further confirmed the successful incorporation of the BuPAG-8 gene, with multiple colonies yielding the expected amplicon (Fig. 1D). These results confirmed the successful construction of the recombinant pET 2 2b(+)-BuPAG-8 expression vector.

Expression of recombinant BuPAG-8

The verified construct was transformed into E. coli BL21 (DE3) Lemo21 for protein expression. Following induction with IPTG, SDS-PAGE analysis revealed a distinct band at ~41 kDa, corresponding to the theoretical molecular weight of BuPAG-8 (Fig. 2). The intensity of this band was markedly higher in induced cultures compared with uninduced controls. However, a faint band of similar size was also detected in uninduced lanes, indicating basal or leaky expression from the T 7 promoter, which is commonly observed in strong expression systems. This confirmed that BuPAG-8 was successfully expressed in E. coli in a controlled, IPTG-inducible manner. SDS-PAGE showing induction and solubility of recombinant BuPAG-8 (41 kDa). Lane M: protein molecular weight marker Lane 1-2: uninduced control. Lanes 3-4: induced whole-cell pellet. Lanes 5: induced cell lysate. Lanes 6-7: induced soluble supernatant. A prominent band of 41 kDa, corresponding to BuPAG-8, is observed in induced fractions compared with the uninduced control.

Solubility analysis of recombinant protein

Fractionation of cell lysates revealed that BuPAG-8 was present in both soluble and insoluble fractions. The recombinant protein was detected predominantly in the whole-cell pellet fraction, suggesting aggregation into inclusion bodies, a common outcome for eukaryotic proteins expressed in bacterial systems. Nevertheless, a weaker band of ~41 kDa was also visible in the soluble supernatant (Fig. 2), indicating that a fraction of the recombinant protein was expressed in a properly folded, soluble form. The recovery of soluble BuPAG-8 is an encouraging result, as it may retain structural features closer to the native protein, providing a suitable basis for antibody generation and future immunodiagnostic applications.

Discussion

Pregnancy-associated glycoproteins (PAGs) are important placental proteins secreted by mononucleate and binucleate trophoblast cells in ruminants (Mohanty et al. 2022; Wiltbank et al. 2023) [13,22] . Their presence in maternal circulation has prompted their use as biomarkers for pregnancy diagnosis in cattle and buffalo. However, the multiplicity of PAG isoforms and variation in their temporal expression patterns complicate the identification of a single reliable diagnostic candidate. Among these, BuPAG-8 has been reported to be highly expressed during early gestation (Lotfan et al. 2018; Wiedemann et al. 2018) [11,21] , making it a promising biomarker for early pregnancy detection. In this study, the BuPAG-8 open reading frame was successfully amplified, cloned into the pET 2 2b(+) vector, and expressed in E. coli BL21 (DE3) Lemo21 cells. Construct verification through PCR and restriction digestion confirmed the integrity of the recombinant plasmid. Following IPTG induction, SDS-PAGE analysis revealed a band at 41 kDa, corresponding to the expected molecular weight of BuPAG-8. Interestingly, a faint band was also visible in uninduced cultures, indicating leaky expression, a well-documented feature of T 7 promoter-driven systems (Studier and Moffatt 1986) [17] . While leaky expression may pose problems for toxic proteins, BuPAG-8 did not appear to hinder bacterial growth, suggesting that it is well tolerated in E. coli. The T 7 promoter-based expression system is one of the most powerful tools for recombinant protein expression in E. coli. For this study, the pET 2 2b + vector was selected which provides a favourable environment for proper folding and disulfide bond formation due to the presence of oxidizing conditions and folding enzymes such as disulfide oxidoreductases and isomerases (Sockolosky and Szoka 2013; Taherian et al. 2019) [16,19] . The vector also carries a C-terminal His-tag that greatly facilitates detection and purification of the recombinant protein through Ni-IMAC, while minimizing the recovery of truncated products that may arise from premature termination of translation (Kashani and Moniri 2015) [8] . The use of a C-terminal tag also allows the native N-terminus to remain unaltered, reducing interference with normal folding pathways. The construct was maintained in E. coli Top10 cells, which offer high transformation efficiency and support a high plasmid copy number. This ensured that sufficient quantities of recombinant plasmid DNA were available for sub-cloning and further manipulations (Liu et al. 2018; Ibrahim et al. 2022) [10,7] . Care was taken to avoid nucleotide insertions or deletions during cloning, as such changes could alter the open reading frame and compromise protein structure. For expression, the construct was introduced into E. coli Lemo21 (DE3), a strain derived from BL21 (DE3) and engineered to lack Lon and Omp proteases. The absence of these proteases prevents degradation of recombinant proteins and enhances overall yield. Optimal expression was achieved with induction using 1 mM IPTG for 4-5 hours at 37 °C in small-scale culture tubes. This condition allowed efficient accumulation of BuPAG-8 protein, consistent with expression strategies commonly employed for mammalian proteins in bacterial systems. The recombinant BuPAG-8 protein was successfully expressed in good quantity and was detected in both soluble and insoluble fractions, as verified using the Qproteome cell lysis system. This confirmed the effectiveness of the chosen vector-host combination and the optimized culture conditions for bacterial expression of BuPAG-8.

Conclusion

The present study successfully demonstrated the subcloning and overexpression of buffalo pregnancy-associated glycoprotein-8 (BuPAG-8) in E. coli using the pET 2 2b + vector system. The recombinant protein was efficiently expressed in both soluble and insoluble fractions, providing the first step toward its purification and further characterization. This work establishes BuPAG-8 as a promising candidate for developing species-specific immunoassays aimed at early pregnancy detection in buffalo, a field where reliable biomarkers are still limited. The production of recombinant BuPAG-8 thus opens opportunities for antibody generation and downstream applications that may enhance reproductive management in buffalo.

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