A Porcine DNMT1 Variant: Molecular Cloning and Generation of Specific Polyclonal Antibody

Abstract

DNA methyltransferase 1 (DNMT1), the first-identified DNA methyltransferase in mammals, has been well studied in the control of embryo development and somatic homeostasis in mice and humans. Accumulating reports have demonstrated that DNMT1 plays an important role in the regulation of differentiation and the activation of immune cells. However, little is known about the effects of porcine DNMT1 on such functional regulation, especially the regulation of the biological functions of immune cells. In this study, we report the cloning of DNMT1 (4833 bp in length) from porcine alveolar macrophages (PAMs). According to the sequence of the cloned DNMT1 gene, the deduced protein sequence contains a total of 1611 amino acids with a 2 amino acid insertion, a 1 amino acid deletion, and 12 single amino acid mutations in comparison to the reported DNMT1 protein. A polyclonal antibody based on a synthetic peptide was generated to study the expression of the porcine DNMT1. The polyclonal antibody only recognized the cloned porcine DNMT1 and not the previously reported protein due to a single amino acid difference in the antigenic peptide region. However, the polyclonal antibody recognized the endogenous DNMT1 in several porcine cells (PAM, PK15, ST, and PIEC) and the cells of other species (HEK-293T, Marc-145, MDBK, and MDCK cells). Moreover, our results demonstrated that all the detected tissues of piglet express DNMT1, which is the same as that in porcine alveolar macrophages. In summary, we have identified a porcine DNMT1 variant with sequence and expression analyses.

Keywords: DNMT1; polyclonal antibody; porcine alveolar macrophages; synthetic peptide.

Figure 1

Multiple-sequence alignments of DNMT1 from different species. Sequences of pig (NP_001027526.1), human (NP_001124295.1), mouse (NP_001300940.1), and the cloned porcine DNMT1 proteins were aligned to determine the levels of homology. Black and gray shading highlights the sequence consistency and similarity across all selected sequences, respectively. The DMAP binding (yellow line), RFD (orange line), -CXXC-type zinc finger (brown line), BAH-1 (green line), BAH-2 (blue line), and Dcm (purple line) domains relative to the porcine DNMT1 (NP_001027526.1) are labeled. The red dashed box indicates the DNMT1 antigenic peptide, which we used for anti-DNMT1 antibody development. Blue and orange dashed boxes indicate inserted and deleted regions relative to the reported porcine DNMT1 (NP_001027526.1). To note that each interval of 10 amino acids is indicated as an asterisk.

Figure 2

Phylogenetic analysis of DNMT1 proteins from various species. The phylogenetic tree was constructed from available DNMT1 proteins with the neighbor-joining method in MEGA version 6.06. Scale bar indicates the genetic distance. The cloned DNMT1 is marked with black dots.

Figure 3

Cross-reactivity of the antibody against DNMT1 in various animal cells. (A) HEK-293T cells were transiently transfected with pFlag14–Vector or pFlag14–DNMT1 (cloned) plasmid. Cell lysates were analyzed with anti-Flag or anti-DNMT1 antibodies by Western blot analysis. (B) DNMT1 abundance in the cells derived from various species was measured using Western blot analysis. (C) Amino acid homology with selected antigenic peptide sequences of DNMT1 from different species. The red color letters indicate amino acid sites that differ from the antigenic peptide sequence.

Figure 4

Specific identification of two different porcine DNMT1s. (A) BHK-21 cells were transiently transfected with pFlag–DNMT1 (NM_001032355.1), pFlag14–Vector, or pFlag14–DNMT1 (cloned) plasmids. The cell lysates were collected for analysis with an anti-Flag antibody or anti-DNMT1 antibody in a Western blot analysis. (B) Lysates of porcine cell lines (PK15, ST, PIEC) and PAMs were harvested to determine their DNMT1 expression with Western blotting. (C) Sequencing analysis of various porcine cells based on the selected antigenic peptide sequences. (D) Sequencing analysis of the inserted and deleted regions in DNMT1 from various porcine cells compared with that in the previously reported porcine DNMT1 (used as the control). (E) Approximately 100 mg of each tissue sample (heart, liver, spleen, lung, kidney, small intestine, thymus, inguinal lymph nodes, submaxillary lymph nodes, mesenteric lymph nodes, tonsils, and hilar lymph nodes) of piglets were obtained. Protein samples were prepared according to the material and method section to determine their DNMT1 expression by Western blotting.