CLONING AND EXPRESSION ANALYSIS OF PROLIFERATING CELL NUCLEAR ANTIGEN GENE DURING REGENERATION IN HYDRA MAGNIPAPILLATA

Freshwater polyp Hydra is an excellent model organism for studying tissue regeneration, and its strong regeneration capacity originates from a large number of stem cell-like cells in the body. However, whether these cells undergo dedifferentiation or mitosis to produce new cells during regeneration is still unclear. Here, we focus on proliferating cell nuclear antigen (PCNA) protein, a marker of proliferating cells. The cDNA sequence of PCNA in Hydra magnipapillata was cloned by rapid amplification of cDNA ends (RACE) method. The total length of the obtained cDNA is 1240 bp, including a 101 bp 5′ non-coding region, a 302 bp 3′ non-coding region and a 837 bp open reading frame (ORF). The PCNA of H. magnipapillata has 278 amino acids with a putative molecular weight of 30.93 kD. H. magnipapillata PCNA ORF fragment was subcloned into the prokaryotic expression plasmid pET-28b(+), and then transformed into E. coli BL21 (DE3) strain, and finally the recombinant PCNA protein was successfully expressed after induction by IPTG (isopropyl-β-d-thiogalactopyranoside). The recombinant protein was used to prepare the rabbit polyclonal antibodies for western blotting assay (WB). The quantitative real-time PCR (qPCR) and the WB results showed that PCNA expression increased at the middle and late regeneration stage in H. magnipapillata. In conclusion, our results suggest that there might be cell proliferation at wound site and nearby areas of hydra at the middle and late regeneration stage.