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A  strikingly upregulated expressed sequence tag was screened from regenerating  rat liver at 8 h in a 0-4-8-12 h short-interval successive partial hepatectomy  model from a previous study. In the present study, a full-length open reading  frame (ORF) corresponding to this expressed sequence tag was predicted through  electronic cloning and was subsequently cloned from an 8-h rat regenerating  liver and deposited in GenBank (accession No. HM448398). Sequence analysis of  HM448398 and the predicted ORF revealed that the two ORFs may be different  transcripts of a gene. The sequence of HM448398 was highly homologous to that  of rat Serpina3n, suggesting that it may be a homolog of Serpina3n. The  pGEX-2TK prokaryotic expression vector for this ORF was constructed, and the  result of sodium dodecyl sulfate polyacrylamide gel electrophoresis manifested  that the recombinant expression vector could express the  glutathione-S-transferase-fused rat homolog of Serpina3n in an insoluble form  in BL21. The target fusion protein was purified with affinity chromatography  and was used as antigen to immunize rabbits for the production of polyclonal  antibodies. Immunohistochemistry and real-time reverse transcription polymerase  chain reaction analysis revealed that the gene was highly expressed in the  priming and termination phases of liver regeneration. These findings lay a  solid foundation for further study of roles of HM448398 using knock-in and RNA  interference methods during liver regeneration.

Cloning and prokaryotic expression of rat homolog of Serpina3n and its expression change during liver regeneration