Cloning And Identification Of Phosphoprotein P₂ Of Cysticercus Cellulosae And Expression In E.coli

Cysticercus cellulosae is a kind of parasite disease that can infect human beings as well as animals. It is caused by the metacestode of Taenia solium- Cysticercus. It spread widely in our country and all over the world. Cysticercus cellulosae not only threatens human beings?health but also restricts the development of animal husbandry . We can solve the difficulty of the limitation in the diagnostic and the immunological antigen resources by searching for the functional gene and expressing in vitro. Acid ribosomal phosphoprotein P2 locates at the 608 subunit of ribosome . It was proved that the P2 protein played an essential role in protein synthesis by subunit deletion and depression experiment. It was reported abroad that the P2 protein of Cysticercus cellulosae can be recognized by the serum of the neurocysticercosis. In this paper, total RNA was extracted from Cysticercus cellulosae, then mRNA was isolated by using oligo(dT) as a probe. The P2 gene specific primers were devised by GOLDkey software. A 366bp specific fragment was amplified by RT-PCR and ligated into PGEM-Teasy vector. It was identified by restriction endonuclease analysis and PCR and sequencing that this fragment contained the complete open reading frame (ORF) of the P2 gene. In comparison with (I3renebank data,the homologies of the nucleotide sequence and amino acid sequence are 99.45% and 98.36%, respectively. After restriction digest the PGEM-P2 and the pProEX-HTh, respectively, subcloned the interested gene P2 into the prokaryotic expression vector pProEX-HTh. Positive clones were selected by restriction endonuclease analysis and PCR identiflcation.The expression was induced by IPTG, then collected the culture in different times and tested them by ELISA and SDS-PAGE. It showed that the P2 gene was expressed successfully in E.coli. The 1 5KD fusion protein can be recognized by the positive serum of Cysticercus cellulosae.