Moleculer Cloning And Expression Of Rio1and Rio2Protein Kinases Encoding Genes From Schistosoma Japonicum

Schistosoma japonicum which causes parasitic zoonosis is currently endemic in the south of China and some tropical or subtropical regions of southeast Asia. Not only human beings but also a series of mammalian hosts including buffaloes, pigs, cattle, sheep, dogs and rodents are in the risk of being infected by S. japonicum in China. In the past six decades, Chinese government has invested an abundant of manpower and financial resources in preventing and controlling schistosomiasis. Three methods, including chemotherapy, snail control and health educatlon, were employed and highlighted for their effects on the control of schistosomiasis japonica. Although the chemotherapy based on Praziquantel(PZQ) inhibited significantly the morbidity of schistosomiasis japonica in China, the emergency of durg resistance of parasite to PZQ suggested that the development of new anti-schistosoma drugs and vaccines is needed for schitosomiasis control in the future.Protein kinases are required for the regulation of a series of cellular processes including DNA replication, transcription, translation and cell cycle progression, and many of them has become extremely significant drug targets. According to their structural differences, protein kinases were divided into two groups, eukaryotic protein kinases (ePKs) and atypical protein kinases (aPKs). RIO kinases, a group of members of atypical protein kinases, were reproted to include13families. RIO kinases, an ATP-dependent serine/threonine protein kinase family, consist of Rio1, Rio2and Rio3. Riol and Rio2exist in various organisms from archaea to human, while Rio3presents only in multicellular eukaryote. With the crystal structure of archaea Riol and Rio2elucidated, we have known that RIO kinases contained a conservative RIO kinase domain. The functional study of yeast Rio1and Rio2demonstrated that RIO kinases are essential for the biosynthesis of the ribosome and the cell cycle process. To date, there are no any articles having been published on the structures and functions of RIO kinases of trematode. Therefore, we investigated Rio1and Rio2of Schistosoma japonicum in the following aspects:1) Obtained the full length cDNAs of S. japonicum Rio1and Rio2and made related bioinformatics analysisThe full length cDNAs of Sj-riol and Sj-rio2protein coding genes is isolated by RACE. The ORF of Sj-riol is1269bp in length, encoding422amino acids. Its coding sequences contain3exons and2introns. The lengthes of3exons are139bp,488bp and642bp, respectively. The introns are3036bp and7188bp, respectively. The ORF of Sj-rio2is1476bp long, encoding491amino acids. Its ORF consists of7exons and6introns. The exons are69bp,154bp,350bp,335bp,96bp and307bp in length, respectively, whereas the introns are38bp,154bp,36bp,4751bp,1406bp and2636bp long, respectively. Evolutionary tree analysis suggests that Sj-riol and Sj-rio2each has the closest genetic relationship with their homologue of S. mansoni, respectively. The genetic relationship with RIO of nematodes is closer than with that of mammals. Alignment of predicted amino acid sequences of Riol and Rio2of S. japonicum with homologues from a range of enkaryotic species, found that Sj-riol and Sj-rio2both contain the conservative domains similar to that of other homologues, respectively. The peptides length of wHTH, P-loop, Hinge and Catalytic domain in rio2of S. japonicum and S. mansoni is greater than the length of corresponding domain in rio2of other species. Protein property forecast analysis shows that Riolp or Rio2p of S. japonicum is a globulin protein and has no signal peptide.2) Northern blot of mRNA of Rio1or Rio2of S. japonicumThe specific probe of Sj-riol and Sj-rio2, the DNA fragments of Sj-riol and Sj-rio2labeled by [y-32P]ATP, hybridized with total RNA of S. japonicum fixed on the nylon membrane, respectively. The result of northern blot shows that a single band is present in Sj-riol and Sj-rio2. It indicates that RNA alternative splicing doesn’t exist in the mRNA processing of Sj-riol and Sj-rio2, namely there is only one transcript in Sj-riol and Sj-rio2.3) Successfully expressed the recombinant proteins of S. japonicum Riol and Rio2in E. coliThe recombinant proteins of S. japonicum Riol or Rio2fused by GST or6×His are obtained by inserting their intact cds into pGEX-6p-1or pET32-a and expressing the recombinant plasmids in E. coli, respectively. Bioinformatics prediction analysis revealed that the254-254amino acids of Sj-riol and the251-491amino acid of Sj-rio2have higher specificity and antigenicity, so the recombinant and truncated proteins of Sj-riol and Sj-rio2fused by6×His tag have also been expressed and purified.4) Preparation and application of the polyclonal antibody of S. japonicum Rio1and Rio2The truncated recombinant proteins of Rio1or Rio2, which have been purified and then emulsified with freund’s adjuvant, are used to immunize mice. After42days, mice were executed and sera were collected, the polyclonal antibodies of anti-Sj-Rio1or Sj-Rio2were obtained. ELISA showed that the titer of these antibodies were both3200. Furthermore, the activity of immune response was identified by Western blot with recombinant proteins. The results indicated that these antibodies may bind specifically with the corresponding target proteins. The antibodies of anti-Rio1or Rio2were also immunobloted with total proteins of S. japonicum adult worms to detect Rio1and Rio2in vivo, respectively. The experimental results showed that Rio1and Rio2of S. japonicum both are low abundant proteins in vivo.In summary, the coding genes of Sj-rio1and Sj-rio2kinase proteins were cloned and expressed in our study, the corresponding polyclonal antibodies were produced, respectively; The structures and functions of Sj-rio1and Sj-rio2were analysed by comparing with the known structures and functions of their homologues in archaea and yeast, suggesting that their main functions may also be the regulation of ribosome biosynthesis and processing of mitosis. The findings provide foundation for further investigating the functions of RIO kinase in S. japonicum.