Cloning, Expressing And Studying On Protective Immunity Of Egg Production Related Genes Of Schistosoma Japonicum (Chinese Strain)

The egg plays a central role in the biology of S. japonicum. It is the primaryagent of pathogenesis in hosts and is responsible for dissemination of the parasite.Therefore, the egg and the development of the female reproductive system thatproduces the egg are potential targets for pharmacological attack and/or vaccinedevelopment. Aimed at identifying an egg production related gene and exploringits function, providing the basis for understanding the molecular mechanisms ofegg formation, development and mature and developing new candidate S.japonicum vaccine antigens~ amplify two genes encoding for Cathepsin L andSjEGP. Design and synthesis specific oligonucleotide primers; total RNA wasisolated from adult worms of Chinese S.japonicum using Trizol Kit. theCathepsin L gene was obtained by amplification of cDNA of Schistosomajaponicum ,chinese strain. By cloning target gene into a high level expressionvector ,pET28a(?,a recombinant pET28a(+)-SjCL was constructed. TransferringpET28a-CL into E. coil BL2 1, the fusion recombinant protein was expressed byIPTG eliciting and was analyzed by SDS-PAGE and Western-blot. The micewere challenged with 40 cercariae simutanously after four times immunized witha dosage of 5Oug pure recombinant Cathepsin L protein plus adjuvant or adjuvantalone respectively The adult worms as well as the eggs from the liver tissues ofmice were counted in 42 days post infection.The size of the amplified CathepsinL gene was in accordance with the expected one matelly inditing the recombinantpET28a(+)-SjCL was successfully constructed. The results of the recombinantCathepsin L protein was expressed in E. coil. SDS-PAGE and western blotrevealed that the molecular weight of recombinant protein was approximately3 lkD and can be specifically recognized by rabbit serum against Schistosomajaponicum adult worm antigen preparation (SWAP) ,showed that the expressedproduct possesses good antigenicity. Compared with the control group, the adultworm burdens and EPG in the livers, deject a reduced 36.04%, 34% and 49%respectively when challenged with 40 cercariae after the vaccination. The gene68encoding Cathepsin L was ampified from cDNA of Schistosoma japonicumchinese strain, a recombinant pET28a-CL was successfully constructed. Therecombinant Cathepsin L protein, was expressed in E. coil and can be used tocarry out further study in pathology an immunology of Schistosomajaponicum .The results confirmed that Cathepsin L might be regarded as thecandidate anti-pathologic vaccine molecule against schistosomiasis.Encoding region gene of SjEGP was amplified by RT-PCR techniques; thefragment from RT-PCR was firstly subcloned into cloning vector pBluescript viaBamH I and HindIII restriction sites and cloned into eukaryotic expression vectorpcDNA3 also via BamH I and HindIII restriction sites; the resulting constructwas determined by PCR and restriction analysis methods. The coding region ofSjEGP gene was specifically amplified by RT-PCR, the size of amplifiedfragment was 639 base pairs, the cloning plasmid construct (pBluescript-SjEGP)and expression plasmid (pCD-SjEGP) contained the amplified fragment. Theresults demonstrated that the amplified fragment was identical with the predicatedone, the eukaryotic expression plasmid successfully constructed and provided the basis for further study on DNA immunization.