Cloning Of Schistosoma Japonicum Ferritin Gene And Its Expression In Brassia Napus

Schistosomasis japonicum threaten Six billion people and infected two billionpeople is serious zoonosis and prevalent in 74 countries through Asia, Africa, America.Schistosoma japonicum vaccine is promising effective method for controlling theSchistosomasis japonicum and become the active area of researches. Mucosa is thefirst line of body. Mucosal immunity can induce mucosal immune response andadaptive immune response (cell-mediated immune response and humoral immuneresponse). Some of the current vaccines, for example the oral poliovaccine, newcastledisease vaccine, have been used in clinical practice. Transgenic plant vaccine is one ofmucosal vaccine vehicle. It is characterized by cheap, safty, convenient. Aim of thisstudy was to prob the possibility of SjFer expression in Brassia napus as vaccinevehicle.1. Cloning and prokaryotic expression of SjFer and preparation of antiseria:Female mice were challenged with 40±S.j miracidium. Forty-five days later, micewere killed and perfused. The adult worm were harvested. Adult worm cDNA librarywere constructed under instruction of cDNA PCR Library Kit and cDNA SynthesisKit. SjFer of 520bp was amplified by PCR from cDNA library and then cloned topMD18-T and sequenced. The results showed the sequence had 100％homology withthat of AF040385 in GenBank. SjFer was inserted into pET32α(+) and transformed inEscherichia coli BL21. The positive clones were ticked and the recombinant plasmidswere determined by PCR, double-ezyme digestion and sequencing. After induced withIPTG, expression of the fragment in E.coli BL21 was analysed by SDS-PAGE andWestern blotting. The results determined by SDS-PAGE showed SjFer couldexpressed rightly. The results detected by Western blotting showed rSjFer had goodantigenicity. The IgG titer of antisera from mice immunizated with rSjFer+Frund'sadjuvant complete was 1:6400.2. The expression of SjFer in Brassia napus: A pair of primers was designedaccording to the SjFer sequence. The SjFer was amplified by PCR with the primers from the cDNA library of Schistosoma japonicum and cloned to pMD18-T. The targetfragment of the positive clones was inserted into pBI121 and transformed inagrobacterium. Then SjFer was transferred again into Brassia napus by agrobacteriummediated system. The regenerated transgenic Brassia napus was selected withkanamycin and confirmed by PCR, Nouthern-blot. The Results showed that SjFer hadtransformed in Brassia napus and was expressed in Transcription. To collect the totalprotein of Brassia napus, Western blotting showed positive. Results showed thatSjFer was expressed in Brassia napus.The results of the research showed that transgenic Brassia napus contained SjFerwere successfully acquired and SjFer was expressed in Brassia napus. This studybuilt foundation for the further study of Schistosoma japonicum vaccine andtransgenic brassia napus vaccine.