Cloning Of The Full-length Cdna Encoding Tβrii From Schistosoma Japonicum And Primary Study Of Protective Immunity Of Its Extracelluar Fragment

Schistosomiasis is a parasitic disease transmitted to man and other mammals. As we know, the main pathologic lesions in patients or animals with schistosomiasis japonica are the granuloma formation around eggs deposited in the host tissues and the subsequent liver and intestines fibrosis.Members of the transforming growth factor-beta(TGF-β) superfamily of secreted polypeptide growth factors play important and diverse roles in cellular growth, differentiation, extracellular matrix formation, and immunosuppression. For Schistosoma japonicum(S.j), TGF-βplays an important role in its own growth and development process or in its interaction with the host. TGF-βsuperfamily consists of a series of growth factor peptides which have similar structure and function. There are many TβRs (TGF-βreceptor), including type I receptor (TβRI), type II receptor (TβRII), type III receptor (TβRIII).They are found in many types of cell membrane. TβRI and TβRII play a leading role in the TGF-βsignal transduction. TβRII with serine / threonine kinase activity binds TGF-βand then recruits the corresponding type I receptor to form an active ligand–receptor complex, in which the type I receptor gets phosphorylated and is activated by the type II receptor. Phosphorylation occurs on the serine and threonine residues in a domain in the juxtamembrane region called the GS domain. It shows that SjTβRII plays an important role in TGF-βsignal transduction. It is speculated that Schistosoma japonicum TβRII (SjTβRII) can provide the basis and new ideas for developing anti-schistosomiasis vaccine.Objective:As SjTβRII could play an important role in the parasite TGF-βsignal transduction and was a potential vaccine antigen, the SjTβRII full length cDNA was obtained firstly and analyzed by the means of molecular biology techniques.Then the extracellular fragment of SjTβRII (SjTβRIIout) was cloned to prokaryotic expression plasmid and expressed. Sequently, the recombinant proteins was investigated its antigenicity and immunogenicity, evaluated the immunoprotectivity as vaccine candidates, and determinated the localization and the transcription level of SjTβRII in female, male, egg, cercariae phases.Methods:1. The full-length cDNA sequence of SjTβRII was obtained by RACE method by Invitrogen Biotechnology Co., Ltd and analyzed by bioinformatics softwares. SjTβRII was amplified from S.japonicum adult worm total RNA by special primers through TA cloning.2. SjTβRIIout was cloned into the prokaryotic expression vector pET28a(+) and expressed in E.coli by IPTG induction. The recombinant protein was detected by SDS-PAGE. SD rats were immunized with the purified recombinant protein in order to obtain the antisera and analyze its immunogenicity and antigenicity by Western blotting.3. SjTβRII location was detected in the parasites by the means of immunohistochemistry and measured the transcription level of SjTβRII in female, male, egg, cercariae phases by fluorescent quantitative PCR technology. 4. The BALB/c female mice were immunized by prokaryotic recombinant proteins of SjTβRIIout. The experiment animals were randomly divided into 3 groups: A group was immunized by SjTβRIIout recombinant protein as the experimental group; B group was immunized by SjGST protein as a positive control group; C group was adjuvant control. The immunoprotections were assessed by worm burden and liver eggs per gram(LEPG) reduction percentages.Results:1. The full length sequence of SjTβRII was 2283bp, encoding 760 amino acids. Its theoretical protein molecular weight was 86.488 kDa. The isoelectric point was 6.47. The deduced amino acid sequence from the full-length cDNA of SjTβRII exhibited identity of 70.7 %,28.6 %,18.4 %,18.3 %,18.1 %,17.9 %,17.9 %,17.8 % to that of corresponding genes of Schistosoma mansoni, Echinococcus multilocularis, Gallus gallus, Pan troglodytes, Mus musculus, Taeniopygia guttata, Homo sapiens, Danio respectively. The nucleotide sequence date of SjTβRII had been submitted to NCBI,the accession number was FJ753578. SjTβRII was amplified by specific primers with S.japonicum adult worm total RNA as a template. The amplification product was about 2300bp, consistent with the expected molecular weight. It showed that it was consistent with the sequence obtained by application of the RACE, indentification by PCR, DNA sequencing.2. The recombinant expression plasmids pET28a(+)-SjTβRIIout were successfully constructed confirmed by enzyme restriction,PCR and sequencing. Induced by IPTG,the recombinant fusion proteins was efficiently expressed. The protein was recognized by serum of mouse infected with Schistosoma japonicum and serum of rat immunized with the recombinant protein by Western-blotting, respectively.3. SjTβRII was expressed in the body wall and the parenchymal cells of S.j adult worms and the parenchymal cells of the miracidium within the eggs. SjTβRII was transcripted in the stages of S. japonicum adult, egg, and cercariae. The transcription level of SjTβRII in the cercariae and egg phases were higher than that in the adult phase.4. The mice were challenged with S.j cercariae after immunized with prokaryotic recombinant proteins and SjGST repectively. The worm reduction rate and the egg reduction rate of SjTβRIIout immunized group were 20.62%,38.23%; The worm reduction rate and the egg reduction rate of SjGST immunized group were 30.18%,45.70%. The two groups have significant difference(p<0.05).Conclusion:1. The full-length cDNA sequence of SjTβRII obtained by the application of RACE was 2283 bp. The homology identity of deduced amino acid sequence was high with Schistosoma mansoni, but low with the hosts of human and mouse.2. The prokaryotic SjTβRII recombinant expression plasmid was suceessfully constructed. The recombinant protein was efficiently expressed in E.coli and had highly antigenicity and immunogenicity.3. SjTβRII was expressed in the body wall and the parenchymal cells of S.j adult worms and the parenchymal cells of the miracidium within the eggs.The transcripted level in the stages of egg and cercariae was higher than that in the stage of S. japonicum adult.4. The recombinant protein of SjTβRIIout could induce partial protective immunity against S.japonicum cercaria challenge infection.