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The Cloing、Expression and Initial Assessment of Immunoprection of Thioredoxin Glutathione Reductase of Schistosomiasis Japonicum

Author: HanYanHui
Tutor: LinJiaoJiao;LiXiangRui
School: Nanjing Agricultural College
Course: Preventive Veterinary Medicine
Keywords: Schistosomiasis japonicum recombinant antigen SjTGR-pET-28a and SjTGR-pET32a cloning and expression immunoprotection immunofluorescence
CLC: R392
Type: Master's thesis
Year: 2010
Downloads: 5
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Abstract


Schistosomiasis caused by schistosome, is a wide spread parasitic zoonosis that causes serious healthy problem to both human and animals.For half a century, in preventing the infection of schistosomiasis, our country have acquired great achievement,however, the sole wide efficient drug for schistosomasis can’t avoid high rates of reinfection after mass treatment. Therefore, it is necessary to develop an efficient safe vaccine and new anthelmintic drugs against schistosome.The thioredoxin glutathione reductase is a specific reductase in Schistosomiaisis japonicum.It is very important to protect the worms from the injury of the reactive oxygen species, which are released from their metabolism and the host effector cells adhering to the antibody-coated worms. In the study, SjTGR was cloned、expression in Ecoli, and the immuno protective efficiency was evalued. All the results will assist us to understand more functions about SjTGR and provide the basis for screening new Schistosomiasis vaccine candidate molecules and drug targets.SjTGR was found on the tegument surface membrane by using biotin-avidin in our laboratory experiments. So, we guessed that SjTGR had very importmant function in schistosome. We got the full length cDNA of the SjTGR according to the amplified methods about the study of SmTGR. Based on bioinformatics analysis, the Open Reading Frame for SjTGR was 1791bp, and encoding 596 amino acids with a molecular weight of 96kD, the isoelectric point was 6.38. SjTGR had 82% similarity with SmTGR. So, we study the SjTGR according to the methods on the study of SmTGR. We successfully got the recombinant expression plasmid SjTGR-pET-28a/32a, the two recombinant proteins were solubility protein in Ecoli (DE3) and the molecular weight were 69kD and 72kD, respectively..Western-blotting revealed that, the two recombinant proteins had antigenicity and immunogenicity. We immuned BALB/c mice with the two purified recombinant protein, and SjTGr-pET-28a induced 91.24% worm and 93.37% liver egg burden reduction, SjTGR-pET-32a induced 42.78% worm and 41.29% liver egg burden reduction compared with the blank control. The change of the specific IgG was measured by ELISA; the induced specific IgG was maintained in a high level. It is show that SjTGR had the potential for the anti-schistosomal drugs.Frozen sections were prepared by collecting the worms of the different stages.The protein of TGR were expressed in all the different stages by the immunofluorescence. And the SjTGR was one protein of schistosome tegument surface membrane. Real-time PCR revealed that mRNA of SjTGR had the transcription in different stages. SjTGR had the high expression level in 35d、42d、the female and the male, and the female had the higher level. The worms got to mature and lay eggs in the stages of 35d and 42d. In the stages, the worms started to release more reactive oxygen species. The high expression of TGR was related to minimizing oxidative stress、growth and development and breeding of the schistosome. The study provided a basis for the deeply study of the function of SjTGR and new idea to select new drugs against schistosomiasis.

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