Studies On Cloning, Expression And Immunological Reaction Of The Gene Encoding Thioredoxin Of Chinese Stain Of Schistosoma Japonicum In Pichia Pastori

Schistosomiasis is a zoonosis causing serious damage to people's health and also is one of the most important infectious diseases to be controlling in China. By 2005, there still are 105 endemic counties(cities, districts) and 38 epidemic-increased counties with 0.798 million infected people and 3.86 billion m² snail habitats in China. Acute schistosomiasis cases are found every year. Schistosomiasis control is still a hard task confronting ChinaTimely and accurate diagnosis of schistosomiasis is one of the key measures for the schistosomiasis control. Pathogenic examine is the only way for the exact diagnosis, but its sensitivity and dependence is low. Compared to it, immunological diagnosis technique of schistosomiasis has a higher sensitivity and specificity, and has been applied to schistosomiasis control in China.The sensitivity for detecting anti-Schistosoma japonicum antibody with schistosomiasis patients is usually higher than 90%. The specificity for healthy people is about 95%～97%. The cross reaction rates for Paragonimusis, Clonorehiasis and Trichinellosis are about 10%～17%, 4%～10% and 8%～13% respectively. The same or similar epitopes between Schistosoma japonicum and other parasites are the main reason for cross reaction.The specificity of immunological diagnostic technique mainly depends on the purity of the antigen. The main antigens used in the diagnosis of schistosomiasis are rough egg or adult worm antigen. They exist certain defects, such as complicated components and higher cross reaction rate. While, the component antigens can not be popularized because the procedure is complicate and the quantity is usually not enough.Along with the rapid development of gene recombine technique and its application in the immunological diagnosis of schistosomiasis, it is possible to improve the diagnosis ability of schistosomiasis. For example, increasing specificity of technique, standardizing and industrial producing the recombinant antigen. Therefore, to screen and clone new recombinant antigen as a specific diagnostic antigen is significant for the control of schistosomiasis.Thioredoxin of Chinese strain of Schistosoma japonicum (SjcTrx) consists of 106 amino acids and its molecular weight is 12kDa. It extensively exists in the worm's body, and has redox modulation and many other biological functions. So, it is a very important protein molecular in the schistosoma' life action. Some experiments showed that thioredoxin of Schistosoma mansoni has a good immunogenicity and immunological reaction, and may become a promising diagnostic antigen.The recombinant antigen produced in E.coli lacks the function of modification and glycosylation epitopes, and its antigenic activity is low. Pichia Pastori is an eukaryotic expressive system, which has been developed recently. It grows much quickly than prokaryotic cells system, and has the function of modification or glycosylation epitopes as mammalian cells. The expressive production has no excessive glycosylation and is easy to be purified. So, it has considerable potential practice value.In order to seeking the possibility of the SjcTrx recombinant antigen as a specific diagnostic antigen of schistosomiasis, the clone, expression and immunological reaction of the gene encoding SjcTrx in Pichia pastori was performed.Firstly, according to cDNA sequence of thioredoxin in Schistosoma japonicum (Phillippine strain), a couple of primers was designed with EcoRâ… restriction endonucleasesite introduced into forward primer with ATG as the start codon and Xbaâ… into reverse prime. Total RNA was extracted from adult worms of Chinese strain of Schistosoma japonicum and the SjcTrx gene was amplified by RT-PCR. Meanwhile, the PCR products and the pPICZαA vector were digested with both EcoRâ… and Xbaâ… , and the target DNA fragments were purified and cloned properly into the expression vector pPICZαA containing AOX1 promoter. After identification by endonuclease digestion, PCR analysis and sequencing, the recombinant vector pPICZαA-SjcTrx was transformed into competent E. coli strain TOP10 and Pichia pastoris strain X-33 respectively, Secreted soluble SjcTrx was produced by induction of the AOX1 promoter with methanol. Recombinant SjcTrx was purified by Ni²⁺-NTA Spin Column Affinity Chromatography. The immunological reactivity of the gene encoding SjcTrx was detected with Western blotting.The research results showed that size of RT-PCR product as judged by agarose gel electrophoresis was around 335 bp, and the same fragments were obtained by restriction enzyme digestion from the recombinant vector pPICZαA -SjcTrx and PCR with the plasmid as a template DNA of the recombinant vector. It were verified again by sequence of SjcTrx fragment from the chromosome. The pPICZαA-SjcTrx was sequenced and shown to be 98. % (313/318)and 70 %(223/318) identical to the DNA sequence, to be 96 %(102/106) and 64 %(68/106) identical to the deduced amino acid sequence of the thioredoxin in Schistosoma japonicum Phillippine strain and Schistosoma mansoni respectively. SDS-PAGE showed the molecular weight of SjcTrx is around 14 kDa and the quantity of expressed protein reach the peak between 48 and 72 hour in BMGY culture media. Western blotting analysis revealed that SjcTrx expressed protein has a good immunological reaction and specificity because it could be recognized by sera from mice infected with Schistosoma japonicum, and not recognized by sera from healthy mice, rat infected with paragonimus, mice infected with Clonorchis sinensis and mice infected with Trichinella spiralis. So, the SjcTrx recombinant antigen has a good immunological reaction and specificityThe clone and expression of gene encoding thioredoxin of Schistosoma japonicum in Pichia pastori has been successful. The recombinant gene product has a good immunological reaction and specificity and is a promise recombinant protein as a specific diagnostic antigen and molecular tool for schistosomiasis study.