Cloning And Expression Of S.japonicum 8kDa Calcium-Binding Protein Gene And The Evaluation For The Immunoreactivity Of The Recombined Protein

Schistosomiasis is prevalent in 76 countries or regions. More than 200 million people are infected and 600 million others are at risk of infection. Among the persons infected by Schistosome, it is estimated that at least 120 million have the symptom of Schistosomiasis, 20 million have the serious result, and 2 million are dead of it every year. There are 12 provinces and 418 counties remaining to be uncontrolled in South of China and the population in the endemic area is about 990 thousand by the end of 2001.Although Schistosomiasis is mainly restrained by killing the snails and chemical treatment, control of this parasite is hampered by the limitations of current chemotherapeutics (toxicity, administration regimes, drug resistance, etc.), frequent re-infection, limited prospects for the rapid introduction of effective vaccines, and the lack of understanding of many biological aspects of this parasite. Gaining knowledge about the genome of these parasites is of importance for understanding their biology, mechanisms of drug resistance and antigenic variation that determine their escape from the host's immune system.We have constructed the S.japonicum cercariae cDNA library previously, and we have screened a new gene from this library coding for the Sj-8kDa calcium-binding protein. PCR fragments of the this genes were got with the template of the phagemid, in the length of 236bp. After purification, the PCR product was linked with pMD18-T vector, and subcloned into pET32a(+). The recombinant plasmid was transformed into BL21 E. coll , and the recombinant BL21 was induced with IPTG.The soluble protein in the supernatant was separated with the precipitate after ultrasonic cytolysis and centrifugation. The BL21 cell and the BL21 transformed with pET32a(+) were used as two controls. After SDS-PAGE, the specific expressed protein fragments can be seen on the gel. The molecular weight of the protein expressed by pET32a(+)/BL21 is 21kDa, and the fused SjSCaBP is as large as 29kDa.The result of Western-blot suggested that the fused SjSCaBP protein had a positive result to 6-His antibody with a specific reactive band, but had a negative result to the serum of the Schistosoma infected rabbits and the UV attenuated cercaria infected rabbits.The fused proteins were purified from the cytolysis with the Ni -NTA agarose. After analysis by SDS-PAGE, the fused Sj-8CaBP had been well purified with the purity of 90%.The result of ELISA showed the negative result of the fused SjSCaBP protein, when reacting with the serum of the S. japoniucm infected rabbits. It was comparative with the result of Western-blot. Such a result may be caused by the fact that SjSCaBP is only expressed at cercariae stage, its expression can't be detected 24h after its penetration into the host's body, and the half-life of it is very short (<24h). Because of its short-term existence in the definitive host, the SjSCaBP can't arouse inspective immunoreactivity.On the base of finding the new gene for SJ-8CBP, the gene has been subcloned into pET32a(+),the protein has been expressed and purified, and the immunoreactivity of this fused protein has been tested by Western-blot and ELISA. It can be concluded from the results that this protein can't be a good diagnostic antigen, but its vaccinal potential is still unclear. The recombinant Sj-SCBP protein can be collected, and prepared for the animal experiment to test its vaccinal value.