Cloning, Expressing And Studying On Protective Immunity Of Sex-specific Genes Of Schistosoma Japonicum (Chinese Strain)

[Objective] Schistosome parasites are unique among the trematodes that the sex are separate. The development and fertility of the female Schistosome is completely dependent on continuous pairing with male .Unpaired female remain stunted and sexually immature .lnterested in this phenomenon, the study attention in this field have been focused on interaction between the male and female worm. In an effort to shed more light on the molecular mechanism of development and sexually mature of Schistosoma and the effective measure for the prevention of Schistosomiasis , our research aim is to study the sexspecific genes adult worm of Schistosoma japonicum (Chinese strain) and further to investigate their protective immunity.[Methods and Resultsl The study on sex-differentiation of proteins between male and female adult worm of Schistosoma was carried out firstly SDSPAGE -and Two-electrophoresis were used to analyse the sex difference of protein. The SDS-PAGE revealed that female has more proteins than male, but the amount of two molecular(79kDa~. 43kDa) are much more in male than those in female .The female,by comparison, has more amount of four protein polypeptides(35kDa 28kDa ~. 25kDa 22kDa) .The significant sexdifference of proteins were further detected by two-dimensional electrophoresis .A smear consisting of several dots with an MW of 43kDa and a isoelectric point (p1) of 5.60 ?5.90 is longer in the male than that in the female. The female has at least 7 specific molecular(38kDa,pI 5.71; 38kDa,pI 5.65; 35kDa,pI 5.70; 35kDa,pI 5.58; 32kDa,p15.72; 22kDa, p1 5.45; l8kDa,pI5.40)which have not been find in male worms .It shown that sex-differentiation of proteins between male and female adult worm of Schistosoma japonicum is significant.109Based on the result of the above, a pair of primers were designed according to the published SmAct2 to amplify a 1131 bp eDNA fragment by RT-PCR from adult Schistosoma japonicum (Chinese strain) mRNASequence analysis indicated that this fragment, named SjAct, with 92% homology to SmAct2, was a complete ORF of eDNA encoding actin of Schistosomajaponicum (Chinese strain). SjAct was cloned into the expression vector pET28a(+) and subsequently expressed in Escerichia co/i. SDS-PAGE, revealing that the expression of the product, named rSiA ci, was very effective. Probed with the rabbit serum immunized against Sj worm antigen preparation, Western Blotting showed that rSiAct has good antigencity. In order to observe the immunity protection induced by the recombinant protein, further study was performed by vaccinating Kuming mice three times with rSjAct. The mice was then challenged with 40 cercariae of Sj I week after the last injection and perfused 7 weeks post-challenge. The reduction rate of worm and liver egg were 28.4% and 29.2% respectively , indicating the significantly protection in animal immunized with rSjA ci.We also designed the primers on the basis of published SmGCP encoding Gynecophoral canal protein, and a 868 bp eDNA fragment was amplified by RT-PCR from adult Schistosomajaponicum (Chinese strain) mRNA .Sequence analysis indicated that this fragment, named SjGCPJ, was a conserved region of the gene encoding Gynecophoral canal protein of Schistosoma japonicum (Chinese strain), and the sequence was 86.6% homology to SmGCP, SjGCP1 was also cloned into the expression vector pET28a(+) and subsequently expressed in Escerichia co/i. . SDS-PAGE revealed that the molecular weight of this expressed product ,named rSjGCP1, was around 29kD. Western Blotting shown that S]GCP1 was recognized by the rabbit serum immunized against SI worm antigen preparation The recombinant protein was used to vaccinate Kuming mice .The vaccinated mice were then challenged with 40 cercariae of 5] and perfused 7 weeks after the challenge. 3 5.2% worm reduction rate and11037.8% liver Egg reduction rate were obtained in the vaccinated mice comparing with the control group. In order to study the immunity effect of Nuclie acid vaccination of S1GCP1, SIGC...