Construction Of The Multivalent DNA Vaccine PVAX1/SjHGPRT·SDISP Against Female Fecundity And Its Immunoprotection Effect

It is proved that the soluble immature egg antigens(SIEA26-28kDa) from Schistosoma japonicum play an important role in anti-embryonation and anti-fecundity immunity.Later,we obtained three highly homologous and immunogenic proteins pots through immunological recognition with anti-sera against SIEA26-28kDa and methods of the peptide mass fingerprintmaps and MALDI-TOF-MS analysis.It include the protein spots relevanted to hypoxanthine-guanine phosphoribosyl transferase(HGPRT),succinate dehydrogenase iron-sulfur protein(SDISP).On this base several DNA recombinant vaccines related to the natural molecular SIEA26-28kDa,such as pcDNA3.0/SjHGPRT,pcDNA3.0/SjSDISP,were constructed which have induced partial protection and be proved that it hardly had the risk of allergization to the host.But monovalent vaccine's protective efficacy needed to be further improved.So we constructed the multivalent DNA vaccine pVAX1/SjHGPRToSDISP and studied their immunoprotection effect.Objective1.Construct the monovalent vaccine pVAX1/SjHGPRT,pVAX1/ SjSDISP and the multivalent vaccine pVAX1/SjHGPRT·SDISP which is the target gene against Schistosoma japonicum.2.Study immunoprotection efficacy of the multivalent vaccine pVAX1/SjHGPRT·SDISP compared with the monovalent vaccine pVAX1/SjHGPRT,pVAX1/SjSDISP which induced in mice. Methods1.Construction of the monovalent vaccine pVAX1/SjHGPRT,pVAX 1/SjSDI SP and the multivalent vaccine pVAX1/SjHGPRT·SDISPSjHGPRT,SjSDISP were amplified with the templates of pcDNA3.0/SjHGPRT,pcDNA3.0/SjSDISP and spliced to SjHGPRT·SDISP by SOE- PCR(splicing by overlap extension),then the SjHGPRT,SjSDISP and SjHGPRToSDISP were cloned into the vector pVAX1 and transformed to the E.coli DH5αcells.Restriction enzyme digestion,PCR and sequencing results confirmed that recombinant expression plasmids pVAX1/SjHGPRT·SDISP were successfully constructed.2.Expression and immunoprotection of the monovalent vaccine pVAX1/SjHGPRT,pVAX1/SjSDISP and the multivalent vaccine pVAX1/SjHGPRT·SDISPSixty mouse are divided to five group in radom.A: pVAX1/SjHGPRT·SDISP,B:pVAX1/SjSDISP,C:pVAX 1/SjHGPRT, D:pVAX1,E:NS.Extracting those plasmids and injecting the plasmids to quadriceps femoris of ten Kunming mise to confirm their successful expression by immunohistochemistry assay.Negative control,the plasmid pVAX1 and NS.Then we largely extracted those plasmids to fifty immune Kunming mise in five groups which followed by being challenged with cercariae of Schistosomajaponicum.Immunoprotection against Schistosoma japonicum in mice was evaluated by reduction of worm burden,intrauterine eggs and liver eggs. Result1.Restriction enzyme digestion,PCR and sequencing results confirmed that recombinant expression plasmids pVAX1/SjHGPRT, pVAX1/SjSDISP and pVAX1/SjHGPRToSDISP were successfully constructed.2.Result of immunohistochemistry showed intense brown particles appeared in the muscle cells of Kunming mise immuned with the multivalent vaccine,while no specific fluorescence or brown particles appeared in muscle of the control groups.These results demonstrated the expression of the recombinant plasmids pVAX1/SjHGPRT, pVAX1/SjSDISP and pVAX1/SjHGPRTo SDISP were successful.3.Compared with the NS group and pVAX1 group,both pVAX1/SjHGPRT·SDISP and monovalent vaccine pVAX1/SjSDISP,pVAX1/SjHGPRT group gained statistically significant protection result. Also statistically significant was induced compared to the monovalent vaccine group on reduction rate in uterus.In the same time statistically significant was attained between pVAX1/SjSDISP group and pVAX1/ SjHGPRT group.While the worm and the liver egg reduction rates between pVAX1/SjHGPRT·SDISP and monovalent vaccine group were no statistically significant.Among all the groups the egg reduction rates are higher than the worm reduction rates.Conclusion1.Both the monovalent vaccine pVAX1/SjHGPRT,pVAX1/SjSDISP and the multivalent vaccine pVAX1/SjHGPRT·SDISP are successfully constructed.Both the monovalent vaccine pVAX1/SjHGPRT,pVAX1/SjSDISP and the multivalent vaccine pVAX1/SjHGPRT·SDISP are successfully expressed in the quadriceps femoris of Kunming mise.2.Both the monovalent vaccine pVAX1/SjHGPRT,pVAX1/SjSDISP and the multivalent vaccine pVAX1/SjHGPRT·SDISP can induced partial protection in mice.The multivalent vaccine pVAX1/SjHGPRT·SDISP can induce significant immunoprotection which is better than the monovalent vaccine on reduction rate in uterus.