| It has been proved in our laboratory that a well anti-embryonation and anti-fecundity immunity in host can be induced after being immunized with soluble immature egg antigen 26-28kDa(SIEA26-28kDa) or adult worm antigen 31-32kDa of Schistosoma japonicum(Sj31-32). But the difficulties in purifying and preparing the natural molecular vaccine in batches hampered the field application.Recently we devoted ourselves to screen and identify the coding gene related to the natural molecular vaccine and constructed several DNA recombinant vaccines such as pcDNA3.0/SjHGPRT,pcDNA3.0/SjSDISP,pcDNA3.0/SjRPS4,pcDNA3.0/SjRPL7 and pcDNA3.0/SjCB which have induced partial protection.But either vaccine's protective efficacy needed to be further improved.Here we constructed the bivalent vaccine pVAX1/SjRPS4·CB, pcDNA3.0/SjRPS4·CB and studied their immunoprotection effect;then we immuned Kunming mise with pcDNA3.0/SjSDISP twice followed by Sj31-32 once so as to rise the protective efficacy and reduce the dosage of the natural molecular vaccine.ObjectiveIn this research we aimed to construct the bivalent vaccine pVAX1/SjRPS4·CB which is the target gene against Schistosoma japonicum as to study its immunoprotection efficacy;we also.immuned the Kunming mice through priming with pcDNA3.0/SjSDISP and boosting with Sj31-32 to study the protective effect.Methods1) Construction of the bivalent vaccine pVAX1/SjRPS4·CB,pcDNA3.0/SjRPS4·CB and pET-32a/SjRPS4·CBSjRPS4,SjCB were amplified with the templates of pcDNA3.0/SjRPS4,pcDNA3.0/SjCB and spliced to SjRPS4-CB by SOE-PCR (splicing by overlap extension),then the SjRPS4·CB were cloned into the vector pVAX1,pcDNA3.0 or pET-32a and transformed the compETent E.coli DH5αor BL21(DE3) cells.Restriction enzyme digestion,PCR and sequencing results confirmed that recombinant expression plasmids pVAX1/SjRPS4·CB,pcDNA3.0/SjRPS4·CB and pET-32a/SjRPS4·CB were successfully constructed.2) Expression and immunoprotection of the bivalent vaccine pVAX1/SjRPS4·CB and pcDNA3.0/SjRPS4·CBExtracting the plasmids pVAX1/SjRPS4·CB,pcDNA3.0/SjRPS4·CB and injecting the plasmids to quadriceps femoris of Kunming mise to confirm their successful expression by indirect fluorescent assay(IFA) and immunohistochemistry assay.Negative control,the plasmid pVAX1,pcDNA3.0,PBS and positive control,the recombinant plasmid pVAX1/SjRPS4 were also tested at the same time.Then we largely extracted the plasmids to immune Kunming mise as following groups: pVAX1/SjRPS4·CB,pVAX1,pcDNA3.0/SjRPS4·CB,pcDNA3.0/ SjRPS4,pcDNA3.0/SjCB,pcDNA3.0 and PBS group,followed by being challenged with cercariae of Schistosoma japonicum.Immunoprotection against Schistosoma japonicum in mice was evaluated by reduction of worm burden,intrauterine eggs and liver eggs.3) pcDNA3.0/SjSDISP prime-Sj31-32 boost combined immuneAdult worms of were collected from Rabbit 45d after being infected with Schistosoma japonicum and Sj31-32kDa antigens were purifed. pcDNA3.0/SjSDISP and nude pcDNA3.0 were largely extracted.Then Kunming mise were immunized as following groups:NS group, Sj31-32kDa group,pcDNA3.0/SjSDISP prime-Sj31-32 boost group, pcDNA3.0/SjSDISP group and pcDNA3 plasmid group,followed by being challenged with cercariae of Schistosoma japonicum. Immunoprotection against Schistosoma japonicum in mice was evaluated by reduction of worm burden,fecal egg,liver eggs,intestine eggs and intrauterine eggs.Result1) Restriction enzyme digestion,PCR and sequencing results confirmed that recombinant expression plasmids pVAX1/SjRPS4·CB,pcDNA3.0/SjRPS4·CB and pET-32a/SjRPS4·CB were successfully constructed.2) Result of IFA and immunohistochemistry showed intense green fluorescence or brown particles appeared in the muscle cells of Kunming mise immuned with the bivalent 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/SjRPS4·CB,pcDNA3.0/SjRPS4·CB successful.3) Compared with the corresponding plasmid group,both pVAX1/SjRPS4·CB and pcDNA3.0/SjRPS4·CB group gained statistically significant protection result.Also statistically significant was induced compared to the monovalent vaccine pcDNA3.0/SjRPS4 and pcDNA3.0/SjCB on reduction rate of LEPG.No significant was induced between pVAX1/SjRPS4·CB and pcDNA3.0/SjRPS4·CB.4) The worm and the egg reduction rates in Sj31-32kDa group and pcDNA3.0/SjSDISP prime-Sj31-32 boost group were all statistically significant compared with the NS group,while no significant existed between Sj31-32kDa group and pcDNA3.0/SjSDISP prime-Sj31-32 boost group.Also significance existed in the pcDNA3.0 and pcDNA3.0/SjSDISP group compared to NS group.But no significant existed between the latter two groups.These results show that "pcDNA3.0/SjSDISP prime-Sj31-32 boost" can bring out a similar immunoprotection to that of Sj31-32kDa nature molecular vaccine;nude pcDNA3.0 plasmid can induce immunological effect to some extent. Conclusion1,The bivalent vaccine pVAX1/SjRPS4·CB,pcDNA3.0/ SjRPS4·CB and pET-32a/SjRPS4·CB are successfully constructed.2,The bivalent vaccine pVAX1/SjRPS4·CB and pcDNA3.0/ SjRPS4·CB are successfully expressed in the quadriceps femoris of Kunming mise.3,The bivalent vaccine pVAX1/SjRPS4·CB and pcDNA3.0/ SjRPS4·CB can induce similar immunoprotection which is better than the monovalent vaccine on reduction rate of liver eggs per gram.The former is based on pVAX1,the vector for human used,which laid the foundation for next study.4,"pcDNA3.0/SjSDISP prime-Sj31-32 boost" can reduce the dosage of the natural molecule and induce a well immunoprotection similar to that of Sj31-32kDa nature molecular vaccine.
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