Construction Of The Schistosoma Japonicum Dna J-like Protein Gene Recombinants And The Protective Immunity Of The DNA Vaccine

ObjectivesAcquire and analyze the new gene of Schistosoma japonicum — Dna J-like protein, and construct eukaryotic and prokaryotic expression vectors for studying the functions of the new gene to find new vaccine candidate for preventing schistosomiasis japonica.MethodsAccording to the c DNA sequence of Sj Dna J-like protein acquired from the adult cDNA library of Schistosoma japonicum (Chinese strain), a pair of primers were synthesized after being designed by PRIMER5.0 software. The Sj DnaJ-like protein gene was amplified by polymerase chain reaction (PCR) and was inserted into the cloning vector of pUCm-T. After cutted by Eco R I + Xba I , Sj DnaJ-like protein was subcloned into eukaryotic expressed vector pcDNA3 .1(+) and prokaryotic expressed vector pWR450-l, pcDNA3 .1(+)/Sj DnaJ-like protein and pWR450-1/ Sj DnaJ-like protein were constructed.The recombinant plasmids were screened by ampicillin and identified with restrictive enzymes, PCR amplification and sequence analysis. The pWR450-1/ Sj DnaJ-like protein was transformed into E.coli DH5α and Sj DnaJ-like protein was expressed in E. coli by IPTG induce. Expressed protein was identitied by SDS-PAGE and Western blotting. Inoculated to several groups of BALB/c mice separately at the quadriceps femoris with the normal sodiumx pcDNA3.1(+) (controls) or recombinantplasmids pcDNA3 .l(+)/S/ DnaJ-like protein (test). After inoculated three times with an interval of 2 weeks, all mice were infected with Schistosoma japonicum cercariaes.During this period, The gene of Sj DnaJ-like protein in quadriceps femoris was indentified by PCR. The expression of Sj DnaJ-like protein in quadriceps femoris was detected withimmuohistochernistry.The changes of serum-antibody ^ lymphocyte proliferation andcytokine were all detected. After 6 weeks, the eggs and adult worms of mice livers were counted respectively.ResultsThe recombinant plasmids pWR450-l/S/ DnaJ-like protein and pcDNA3 .1(+)/ S/ DnaJ-like protein were constructed successfully. pWR450-l/S/ DnaJ-like protein was transfected into E.coli, and Sj DnaJ-like protein expression were observed by SDS-PAGE and Western blotting. pcDNA3 .1(+)/ Sj DnaJ-like protein was constructed too. The immuohistochemistry analysis showed that Sj DnaJ-like protein gene was expressed in the local tissue of the test group mice. Detect the content of NO in serum, the test group's was significantly higher than the others. After stimulated with SWA, splenic lymphocyte multiplication of the test group mice was significantly higher than the others. Inoculation of the mice with pcDNA3 .1(+)/ Sj DnaJ-like protein induced high titers of specific IgG antibodies. The test group emerged high-level specific antibody after three times immunization.Before and after c hallenge infection,the contents oflFN—y in the splenic lymphocyte cell culture supernatant of test group mice was higher than the others. Compared with the control groups, the vaccination group obtain significant reduction in worm burden and eggs in the livers.Conclusion1 > Sj DnaJ-like protein gene was amplified . The recombinant plasmid pWR450-l/S/DnaJ-like protein was constructed. Sj DnaJ-like protein gene was expressed in E.coli.2 ■> pcDNA3 .1 (+)/ Sj DnaJ-like protein gene was constructed.