Construction Of Expression Plasmids With Cathepsin B Endopeptidase Of Schistosoma Japonicum And Study The Protective Immunity Induced By Nucleic Acid Vaccine Of PcDNA3.1(+)/Sjcb2 In Mice

Objectives:To construct the prokaryotic expression plasmid pWR450-l/Sjcb2 containing cathepsin B endopeptidase gene of Schistosoma japonicum and to express the fusion protein in E.coli DH5a.To construct the eukaryotic expression plasmid pcDNA3.1(+)/Sjcb2,then the recombinant plasmid was transfected into HeLa cells,and was inoculated in quadriceps femoris of BALB/c mice.And to observe immune response induced by nucleic acid vaccine pcDNA3.1(+)/Sjcb2 in mice and evaluate its utility for DNA immunization.The main aim was to obtain experiment data for preparation of new nucleic acid vaccine to anti-schistosomiasis.Methods:A pair of primers was synthesized after being designed by PRIMER5.0 software according to the cDNA sequence of Sjcb2 gene. The Sjcb2 gene was amplified by polymerase chain reaction (PCR) and was inserted into the cloning vector of pUCm-T. The gene fragment of Sjcb2 inserted in pUCm-T was subcloned into prokaryotic expressed vector pWR450-l. pWR450-l/Sjcb2 was constructed, the recombinant plasmid was identified with restrictive enzymes, PCR amplification and being sequenced. It was transformed into E.coli and Sjcb2 was expressed in E.coli DH5a by IPTG induce. Expressed fusion protein was identitied with SDS-PAGE and western blotting. And fragment of Sjcb2 inserted in pUCm-T was also subcloned into eukaryotic expression plasmid pcDNA3.1(+) to construct recombinant plasmid pcDNA3.1(+)/ Sjcb2 that was identified with restrictive enzymes, PCR amplification and sequence analysis. The recombinant plasmids were transfected into HeLa cells using electroporation. The expressed protein was identified with immunocytochemistry. 4-6 weeks old BALB/c mice were immunized with pcDNA3.1(+)/Sjcb2 or pcDNA3.1(+) to the quadriceps femoris at 2-week interval for 3 times, and each mouse was challengeinfected with cercariaes of Schistosoma japonicum after 2 weeks since final inoculation. After 42 days, the eggs of every mice liver were counted respectively, and the quantity of adult worm burden of each mice were counted ,also. The gene of Sjcb2 in quadriceps femoris was indentified by PCR. The expression of Sjcb2 in quadriceps femoris was observed with immunohistochemistry. The proliferation response of spleen cells was detected by MTT. Enzyme-linked immunosorbent assay(ELISA) was used for the quantitative detection of the cytokines IFN-y and IL-4 in murine spleen lymphocyte culture medium after stimulating.Results:The recombinant plasmids pWR450-l/Sjcb2 was constructed successfully. And the fusion protein about 86KDa was expressed in E.coli DH5a, Sjcb2 gene expression products were identified by SDS-PAGE and western blotting.The eukaryotic expression recombinant pcDNA3.1(+)/Sjcb2 was successfully constructed, and Sjcb2 was expressed in HeLa cells and located at cytoplasm. Sjcb2 gene could be successfully amplified by PCR in the following days,l,3,7,10,14 days after the first immunization, 14 days after the second immunization, and 14 days after the third immunization.The immunohistochemistry analysis showed that the Sjcb2 was expressed in the local tissue of the nucleic acid vaccine pcDNA3.1(+)/Sjcb2 group mice. When SWAP or PHA as stimulator,the proliferation response of spleen cells of nucleic acid vaccine was significantly higher than those of pcDNA3.1(+) and NS group(P<0.05).Before challenge infection, the quantity of IFN-y released from pcDNA3.1(+)/Sjcb2-immunity spleen cells were significantly higher than those of pcDNA3.1(+) and NS group(P<0.05) when SWAP or PHA as stimulator. But the quantity of IL-4 released from spleen cells of nucleic acid vaccine were no difference with pcDNA3.1(+) and NS group(P>0.05). The level of IFN-y in post challenge decreased in nucleic acid vaccine group and pcDNA3.1(+) and NS group. The level of IL-4 in pcDNA3.1(+) and NS group were elevated following challenge(P<0.05), and were significantly higher than those of pcDNA3.1(+)/Sjcb2 group. The worm burden reduction rate and the reduction rate of liver egg in pcDNA3.1(+)/Sjcb2 group were significantly higher than those of the pcDNA3.1(+) and NS group(/><0.05).Compared with the pcDNA3.1(+) and NS group, the worm burden reduction rate was 36.32%, and the reduction rate of liver egg was 60.61%.