日本血吸虫DNA多价疫苗pVIVO_2SjFABP-23的构建及其保护性免疫

目的构建日本血吸虫多价DNA疫苗pVIVO2SjFABP-23,并观察其在小鼠抗血吸虫感染中的免疫保护作用。方法根据Sj FABP和Sj23的基因序列合成两对特异性引物,利用重组PCR技术将日本血吸虫Sj FABP和Sj23的基因片段进行拼接,得到融合基因SjFABP-23,再将该片段重组于p GEM-T载体中,进行PCR扩增及DNA序列测定。然后经双酶切将目的片段SjFABP-23克隆到pVIVO2的一个多克隆位点上转化E.coliGT 110获得重组质粒pVIVO2SjFABP-23,免疫小鼠进行免疫保护性实验。结果PCR扩增出一条大小约为1.1Kb的目的片段。免疫小鼠获得52.14%减虫率(P<0.01)和60.93%的减卵率(P<0.01),且均高于单价疫苗pVIVO2Sj23(P<0.05)。结论PCR成功扩增SjFABP-23的基因片段,血吸虫DNA多价疫苗pVI-VO2SjFABP-23构建成功,该疫苗在小鼠抗血吸虫感染中具有比单价疫苗pVIVO2Sj23更好的免疫保护作用。

Construction of the multivalent DNA vaccine pVIVO2SjFABP-23 and its immuno-protection in mice

The multivalent DNA vaccine pVIVO_2SjFABP-23 was constructed and its immuno-protection to Schistosoma japonicum was investigated in mice,in which two couples of specific primers were designed according to gene sequences of Sj-FABP(fatty acid binding protein) and Sj-23(23kDa membrane protein),and these two gene fragments were spliced through a linker sequence by recombinant PCR technique to obtain the fusion gene Sj-FABP-23,which was inserted into vector pGEM-T,identified by restriction analysis digested with BamHI and EcoR1 and then cloned into expression plasmid pVIVO_2/MCS and transformed to E.coli GT110.The recombinant plasmid pVIVO_2-SjFABP-23 was extracted,purified and then inoculated to BALB/c mice through intramuscular route.Mice were challenged with cercariae of S.japonicum 4 weeks after inoculation,and killed to get specimens to count the adult worms and eggs.The experimental results showed that a target fragment with an obvious band of 1.1 kb demonstrated by electrophoresis of SjFABP-23 on 0.7% agarose gel could be amplified by PCR,and inoculation with this fragment into mice induced the reduction of the adult worm counts up to 52.14% and those of eggs up to 60.93% in comparison with those of control group of mice inoculated with blank plasmid pVIVO_2 intrtamuscularly at the same dosage of 100 μ per mouse.It is concluded that the multivalent DNA vaccine expressing SjFABP-23 was successfully constructed and this DNA vaccine demonstrates certain immuno-protection to S.japonicum infection in mice.