摘要
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。
The multivalent DNA vaccine pVIVO2SjFABP-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 Barn HI and EcoR1 and then cloned into expression plasmid pVIVO2/MCS and transformed to E. coli GT110. The recombinant plasmid pVIVO2-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 96 and those of eggs up to 60.93 96 in comparison with those of control group of mice inoculated with blank plasmid pVIVO2 intrtamuscularly at the same dosage of 100μ per mouse. It is concluded that the multivalent DNA vaccine expressing SjFABP-23 was successfully constructed and this DNA vaccine demonstrates certain immunoprotection to S .japonicum infection in mice.
出处
《中国人兽共患病学报》
CAS
CSCD
北大核心
2006年第2期122-126,共5页
Chinese Journal of Zoonoses
基金
国家863(2004AA2Z3212)项目
