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目的构建遗传减毒伯氏疟原虫ANKA株(Plasmodium berghei ANKA,P.b ANKA),并用此减毒疟原虫株免疫C57BL/6J小鼠观察免疫保护效果。方法分别扩增UIS3基因编码序列两端的非编码区5′UTR和3′UTR及用于筛选的抗性标记h DHFR,然后利用融合PCR原理,扩增全长同源重组片段(5′UTR+h DHFR+3′UTR),最后常规PCR扩增获得大量线性化同源重组片段并纯化。体外培养富集伯氏疟原虫ANKA株的成熟裂殖体,将线性化同源重组片段通过电转的方式导入裂殖体中并接种到小鼠体内,再对转化后的疟原虫进行筛选和鉴定,最后用构建成功的减毒伯氏疟原虫ANKA株免疫小鼠并攻击,观察减毒株的免疫保护效力。结果成功扩增3个独立片段5′UTR、h DHFR和3′UTR,长度分别为798、1 628和759 bp,后经融合PCR反应形成全长重组片段5′UTR+h DHFR+3′UTR,长度为3 185 bp。转化至裂殖体后经筛选鉴定获得遗传减毒伯氏疟原虫ANKA株,将此遗传减毒株免疫小鼠后攻击,小鼠红内期原虫率为0%,脑型疟发生率为0%,存活率为100%,可使其完全抵御野生型疟原虫感染。结论成功构建遗传减毒伯氏疟原虫ANKA株,此减毒株对小鼠的免疫保护率为100%,此模型的建立可为研究红前期疫苗免疫保护机制奠定基础。
Objective To construct an attenuated Plasmodium berghei ANKA strain (Plasmodium berghei ANKA, P. b ANKA) and immunize C57BL / 6J mice with the attenuated Plasmodium strain to observe the protective effect. Methods The 5 ’UTR and 3’ UTR of the UIS3 gene coding region and the h DHFR marker for resistance screening were amplified by polymerase chain reaction (PCR). The full length homologous recombination fragment (5’UTR + h DHFR + 3’UTR). Finally, a large number of linearized homologous recombination fragments were obtained by routine PCR and purified. Mature schizonts enriched in ANKA strain of Plasmodium berghei were cultured in vitro. The linearized homologous recombination fragments were introduced into the schizonts by electroporation and inoculated into mice, and the transformed plasmids were screened and identified Finally, the mice were immunized with the constructed ANKA strain of attenuated P. berghei and challenged to observe the immunoprotective efficacy of the attenuated strain. Results The 3 independent fragments of 5’UTR, h DHFR and 3’UTR were successfully amplified and their length were 798,1 628 and 759 bp, respectively. The full-length recombinant fragment 5’UTR + h DHFR + 3’UTR , Length of 3 185 bp. After transformed into schizont, screening and identification of the genetically attenuated Plasmodium berghei ANKA strain, the genetic attenuated strains were challenged in mice after the red end of the mouse parasite rate was 0%, the incidence of cerebral malaria was 0 %, The survival rate of 100%, make it completely resistant to wild type Plasmodium infection. Conclusion The ANKA strain of Plasmodium berghei was successfully constructed. The immune protection rate of this attenuated strain to mice was 100%. The establishment of this model could lay the foundation for the study of immune protection mechanism of pre-red vaccine.