瘦素蛋白转基因约氏疟原虫对小鼠体质量的影响

发布时间：2018-05-26 00:06

本文选题：CRISPR/Cas技术 + 转基因　；参考：《中国寄生虫学与寄生虫病杂志》2017年01期

【摘要】：目的探讨含瘦素(leptin)蛋白转基因约氏疟原虫(Plasmodium yoelli)对感染小鼠体质量的影响。方法设计并构建含小鼠瘦素基因的疟原虫CRISPR/Cas9重组质粒,该质粒两端带有约氏疟原虫17XNL株巨噬细胞迁移抑制因子(macrophage migration inhibitory factor,MIF)的5′和3′同源序列,将外源的小鼠瘦素基因经同源重组插入至MIF基因编码区下游,构建重组质粒PYC-MIF-Leptin。将该重组质粒电转入体外培养的约氏疟原虫成熟裂殖体内,通过尾静脉注射该裂殖体感染雌性昆明小鼠1只,经乙胺嘧啶筛选和PCR鉴定获得转基因约氏疟原虫克隆。将转基因疟原虫和野生型疟原虫感染C57BL/6小鼠各1只,取眼球血和尾静脉血,通过RTPCR和免疫荧光检测瘦素在疟原虫内是否成功表达。将含转基因疟原虫和野生型疟原虫(1×104接种量)的200μl PBS尾静脉注射感染C57BL/6小鼠各5只,阴性对照组注射等量PBS。每两天记录并统计小鼠原虫血症及体质量变化。采用SPSS 19.0软件进行统计学分析。结果构建了含瘦素基因和疟原虫MIF同源重组序列的重组质粒PYC-MIF-Leptin。转基因疟原虫DNA测序结果证实,瘦素基因整合入MIF基因下游,并在疟原虫中成功转录。免疫荧光实验结果表明,转基因疟原虫能够表达小鼠瘦素蛋白。转基因疟原虫组17 d体质量下降尤其明显,为(17.26±1.40)g。野生型疟原虫组和阴性对照组小鼠体质量无明显变化,而转基因疟原虫组体质量下降达10.7%(P0.05)。两种疟原虫都在原虫血症达到10%左右时开始下降,但是转基因疟原虫增殖速度较快,最终在23 d左右均消失。结论表达瘦素基因的转基因约氏疟原虫可降低感染小鼠的体质量。
[Abstract]:Objective to investigate the effect of Plasmodium yoellii (Plasmodium yoellii) on the body mass of infected mice. Methods the recombinant plasmids of Plasmodium CRISPR/Cas9 containing mouse leptin gene were designed and constructed. The recombinant plasmids contained 5 'and 3' homologous sequences of macrophage migration inhibitory factor-MIF, a macrophage migration inhibitor of Plasmodium yoelii 17XNL strain. The recombinant plasmid PYC-MIF-Leptin was constructed by inserting the exogenous mouse leptin gene into the downstream of MIF gene coding region. The recombinant plasmids were transfected into mature Plasmodium yoelii cultured in vitro. One female Kunming mouse was infected with the recombinant plasmid by tail vein injection. The cloned plasmodium yoelii was obtained by ethylamine screening and PCR identification. C57BL/6 mice were infected with transgenic Plasmodium falciparum and wild-type Plasmodium falciparum respectively. Eyeball blood and caudal vein blood were collected. Leptin expression in Plasmodium was detected by RTPCR and immunofluorescence. Two hundred 渭 l PBS containing 1 脳 10 ~ 4 transgenic plasmodium and wild plasmodium were injected intravenously into C57BL/6 mice, and the negative control group were injected with the same amount of PBSs. The changes of protozoemia and body mass of mice were recorded and counted every two days. SPSS 19.0 software was used for statistical analysis. Results the recombinant plasmid PYC-MIF-Leptin containing leptin gene and plasmodium MIF homologous recombination sequence was constructed. The DNA sequencing of the transgenic plasmodium confirmed that leptin gene was integrated into the downstream of MIF gene and successfully transcribed in Plasmodium falciparum. Immunofluorescence assay showed that the transgenic plasmodium could express leptin protein in mice. The body weight of transgenic plasmodium group was 17.26 卤1.40g. There was no significant difference in body mass between wild type malaria parasite group and negative control group, but the body weight of transgenic malaria parasite group decreased to 10.7% (P 0.05). The two plasmodium species began to decrease when the parasitemia reached about 10%, but the growth rate of the transgenic plasmodium was faster, and finally disappeared at about 23 days. Conclusion the transgenic plasmodium yoelii expressing leptin gene can reduce the body mass of infected mice.
【作者单位】：第三军医大学基础医学部病原生物教研室;
【基金】：国家自然科学基金(No.81471976)~~
【分类号】：R382.31

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