瘦素蛋白转基因约氏疟原虫对小鼠体质量的影响

摘要/Abstract

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RT-PCR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200 μl 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%（P < 0.05）。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。

关键词: CRISPR/Cas9技术, 转基因, 约氏疟原虫, 瘦素, 体质量下降

Abstract:

Objective To investigate the effect of leptin transgenic Plasmodium yoelii on mouse body weight. Methods To construct the leptin gene-containing CRISPR/Cas9 recombinant plasmid which had the 5′UTR and 3′UTR of MIF（macrophage migration inhibitory factor） of Plasmodium yoelli 17XNL strain at two ends, the exogenous mouse leptin gene was inserted downstream of MIF coding region through homologous recombination, resulting in the PYC-MIF-Leptin recombinant plasmid. The recombinant plasmid was then electroporated into P.y 17XNL mature schizonts, and the transgenic schizonts were used to infect a Kunming mouse via tail vein injection. The trangenic P.y clone was screened by pyrimethamine selection and identified by PCR. The trangenic or wild-type P.y was used to infect a C57BL/6 mouse respectively. Blood sample was collected through eye ball and tail vein, and immunofluorescence and RT-PCR were performed to determine the expression of leptin protein in the parasites. Finally, PBS （200 μl） containing trangenic or wild-type P.y （1 × 10⁴） was injected through the tail vein into C57BL/6 mice（n = 5 respectively）. The negative control received a same volume of PBS. The changes of parasitemia and body weight were recorded every two days. Results The leptin-expressing recombinant plasmid PYC-MIF-Leptin was constructed successfully. Results of DNA sequencing of transgenic parasites confirmed the integration of leptin gene at the downstream of MIF gene and successful transcription. Immunofluorescence results indicated successful expression of mouse leptin protein. The weight loss was significant in mice infected with transgenic parasites on day 17（17.26 ± 1.40）g, decreased by 10.7%, but not in the other two groups. Both transgenic and wild-type parasites began to decline when parasitemia reached about 10%, but the transgenic parasites proliferated more rapidly. Both disappeared at 23 days. Conclusion Infection with leptin transgenic parasites decreases the body weight of the infected mice.

Key words: CRISPR/Cas9 technique, Transgenic, Plasmodium yoelli 17XNL, Leptin, Weight loss

中图分类号:

R382.319

李楷, 郑鸿, 朱锋, 付雍, 徐文岳. 瘦素蛋白转基因约氏疟原虫对小鼠体质量的影响[J]. 中国寄生虫学与寄生虫病杂志, 2017, 35(1): 36-41.

Kai LI, Hong ZHENG, Feng ZHU, Yong FU, Wen-yue XU. The effect of leptin transgenic Plasmodium yoelii on mouse body weight[J]. Chinese Journal of Parasitology and Parasitic Diseases, 2017, 35(1): 36-41.

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