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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%(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。
Objective To investigate the effect of leptin-containing gene Plasmodium yoelli on the body weight of infected mice. Methods The plasmodium CRISPR / Cas9 recombinant plasmid containing mouse leptin gene was designed and constructed. Both ends of the plasmids contained 5 ’and 3’ of macrophage migration inhibitory factor (MIF) of 17XNL strain of Plasmodium yoelii ’Homologous sequence. The exogenous mouse leptin gene was inserted into the downstream of the MIF gene coding region by homologous recombination to construct the recombinant plasmid PYC-MIF-Leptin. The recombinant plasmids were electroporated into mature Schizosaccharomyces pallidum Schistosome schizonts in vitro, and one female Kunming mouse was infected via tail vein injection of the schizonts. After screening with pyrimethamine and PCR, the transgenic P. yoelii clone . Plasmodium falciparum and wild-type Plasmodium were inoculated into one of C57BL / 6 mice respectively. The blood and tail venous blood were collected. The expression of leptin in Plasmodium was detected by RTPCR and immunofluorescence. Five C57BL / 6 mice were each injected intravenously with 200 μl of PBS containing the plasmodium malariae and wild-type Plasmodium (1 × 104 inoculum), and the negative control group was injected with an equal volume of PBS. The parasitemia and body weight 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 MIF homologous recombination sequence was constructed. Plasmodium DNA sequencing confirmed that the leptin gene was integrated into the downstream of the MIF gene and successfully transcribed in the parasite. Immunofluorescence results show that the transgenic Plasmodium can express mouse leptin protein. The body mass of Plasmodium falciparum 17 d decreased especially significantly (17.26 ± 1.40) g. There was no significant change in the body weight of the mice in the wild-type Plasmodium group and the negative control group, while the body mass of the Plasmodium falciparum group was reduced by 10.7% (P <0.05). Both Plasmodium species began to decline when parasitemia reached about 10%, but the multiplication rate of the transgenic Plasmodium was faster and eventually disappeared around 23 days. Conclusion Transgenic P. yoelii expressing leptin gene can reduce the body weight of infected mice.