日本血吸虫P14基因纳米微球-DNA疫苗的初步研究
发布时间：2018-10-31 15:16
【摘要】：目的:pcDNA3.1(+)-SjP14真核表达载体的提取及鉴定,并利用重组质粒和纳米微球-DNA疫苗免疫小鼠,探讨其对小鼠血吸虫感染的保护作用和纳米微球的增强作用。 方法:1. pcDNA3.1(+)-SjP14真核表达载体的提取和鉴定:从重组质粒菌株中提取质粒并进行双酶切鉴定,利用该重组质粒转染Cos-7细胞,RT-PCR和Western blotting检测其体外表达,以验证其能表达出重组蛋白。2. DNA疫苗的制备及保护作用研究:制备无内毒素DNA疫苗及纳米微球-DNA疫苗,用于免疫小鼠。将雌性BALB/c小鼠(6-8周龄,体重18-22g)随机分为3组,每组10只,分别为生理盐水对照组、pcDNA3.1(+)-SjP14组及纳米微球-DNA疫苗组。各组小鼠分别肌肉注射100μg/鼠/次,每2周免疫一次,共3次。末次免疫后2周,经腹部皮肤感染日本血吸虫尾蚴(30±1)条/鼠。尾蚴攻击6w后用颈椎脱臼法处死小鼠,收集成虫,计算减虫率;同时留取肝脏,部分消化后在显微镜下行虫卵计数,计算减卵率,部分肝脏用于组织病理学分析(HE染色法),观察肝细胞变化及肉芽肿情况。 结果:本实验成功提取了pcDNA3.1(+)-SjP14重组质粒,进行BamHⅠ和XhoⅠ双酶切,得到一个大小与PCR扩增产物一致的插入片段;纯化上述质粒,转染至Hela细胞,经新霉素筛选得出阳性单克隆细胞株,扩大培养。RT-PCR和Western blotting结果显示,两种质粒转染至Hela细胞内均可表达。分别制备无内毒素pcDNA3.1(+)-SjP14和纳米微球-DNA疫苗。以该疫苗免疫BALB/c小鼠,结果表明:pcDNA3.1(+)-Sj P14p核酸疫苗能明显提高小鼠的抗血吸虫感染能力,减虫率和减卵率分别达到44.6%和61.6%;纳米微球-DNA疫苗免疫能进一步提高小鼠抗血吸虫感染作用,减虫率和减卵率分别提高至56.2%和73.5%。大体解剖可见,生理盐水组肝脏呈暗褐色,质硬,表面可见弥漫性粟粒样虫卵结节分布,结节隆起密集,体积较大。pcDNA3.1(+)-SjP14组小鼠肝脏色泽略带暗红色,质地较软,表面较光滑,隐约可见少量灰白色小点,虫卵结节较少;纳米微球-DNA疫苗组,肝脏颜色呈鲜红色,表面光滑,虫卵结节少,质软。肝组织切片镜下显示,生理盐水组小鼠肝组织中可见大量的虫卵肉芽肿形成,肉芽肿体积较大,周围有大量炎细胞浸润,大量的肝细胞变性坏死,pcDNA3.1(+)-SjP14组肝脏病变较生理盐水组明显减轻,pcDNA3.1(+)-SjP14纳米微球组肝脏损伤程度最轻,肝小叶结构基本完整,虫卵肉芽肿周围炎症反应轻,肉芽肿面积较小。 结论:本研究成功地构建pcDNA3.1(+)-SjP14-纳米微球复合物, pcDNA3.1(+)-SjP14核酸疫苗有一定程度的抗血吸虫感染作用,pcDNA3.1(+)-SjP14-纳米微球复合物疫苗免疫能增强小鼠对血吸虫感染的保护性免疫力。
[Abstract]:Aim: to extract and identify the eukaryotic expression vector of pcDNA3.1 ()-SjP14 and immunize mice with recombinant plasmid and nano-microspheres-DNA vaccine to investigate its protective effect on schistosomiasis infection in mice and enhance the effect of nanoparticles. Methods: 1. Extraction and identification of pcDNA3.1 ()-SjP14 eukaryotic expression vector: the recombinant plasmid was extracted from the recombinant plasmid strain and identified by double enzyme digestion. The recombinant plasmid was transfected into Cos-7 cells, and its expression in vitro was detected by RT-PCR and Western blotting. To verify the expression of recombinant protein. 2. 2. Preparation and Protective effect of DNA Vaccine: preparation of endotoxin-free DNA vaccine and nano-microsphere DNA vaccine for immunizing mice. The female BALB/c mice (6-8 weeks old, weight 18-22 g) were randomly divided into 3 groups: normal saline control group, pcDNA3.1 () SjP14 group and nano-microsphere DNA vaccine group. Mice in each group were injected intramuscularly with 100 渭 g / mouse for 3 times every 2 weeks. 2 weeks after the last immunization, cercariae of Schistosoma japonicum were infected with (30 卤1) cercariae per mouse through abdominal skin. After cercariae were attacked for 6 weeks, the mice were killed by cervical dislocations, the adult worms were collected and the worm reduction rate was calculated. At the same time, the liver was retained, and the egg count was counted under microscope after partial digestion, and the egg reduction rate was calculated. The liver was used for histopathological analysis (HE staining) to observe the changes of hepatocytes and granuloma. Results: the recombinant plasmid of pcDNA3.1 ()-SjP14 was successfully extracted and digested with BamH 鈪，
本文编号：2302664
[Abstract]:Aim: to extract and identify the eukaryotic expression vector of pcDNA3.1 ()-SjP14 and immunize mice with recombinant plasmid and nano-microspheres-DNA vaccine to investigate its protective effect on schistosomiasis infection in mice and enhance the effect of nanoparticles. Methods: 1. Extraction and identification of pcDNA3.1 ()-SjP14 eukaryotic expression vector: the recombinant plasmid was extracted from the recombinant plasmid strain and identified by double enzyme digestion. The recombinant plasmid was transfected into Cos-7 cells, and its expression in vitro was detected by RT-PCR and Western blotting. To verify the expression of recombinant protein. 2. 2. Preparation and Protective effect of DNA Vaccine: preparation of endotoxin-free DNA vaccine and nano-microsphere DNA vaccine for immunizing mice. The female BALB/c mice (6-8 weeks old, weight 18-22 g) were randomly divided into 3 groups: normal saline control group, pcDNA3.1 () SjP14 group and nano-microsphere DNA vaccine group. Mice in each group were injected intramuscularly with 100 渭 g / mouse for 3 times every 2 weeks. 2 weeks after the last immunization, cercariae of Schistosoma japonicum were infected with (30 卤1) cercariae per mouse through abdominal skin. After cercariae were attacked for 6 weeks, the mice were killed by cervical dislocations, the adult worms were collected and the worm reduction rate was calculated. At the same time, the liver was retained, and the egg count was counted under microscope after partial digestion, and the egg reduction rate was calculated. The liver was used for histopathological analysis (HE staining) to observe the changes of hepatocytes and granuloma. Results: the recombinant plasmid of pcDNA3.1 ()-SjP14 was successfully extracted and digested with BamH 鈪，
本文编号：2302664
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