【摘要】：目的:pcDNA3.1(+)-SjP14真核表達(dá)載體的提取及鑒定,并利用重組質(zhì)粒和納米微球-DNA疫苗免疫小鼠,探討其對小鼠血吸蟲感染的保護(hù)作用和納米微球的增強(qiáng)作用。 方法:1. pcDNA3.1(+)-SjP14真核表達(dá)載體的提取和鑒定:從重組質(zhì)粒菌株中提取質(zhì)粒并進(jìn)行雙酶切鑒定,利用該重組質(zhì)粒轉(zhuǎn)染Cos-7細(xì)胞,RT-PCR和Western blotting檢測其體外表達(dá),以驗(yàn)證其能表達(dá)出重組蛋白。2. DNA疫苗的制備及保護(hù)作用研究:制備無內(nèi)毒素DNA疫苗及納米微球-DNA疫苗,用于免疫小鼠。將雌性BALB/c小鼠(6-8周齡,體重18-22g)隨機(jī)分為3組,每組10只,分別為生理鹽水對照組、pcDNA3.1(+)-SjP14組及納米微球-DNA疫苗組。各組小鼠分別肌肉注射100μg/鼠/次,每2周免疫一次,共3次。末次免疫后2周,經(jīng)腹部皮膚感染日本血吸蟲尾蚴(30±1)條/鼠。尾蚴攻擊6w后用頸椎脫臼法處死小鼠,收集成蟲,計(jì)算減蟲率;同時留取肝臟,部分消化后在顯微鏡下行蟲卵計(jì)數(shù),計(jì)算減卵率,部分肝臟用于組織病理學(xué)分析(HE染色法),觀察肝細(xì)胞變化及肉芽腫情況。 結(jié)果:本實(shí)驗(yàn)成功提取了pcDNA3.1(+)-SjP14重組質(zhì)粒,進(jìn)行BamHⅠ和XhoⅠ雙酶切,得到一個大小與PCR擴(kuò)增產(chǎn)物一致的插入片段;純化上述質(zhì)粒,轉(zhuǎn)染至Hela細(xì)胞,經(jīng)新霉素篩選得出陽性單克隆細(xì)胞株,擴(kuò)大培養(yǎng)。RT-PCR和Western blotting結(jié)果顯示,兩種質(zhì)粒轉(zhuǎn)染至Hela細(xì)胞內(nèi)均可表達(dá)。分別制備無內(nèi)毒素pcDNA3.1(+)-SjP14和納米微球-DNA疫苗。以該疫苗免疫BALB/c小鼠,結(jié)果表明:pcDNA3.1(+)-Sj P14p核酸疫苗能明顯提高小鼠的抗血吸蟲感染能力,減蟲率和減卵率分別達(dá)到44.6%和61.6%;納米微球-DNA疫苗免疫能進(jìn)一步提高小鼠抗血吸蟲感染作用,減蟲率和減卵率分別提高至56.2%和73.5%。大體解剖可見,生理鹽水組肝臟呈暗褐色,質(zhì)硬,表面可見彌漫性粟粒樣蟲卵結(jié)節(jié)分布,結(jié)節(jié)隆起密集,體積較大。pcDNA3.1(+)-SjP14組小鼠肝臟色澤略帶暗紅色,質(zhì)地較軟,表面較光滑,隱約可見少量灰白色小點(diǎn),蟲卵結(jié)節(jié)較少;納米微球-DNA疫苗組,肝臟顏色呈鮮紅色,表面光滑,蟲卵結(jié)節(jié)少,質(zhì)軟。肝組織切片鏡下顯示,生理鹽水組小鼠肝組織中可見大量的蟲卵肉芽腫形成,肉芽腫體積較大,周圍有大量炎細(xì)胞浸潤,大量的肝細(xì)胞變性壞死,pcDNA3.1(+)-SjP14組肝臟病變較生理鹽水組明顯減輕,pcDNA3.1(+)-SjP14納米微球組肝臟損傷程度最輕,肝小葉結(jié)構(gòu)基本完整,蟲卵肉芽腫周圍炎癥反應(yīng)輕,肉芽腫面積較小。 結(jié)論:本研究成功地構(gòu)建pcDNA3.1(+)-SjP14-納米微球復(fù)合物, pcDNA3.1(+)-SjP14核酸疫苗有一定程度的抗血吸蟲感染作用,pcDNA3.1(+)-SjP14-納米微球復(fù)合物疫苗免疫能增強(qiáng)小鼠對血吸蟲感染的保護(hù)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 鹵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，

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