【摘要】：弓形蟲(chóng)病是由剛地弓形蟲(chóng)(Toxoplasma gondii)引起的一種危害嚴(yán)重的人獸共患寄生蟲(chóng)病,呈世界性分布。弓形蟲(chóng)是一種專(zhuān)性細(xì)胞內(nèi)寄生原蟲(chóng),可感染大部分哺乳動(dòng)物和鳥(niǎo)類(lèi)。對(duì)于免疫抑制或免疫功能缺陷者,患弓形蟲(chóng)病可能會(huì)造成生命威脅。孕婦感染后可造成流產(chǎn)、死胎,或胎兒出生后先天性缺陷。動(dòng)物弓形蟲(chóng)病給畜牧業(yè)造成極大的損失,并可引起嚴(yán)重的公共衛(wèi)生問(wèn)題。因此,弓形蟲(chóng)病的研究對(duì)于保障人類(lèi)公共安全和畜牧業(yè)健康發(fā)展具有十分重要的意義。為研究剛地弓形蟲(chóng)硫氧還蛋白樣蛋白(thioredoxin like,TRXL)和烯醇酶2蛋白(Enolase 2,ENO2)的生物學(xué)特性及其作為抗弓形蟲(chóng)疫苗的候選抗原的潛力,本研究針對(duì)TRXL和ENO2基因序列分別設(shè)計(jì)一對(duì)特異性引物,通過(guò)RT-PCR法擴(kuò)增了弓形蟲(chóng)GJS株TRXL和ENO2基因,連接至pET-30a(+)原核表達(dá)載體。經(jīng)IPTG誘導(dǎo)表達(dá)后,分析rTRXL和rENO2蛋白的反應(yīng)原性。結(jié)果顯示,弓形蟲(chóng)TRXL基因全長(zhǎng)1 275bp,共編碼424個(gè)氨基酸,與GenBank中下載的弓形蟲(chóng)ME49株TRXL參考序列的核苷酸序列完全一致。SDS-PAGE分析表明,誘導(dǎo)表達(dá)的rTRXL大小約48 kD,與預(yù)期大小相符,主要以包涵體形式存在。弓形蟲(chóng)ENO2基因全長(zhǎng)全長(zhǎng)1 335 bp,可編碼444個(gè)氨基酸,與Gen Bank中下載的弓形蟲(chóng)ME49株ENO2核酸序列的一致性為99.9%。SDS-PAGE分析表明,誘導(dǎo)表達(dá)的rENO2大小約49 kD,與預(yù)期大小相符,主要以可溶蛋白形式存在。Western Blotting結(jié)果顯示,重組蛋白rTRXL和重組蛋白rENO2均能被感染弓形蟲(chóng)的豬陽(yáng)性血清識(shí)別,具有良好的反應(yīng)原性。進(jìn)一步將TRXL基因和ENO2基因大量原核表達(dá)并純化,以弗氏佐劑為佐劑,分別應(yīng)用rTRXL,rENO2重組蛋白及二者的混合蛋白rTRXL+rENO2制成蛋白疫苗免疫小鼠。三個(gè)蛋白疫苗均可刺激小鼠機(jī)體產(chǎn)生較強(qiáng)的免疫應(yīng)答反應(yīng)和一定抗弓形蟲(chóng)急性感染效果,說(shuō)明三個(gè)抗原具有良好的免疫原性和部分免疫保護(hù)力,可作為研制新型弓形蟲(chóng)疫苗的候選抗原。
[Abstract]:Toxoplasma gondii (Toxoplasma gondii) is a serious zoonotic parasitic disease caused by Toxoplasma gondii (Toxoplasma gondii). Toxoplasma gondii is a specific intracellular parasite that infects most mammals and birds. Toxoplasma gondii may be life-threatening for immunosuppressive or immunodeficient persons. Pregnancy infection can cause miscarriage, stillbirth, or congenital defects after birth. Animal toxoplasmosis causes great losses to animal husbandry and can cause serious public health problems. Therefore, the study of Toxoplasma gondii disease is of great significance to ensure human public safety and healthy development of animal husbandry. To study the biological characteristics of thioredoxin like protein (thioredoxin like,TRXL) and enolase 2 protein (Enolase 2ENO2) of Toxoplasma gondii and their potential as candidate antigens for Toxoplasma gondii vaccine. The TRXL and ENO2 genes of GJS strain of Toxoplasma gondii (Toxoplasma gondii) were amplified by RT-PCR and ligated to the prokaryotic expression vector of pET-30a (). The reactivity of rTRXL and rENO2 protein was analyzed after IPTG induced expression. The results showed that the total length of TRXL gene of Toxoplasma gondii was 1275 BP, encoding 424 amino acids, which was consistent with the nucleotide sequence of TRXL reference sequence of Toxoplasma gondii ME49 strain downloaded from GenBank. SDS-PAGE analysis showed that the induced expression of rTRXL was about 48 kD,. In accordance with the expected size, mainly in the form of inclusion body. The total length of ENO2 gene of Toxoplasma gondii 1 335 bp, can encode 444 amino acids. The sequence of ENO2 nucleic acid of Toxoplasma gondii ME49 strain downloaded from Gen Bank is consistent with 99.9%.SDS-PAGE analysis. The result of 99.9%.SDS-PAGE analysis shows that the length of rENO2 induced expression is about 49 kD,. The results of. Western Blotting in the form of soluble protein showed that both the recombinant protein rTRXL and the recombinant protein rENO2 could be recognized by the porcine positive sera infected with Toxoplasma gondii. TRXL gene and ENO2 gene were expressed in prokaryotic expression and purified. Freund's adjuvant was used as adjuvant to immunize mice with rTRXL,rENO2 recombinant protein and their mixed protein rTRXL rENO2 respectively. The three protein vaccines could stimulate the mice to produce a strong immune response and a certain anti-toxoplasmosis acute infection effect, indicating that the three antigens have good immunogenicity and partial immune protection. It can be used as a candidate antigen for the development of new Toxoplasma vaccine.
【學(xué)位授予單位】：中國(guó)農(nóng)業(yè)科學(xué)院
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類(lèi)號(hào)】：S852.7

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本文編號(hào)：2318865