重组弓形虫TRXL蛋白和EN02蛋白免疫保护力的评价
发布时间：2018-11-08 15:14
【摘要】：弓形虫病是由刚地弓形虫(Toxoplasma gondii)引起的一种危害严重的人兽共患寄生虫病,呈世界性分布。弓形虫是一种专性细胞内寄生原虫,可感染大部分哺乳动物和鸟类。对于免疫抑制或免疫功能缺陷者,患弓形虫病可能会造成生命威胁。孕妇感染后可造成流产、死胎,或胎儿出生后先天性缺陷。动物弓形虫病给畜牧业造成极大的损失,并可引起严重的公共卫生问题。因此,弓形虫病的研究对于保障人类公共安全和畜牧业健康发展具有十分重要的意义。为研究刚地弓形虫硫氧还蛋白样蛋白(thioredoxin like,TRXL)和烯醇酶2蛋白(Enolase 2,ENO2)的生物学特性及其作为抗弓形虫疫苗的候选抗原的潜力,本研究针对TRXL和ENO2基因序列分别设计一对特异性引物,通过RT-PCR法扩增了弓形虫GJS株TRXL和ENO2基因,连接至pET-30a(+)原核表达载体。经IPTG诱导表达后,分析rTRXL和rENO2蛋白的反应原性。结果显示,弓形虫TRXL基因全长1 275bp,共编码424个氨基酸,与GenBank中下载的弓形虫ME49株TRXL参考序列的核苷酸序列完全一致。SDS-PAGE分析表明,诱导表达的rTRXL大小约48 kD,与预期大小相符,主要以包涵体形式存在。弓形虫ENO2基因全长全长1 335 bp,可编码444个氨基酸,与Gen Bank中下载的弓形虫ME49株ENO2核酸序列的一致性为99.9%。SDS-PAGE分析表明,诱导表达的rENO2大小约49 kD,与预期大小相符,主要以可溶蛋白形式存在。Western Blotting结果显示,重组蛋白rTRXL和重组蛋白rENO2均能被感染弓形虫的猪阳性血清识别,具有良好的反应原性。进一步将TRXL基因和ENO2基因大量原核表达并纯化,以弗氏佐剂为佐剂,分别应用rTRXL,rENO2重组蛋白及二者的混合蛋白rTRXL+rENO2制成蛋白疫苗免疫小鼠。三个蛋白疫苗均可刺激小鼠机体产生较强的免疫应答反应和一定抗弓形虫急性感染效果,说明三个抗原具有良好的免疫原性和部分免疫保护力,可作为研制新型弓形虫疫苗的候选抗原。
[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.
【学位授予单位】：中国农业科学院
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S852.7
本文编号：2318865
[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.
【学位授予单位】：中国农业科学院
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S852.7
【参考文献】
相关期刊论文 前1条
1 曹建平,徐馀信,韩海勃,宋光承,李小红,卢潍媛,沈玉娟,刘述先;血吸虫硫氧还蛋白免疫原性研究 Ⅱ日本血吸虫硫氧还蛋白DNA疫苗小鼠保护性免疫研究[J];中国血吸虫病防治杂志;2005年04期
相关硕士学位论文 前4条
1 贾佳;硫氧还蛋白系统在衰老大鼠心肌缺血/再灌注损伤中的作用[D];山西医科大学;2007年
2 李永光;多头带绦虫硫氧还蛋白过氧化物酶基因的克隆、表达及抗原性研究[D];甘肃农业大学;2009年
3 盖文燕;旋毛虫丝氨酸蛋白酶抑制因子基因的克隆、表达及抗原性研究[D];中国农业科学院;2010年
4 叶强;弓形虫排泄分泌抗原的功能组分分析与鉴定[D];中国农业科学院;2013年
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