重组GRA5蛋白用于弓形虫病免疫诊断的研究

发布时间：2018-04-16 17:11

  本文选题：刚地弓形虫 + 重组致密颗粒蛋白GRA5　； 参考：《安徽医科大学》2017年硕士论文

【摘要】：目的原核表达和鉴定刚地弓形虫致密颗粒蛋白5(GRA5),以期获得大量与天然抗原活性相似的弓形虫重组GRA5蛋白抗原,将获得的重组蛋白进行纯化,应用纯化的GRA5重组蛋白抗原包被在96孔板上,建立ELISA法检测弓形虫感染。方法从GenBank中查到弓形虫GRA5基因序列,根据基因序列设计合成一对引物,用RT-PCR方法将GRA5基因扩增,构建pET28a-GRA5原核表达载体,双核酸内酶切及序列测定进行鉴定,IPTG诱导pET28a-GRA5转化的BL21/DE3菌,SDS-PAGE和Western-blotting分析表达产物并鉴定弓形虫GRA5是否表达。建立以纯化的重组蛋白的间接ELISA法,检测收集样本血清中弓形虫特异性抗体。结果成功构建刚地弓形虫GRA5基因原核表达质粒,PCR反应扩增出为363 bp大小的GRA5基因,所构建的pET28a-GRA5原核表达载体经双酶切显示插入片段大小与上相符,DNA测序结果表明与GenBank中录入的GRA5基因经Blast比对序列同源性100%,原核细胞表达的该重组蛋白在SDS-PAGE和Western blot中均有显示(约14 ku)。将重组GRA5蛋白作为抗原包被在96孔板中,建立了检测弓形虫感染的ELISA方法,ELISA法经过优化后最终确定:最佳抗原包被浓度为10ug/ml、最佳条件为在37℃作用2h后,在4℃包被过夜、弓形虫血清最佳稀释度为1:25,最佳封闭条件为用5%脱脂奶粉在37℃作用2h,二抗最佳工作浓度为1:20000,底物的最佳反应时间为20min。本实验建立的ELISA法检测的100例弓形虫感染病人(血清学阳性)血清中有73例呈阳性,阳性率为73%(73/100),其中40例IgG阳性标本的阳性率为72.5%(29/40),30例IgM阳性标本的阳性率为53.3%(16/30),30例IgG、IgM均阳性标本的阳性率为93.3%(28/30),30例阴性血清标本的阳性率仅为6.7%(2/30)
[Abstract]:Objective to express and identify Toxoplasma gondii dense granuloprotein 5 (GRA5) in order to obtain a large number of recombinant GRA5 protein antigens similar to those of natural antigens, and to purify the recombinant proteins obtained from Toxoplasma gondii (Toxoplasma gondii).The purified GRA5 recombinant protein antigen was coated on 96 well plate to establish ELISA assay for detection of Toxoplasma gondii infection.Methods the GRA5 gene sequence of Toxoplasma gondii was identified from GenBank. A pair of primers were designed and synthesized according to the sequence of Toxoplasma gondii. The GRA5 gene was amplified by RT-PCR method to construct the prokaryotic expression vector of pET28a-GRA5.DNA endonuclease digestion and sequencing were performed to identify the expression products of BL21/DE3 transformed by BL21/DE3 induced by IPTG and Western-blotting analysis, and to identify the expression of Toxoplasma gondii GRA5 (Toxoplasma gondii).An indirect ELISA assay was established to detect Toxoplasma gondii specific antibodies in serum of purified recombinant proteins.Results We successfully constructed the prokaryotic expression plasmid of Toxoplasma gondii GRA5 gene and amplified the 363bp GRA5 gene.The construction of pET28a-GRA5 prokaryotic expression vector was detected by double enzyme digestion. The result of sequencing showed that the inserted GRA5 gene was homologous to the GRA5 gene input in GenBank by Blast alignment sequence. The recombinant protein expressed by prokaryotic cells was expressed in SDS-PAGE and Western blot.All of them were displayed (about 14 kus).The recombinant GRA5 protein was coated in 96-well plate. A ELISA method for detecting Toxoplasma gondii infection was established. The optimized Elisa method was determined as follows: the best antigen coating concentration was 10ugrml, and the best condition was that the antigen was coated overnight at 4 鈩，

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