重组GRA5蛋白免疫诊断弓形虫病价值的研究

发布时间：2018-11-20 06:01

【摘要】：目的原核表达的刚地弓形虫致密颗粒蛋白5(rGRA5)免疫诊断价值的探讨。方法 GRA5基因在RTPCR被扩增,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,pET28a-GRA5IPTG诱导表达后,SDS-PAGE和Western blot法检测该重组蛋白的表达。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 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Gen Bank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)。本实验建立的ELISA法检测的100例弓形虫感染者(血清学阳性)血清中有73例呈阳性,阳性率为73.0%(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)。结论本实验获得的原核表达的rGRA5具有一定的弓形虫病免疫诊断的潜在价值。
[Abstract]:Objective to investigate the immunological diagnostic value of dense granule protein 5 (rGRA5) of Toxoplasma gondii expressed in prokaryotes. Methods the GRA5 gene was amplified by RTPCR, and the prokaryotic expression vector of pET28a-GRA5 was constructed. The expression of the recombinant protein was detected by SDS-PAGE and Western blot methods after the expression of the recombinant protein was identified by endonuclease digestion and sequencing. After the expression was induced by pET28a-GRA5IPTG, the expression of the recombinant protein was detected by SDS-PAGE and Western blot methods. An indirect ELISA assay was established to detect Toxoplasma gondii specific antibodies in serum of purified recombinant proteins. Results the GRA5 gene with the size of 363 bp was amplified by PCR reaction, and the pET28a-GRA5 prokaryotic expression vector was constructed by double enzyme digestion. The DNA sequencing results showed that the length of the inserted GRA5 gene was 100 homology with the GRA5 gene entered in Gen Bank by Blast comparison. The recombinant protein expressed by prokaryotic cells was displayed in both SDS-PAGE and Western blot (about 14 ku). Of the 100 cases of Toxoplasma gondii infection (seropositive) detected by ELISA method, 73 cases were positive, the positive rate was 73.0% (73 / 100), and the positive rate of 40 IgG positive specimens was 72.5% (29 / 40). The positive rate of 30 IgM positive specimens was 53.3% (16 / 30), the positive rate of 30 IgG,IgM positive samples was 93.3% (28 / 30), and the positive rate of 30 negative serum samples was only 6.7% (2 / 30). Conclusion the prokaryotic expression of rGRA5 obtained in this study has a potential value in immunological diagnosis of toxoplasmosis.
【作者单位】：安徽医科大学病原生物学教研室;太和县中医院检验科;
【基金】：安徽省高校省级自然科学研究重点项目(编号:KJ2016A353)
【分类号】：R531.8

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