重组GRA5蛋白免疫诊断弓形虫病价值的研究

Study of recombinant GRA5 protein on immuno-diagnosis of Toxoplasma infection

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。

Objective To investigate the immunodiagnostic valueofrecombinant granulocvte protein 5（rGRA5） protein in prokaryotic Toxoplasma gondii. Methods The PCR was used to ampliify GRA5 gene,then insert the target gene into the prokaryotic expression vector pET28 a,and identified by double digestion and sequencing,then transfecte the correct targert gene into BL21 competent cells,SDS-PAGE and Western blot were used to detect the expression of the recombinant protein in BL21. ELISA was used to detect the serum of 100 cases of serologically Toxoplasma-positive individuals. Results The products of GRA5 gene of 363 bp in length was successfully amplified by PCR,the prokaryotic expression vector pET28a-GRA5 was constructed successfully,the result of double digestion showed that the insert fragment size was correct,and DNA sequencing results showed that the homology of GRA5 gene with Gen Bank was 100%. Also the expression of GRA5 protein was successfully detected in BL21 by Western blot（about 14 ku）. ELISA method was used to detect 100 cases of patients with 73 cases showed positive results,the positive diagnosis rate was 73. 0%. Among them,the positive detection rate of IgG positive samples was 72. 5% in 40 cases,the positive detection rate of IgM positive samples was 53. 3% in 30 cases,the positive detection rate of IgG and IgM positive samples was 93. 3% in 30 cases. Conclusion The prokaryotic expression vector pET28a-GRA5 is constructed successfully,and the recombinant protein has potential for immunological diagnosis of toxoplasmosis.