弓形虫GRA3基因的原核表达及单克隆抗体的制备与初步应用研究
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摘要
弓形虫病(toxoplasraosis),又称弓形体病,它是由一种球虫,即弓形体(Toxoplasma gandii)所引起的一种寄生原虫病。弓形虫体内存在一种重要的分泌抗原致密性颗粒蛋白,经临床人体和动物试验结果显示,致密性颗粒蛋白的反应原性和免疫原性均很高,而目前对GRA3抗原的研究显示,该抗原可以诱发机体产生体液免疫,所以该抗原对于将来用于该病的临床诊断及其疫苗的研究均具有很大价值。本研究旨在成功构建致密性颗粒蛋白GRA3(Dense granle protein GRA3)原核表达载体,进一步利用表达的重组GRA3蛋白抗原制备抗重组GRA3的单克隆抗体,并进行单克隆抗体的鉴定及应用,进一步的研究该抗原的应用价值,为弓形虫病的诊断及亚单位疫苗的研制奠定基础。
本研究根据GenBank AF414079已发表的弓形虫GRA3基因序列设计了一对特异性的引物,利用PCR技术成功扩增出了一段687bp的基因片段,对该基因进行了序列分析,结果显示该基因片段为弓形虫GRA3基因；并成功构建了原核表达载体pGEX-GRA3;将构建好的表达载体,经IPTG诱导,在大肠杆菌BL21中进行了表达,经SDS-聚丙烯酰胺凝胶电泳显示,获得了分子量为50ku的融合蛋白GST-GRA3,经分析大部分以可溶性蛋白的形式存在。Western-Blotting免疫印迹分析,该重组蛋白与弓形虫阳性血清发生特异性反应,说明该蛋白具有一定的反应原性。
为进一步探讨重组蛋白作为诊断抗原的可能性。本研究利用高效表达的重组GRA3蛋白免疫BALB/c小鼠,采用细胞融合技术,经间接ELISA方法筛选和有限稀释法克隆后获得一株抗重组GRA3蛋白的杂交瘤细胞株(5E4)。经鉴定,其McAb的亚型为IgGl型。ELISA和western blotting试验结果表明,获得的5E4McAb可特异性的识别弓形虫GRA3蛋白,表明5E4McAb识别的表位可能位于GRA3蛋白的保守区域。
并以兔抗弓形虫IgG为捕获抗体,利用抗重组GRA3蛋白单克隆抗体为检测抗体,成功建立了用于弓形虫病检测的单抗双夹心ELISA方法,该方法对弓形虫检病检测灵敏度达0.154ug/mL。用该法对延边某猪场152头猪进行检测,结果阳性率为13.2%。并与常规ELISA相比较,该法更加敏感、特异,更有利于准确诊断。为临床研制弓形虫诊断试剂、治疗试剂奠定了基础,为制备疫苗的候选基因的筛选提供依据,同时为弓形虫的检测和区域流行病学调查提供了一种简便快速的诊断方法。
The obligate intracelllar protozoan parasite Toxoplasma gondii (T. gondii)is worldwide spread, and is responsible for toxoplasmosis and able to infect all species of mammals (inclde human) and birds. Nearly one-third of the human popluation is exposed to the threat of this parasite,It is a severe zoonosis and genetic disorders disease. It can case considerable economic loss on the people health and farming losses. Therefore, the effective prevention and timely diagnosis of toxoplasmosis is particlarly important. At present, the effective protection of gene of Toxoplasma gondii screening work is studying at home and abroad. While the dense granle protein is a kind of important secretion of metabolic antigen of Toxoplasma gondii, it has strong Original reaction and immunogenicity in human and animal experimental infection. So far, it have been reported to have10different tachyzoites dense granle protein (GRA1-GRA10). In the diagnosis of toxoplasmosis and immune prevention has potential value. Where GRA3can stimulate the body to generate humoral immunity,The antigen have potential application value for diagnosis of toxoplasmosis and as a vaccine candidate molecular.This study aims is main in constructing GRA3prokaryotic expression vector, And use the expression of recombinant GRA3protein to prepare GRA3monoclonal antibody, and it is going to identify and applicate GRA3monoclonal antibody. The antigen has potential application value for diagnosis of toxoplasmosis and as a vaccine candidate molecular
This study according to the GenBank AF414079published in Toxoplasma gondii GRA3gene seqences. The687bp RH strain GRA3gene of Toxoplasma gondii were cloned by polymerase chain reaction (PCR), On the gene seqence analysis, the result shows that the gene fragment is GRA3gene of toxoplasma; And it is Succeed in construct the prokaryotic expression vector pGEX-GRA3; induced by IPTG,they were expressed in Ecoli BL21, by means of SDS polyacrylamide gel electrophoresis display,that obtained the moleclar weight of50ku fusion protein GST-GRA3, it is most of the soluble protein. Western-Blotting immunoblotting analysis, fusion protein reacted with Toxoplasma gondii positive serum specific.The results show the protein has some antigenicity and specificity.
For the further study of recombinant proteins is as novel diagnostic antigen of possibility. This study is the efficient expression of recombinant GRA3protein immunized BALB/c mice, sing cell fusion technology, after indirect ELISA method screened and limited dilution, obtained a hybridoma cell strain5E4. After identification, the McAb subtype of type is IgGl. ELISA and Western blotting test results show that,5E4MAb can distingished Toxoplasma gondii GRA3protein,.It is suggested5E4McAb recognition epitopes may be located in the GRA3protein conserved region.
And the Rabbit anti-Toxoplasma gondii IgG is as coated antibody against recombinant GRA3protein, monoclonal antibody is as the detection of antibodies, Antibody sandwich ELISA method was Successed in establishing of the detection of Toxoplasma gondii, the method was for detection of Toxoplasma gondii in sensitivity that is to0.154g/ml. By the method to a pig farm in Yanbian152pigs were detected, the positive rate is13.2%.
The study cloned and expresed the GRA3gene of Toxoplasma gondii and prepared of recombinant GRA3protein McAb, that established a monoclonal antibody sandwich ELISA method. For clinical research of Toxoplasma gondii diagnostic reagents, reagent treatment laid the foundation, at the same time for detection of Toxoplasma gondii and the regional epidemiological investigation provided a simple and rapid diagnosis method.
本研究根据GenBank AF414079已发表的弓形虫GRA3基因序列设计了一对特异性的引物,利用PCR技术成功扩增出了一段687bp的基因片段,对该基因进行了序列分析,结果显示该基因片段为弓形虫GRA3基因；并成功构建了原核表达载体pGEX-GRA3;将构建好的表达载体,经IPTG诱导,在大肠杆菌BL21中进行了表达,经SDS-聚丙烯酰胺凝胶电泳显示,获得了分子量为50ku的融合蛋白GST-GRA3,经分析大部分以可溶性蛋白的形式存在。Western-Blotting免疫印迹分析,该重组蛋白与弓形虫阳性血清发生特异性反应,说明该蛋白具有一定的反应原性。
为进一步探讨重组蛋白作为诊断抗原的可能性。本研究利用高效表达的重组GRA3蛋白免疫BALB/c小鼠,采用细胞融合技术,经间接ELISA方法筛选和有限稀释法克隆后获得一株抗重组GRA3蛋白的杂交瘤细胞株(5E4)。经鉴定,其McAb的亚型为IgGl型。ELISA和western blotting试验结果表明,获得的5E4McAb可特异性的识别弓形虫GRA3蛋白,表明5E4McAb识别的表位可能位于GRA3蛋白的保守区域。
并以兔抗弓形虫IgG为捕获抗体,利用抗重组GRA3蛋白单克隆抗体为检测抗体,成功建立了用于弓形虫病检测的单抗双夹心ELISA方法,该方法对弓形虫检病检测灵敏度达0.154ug/mL。用该法对延边某猪场152头猪进行检测,结果阳性率为13.2%。并与常规ELISA相比较,该法更加敏感、特异,更有利于准确诊断。为临床研制弓形虫诊断试剂、治疗试剂奠定了基础,为制备疫苗的候选基因的筛选提供依据,同时为弓形虫的检测和区域流行病学调查提供了一种简便快速的诊断方法。
The obligate intracelllar protozoan parasite Toxoplasma gondii (T. gondii)is worldwide spread, and is responsible for toxoplasmosis and able to infect all species of mammals (inclde human) and birds. Nearly one-third of the human popluation is exposed to the threat of this parasite,It is a severe zoonosis and genetic disorders disease. It can case considerable economic loss on the people health and farming losses. Therefore, the effective prevention and timely diagnosis of toxoplasmosis is particlarly important. At present, the effective protection of gene of Toxoplasma gondii screening work is studying at home and abroad. While the dense granle protein is a kind of important secretion of metabolic antigen of Toxoplasma gondii, it has strong Original reaction and immunogenicity in human and animal experimental infection. So far, it have been reported to have10different tachyzoites dense granle protein (GRA1-GRA10). In the diagnosis of toxoplasmosis and immune prevention has potential value. Where GRA3can stimulate the body to generate humoral immunity,The antigen have potential application value for diagnosis of toxoplasmosis and as a vaccine candidate molecular.This study aims is main in constructing GRA3prokaryotic expression vector, And use the expression of recombinant GRA3protein to prepare GRA3monoclonal antibody, and it is going to identify and applicate GRA3monoclonal antibody. The antigen has potential application value for diagnosis of toxoplasmosis and as a vaccine candidate molecular
This study according to the GenBank AF414079published in Toxoplasma gondii GRA3gene seqences. The687bp RH strain GRA3gene of Toxoplasma gondii were cloned by polymerase chain reaction (PCR), On the gene seqence analysis, the result shows that the gene fragment is GRA3gene of toxoplasma; And it is Succeed in construct the prokaryotic expression vector pGEX-GRA3; induced by IPTG,they were expressed in Ecoli BL21, by means of SDS polyacrylamide gel electrophoresis display,that obtained the moleclar weight of50ku fusion protein GST-GRA3, it is most of the soluble protein. Western-Blotting immunoblotting analysis, fusion protein reacted with Toxoplasma gondii positive serum specific.The results show the protein has some antigenicity and specificity.
For the further study of recombinant proteins is as novel diagnostic antigen of possibility. This study is the efficient expression of recombinant GRA3protein immunized BALB/c mice, sing cell fusion technology, after indirect ELISA method screened and limited dilution, obtained a hybridoma cell strain5E4. After identification, the McAb subtype of type is IgGl. ELISA and Western blotting test results show that,5E4MAb can distingished Toxoplasma gondii GRA3protein,.It is suggested5E4McAb recognition epitopes may be located in the GRA3protein conserved region.
And the Rabbit anti-Toxoplasma gondii IgG is as coated antibody against recombinant GRA3protein, monoclonal antibody is as the detection of antibodies, Antibody sandwich ELISA method was Successed in establishing of the detection of Toxoplasma gondii, the method was for detection of Toxoplasma gondii in sensitivity that is to0.154g/ml. By the method to a pig farm in Yanbian152pigs were detected, the positive rate is13.2%.
The study cloned and expresed the GRA3gene of Toxoplasma gondii and prepared of recombinant GRA3protein McAb, that established a monoclonal antibody sandwich ELISA method. For clinical research of Toxoplasma gondii diagnostic reagents, reagent treatment laid the foundation, at the same time for detection of Toxoplasma gondii and the regional epidemiological investigation provided a simple and rapid diagnosis method.
引文
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