呼肠孤病毒3型S1基因的原核表达及间接ELISA检测方法的建立
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摘要
呼肠孤病毒3型(reovirus type 3,Reo-3)属于呼肠孤病毒科(Reoviridae)、正呼肠孤病毒属(Orthoreovirus)、双链RNA病毒[1]。呼肠孤病毒3型是实验动物SPF级大鼠、小鼠、豚鼠、地鼠的必须检测项目[2]。其自然感染宿主比较广泛[3],如人类、小鼠、大鼠、猴、牛等,可隐性感染动物并在体内产生抗体,给血液制品、单克隆抗体、细胞培养等外来致病因子的检定及动物试验带来一定的困难[4]。因此建立快速诊断方法十分必要。
目前,检测呼肠孤病毒3型主要用酶联免疫吸附法(ELISA)检测,其抗原获得方法主要为细胞培养,但很难获得高滴度的病毒;病毒虽可在鸡胚内增殖,但没有规律性[5]。本研究利用大肠杆菌表达系统pQE31表达呼肠孤病毒δ1蛋白抗原部分,用纯化的融合蛋白作为包被抗原建立ELISA方法,可用于呼肠孤病毒血清抗体的检测。
用生物学软件DNAstar分析δ1蛋白,根据GenBank中报道的Reo-3 S1基因序列(gi:61780)设计特异性引物,用一步法RT-PCR方法扩增S1基因的主要抗原片段SR,将其插入到pMD18-T载体中,酶切鉴定后测序。将阳性重组质粒用Sph I+Sal I双酶切后连接到同样双酶切的表达载体pQE-31上,转化E.coli M15(pREP-4),IPTG诱导表达。对表达蛋白进行SDS-PAGE电泳和Western-blot的结果表明,SR基因在E.coli M15中获得表达,表达蛋白SR-δ1分子质量约为32 kD,与预期大小相符,能与Reo-3阳性血清发生特异性反应,说明SR-δ1有很高的抗原性。
把表达的SR-δ1重组蛋白经初步纯化后用作包被抗原,建立了检测Reo-3血清抗体的间接ELISA方法,并确定了最适抗原包被浓度(0.6μg/mL)、最适血清稀释度(1:100)、血清最适作用时间(150 min)、最适封闭液(8%脱脂奶粉)、最适封闭时间(45min)、酶标抗体最适稀释度(1:8000)、酶标二抗最适作用时间(60min)、底物最适显色时间(10 min)和判定界值(0.292)。包被的重组抗原不与EcT(鼠痘病毒)、SeV(仙台病毒)、MHV(小鼠肝炎病毒)、PVM(小鼠肺炎病毒)阳性血清发生交叉反应,表明建立的ELISA诊断方法具有良好的特异性。批内重复试验和批间重复试验变异系数均小于10%,说明该方法具有很好的重复性。该诊断方法与国家实验动物检测中心建立的ELISA检测试剂盒比较,特异性、敏感性和符合率分别为98.5%、100%和98.7%。上述结果表明该诊断方法具有良好的特异性和敏感性,显示了良好的应用前景。
本研究成功克隆了Reo-3 S1基因,进行了表达。利用表达的SR-δ1蛋白成功建立了快速简便的ELISA诊断方法,为实验动物的质量控制、开发商品化试剂盒提供了技术支持。
Reovirus type 3(Reo-3), a orthoreovirus of the Reoviridae, has a linear double -stranded RNA genome. Reo-3 is the must detection project for the SPF level laboratory animal, such as rat, mouse, Cavia procellus and hamster. Its nature infection host is quite widespread, such as human, rat, mouse, monkey, cattle and so on, and can silent infect animal and produce the antibody in vivo. It brings difficulty for animal test and the external pathogenic factor examination such as blood product, monoclonal antibody, cell culture etc. Thus, it is necessary to establish a quick and accurate diagnostic method.
Serological methods, such as the enzyme-linked immunosorbent assay (ELISA), are commonly used to determine whether laboratory animals are infected with Reo-3. The antigen gain method mainly is the cell culture, but is very difficult to obtain the high titer the virus; Although may multiply in the chicken embryo, but does not have the regularity. In our study ,δ1 is expressed by prokaryotic expression system pQE31 and we establishes new ELISA method using the purified recombinant antigen .The serum antibodies of Reo-3 can be detected easily and quickly.
Based on hydrophilicity and antigenity analysis of the amino acids ofδ1 using biosoftware DNAstar, PCR primers were designed according to gene order of Reo-3 S1(gi:61780). SR gene was amplified by one step reverse transcription polymerase chain reaction (RT-PCR), and was cloned into pMD18-T vector for sequence analysis .The masculine recombinant plasmid was digested by restriction enzymes Sph I+Sal I and then was sub-cloned into prokaryotic expression vector pQE-31, and was induced expression in E.coli M15(pREP-4).The expressed SR-δ1 protein was identified by SDS-PAGE and Western-blot analysis. The results showed that the molecular weight of the expressed protein was 32 kD and the protein could specifically react with antiserum against Reo-3.
The indirect ELISA for detection of antibodies of Reo-3 was established using SR-δ1 as coated antigen with the optimal working parameters, including 0.6μg/mL of the SR-δ1 protein antigen for coating, testing sera dilution at 1:100, second antibody (Sigma) dilution at 1:8000, the standard of determining as positive sample S/P≥0.292 and others. It revealed a negative reaction with the positive sera of EcT, SeV, MHV and PVMT. demonstrating specificity of the diagnosis method is excellent. The variation coefficient of inter- and inner- batch antigen production testing quality control serum which can guarantee the quality of antigen and the accuracy of testing results is below 10 percents, demonstrating that repeatability of the antigen is good. Compared with the Reo-3 ELISA kit by national monitoring center of the quality laboratory animals, the two methods had 98.7% agreement by detecting 75 samples. the speciality of the test is 98.5%, the sensitivity, 100%. The result shows that the ELISA assay has excellent specificity , high sensitivity and excellent reproducibility, and could be used to detect the antibody against Reo-3.
In conclusion, Reo-3 S1 gene was successfully cloned and expressed in E.coli and purified. They lay the foundation for further studies of the protein structure and function ofδ1. The simple and fast diagnosis method using SR-δ1 expressed as diagnosis antigen can be a technique support for further developing the commercial kit of diagnosis and quality control for Laboratory Animals.
目前,检测呼肠孤病毒3型主要用酶联免疫吸附法(ELISA)检测,其抗原获得方法主要为细胞培养,但很难获得高滴度的病毒;病毒虽可在鸡胚内增殖,但没有规律性[5]。本研究利用大肠杆菌表达系统pQE31表达呼肠孤病毒δ1蛋白抗原部分,用纯化的融合蛋白作为包被抗原建立ELISA方法,可用于呼肠孤病毒血清抗体的检测。
用生物学软件DNAstar分析δ1蛋白,根据GenBank中报道的Reo-3 S1基因序列(gi:61780)设计特异性引物,用一步法RT-PCR方法扩增S1基因的主要抗原片段SR,将其插入到pMD18-T载体中,酶切鉴定后测序。将阳性重组质粒用Sph I+Sal I双酶切后连接到同样双酶切的表达载体pQE-31上,转化E.coli M15(pREP-4),IPTG诱导表达。对表达蛋白进行SDS-PAGE电泳和Western-blot的结果表明,SR基因在E.coli M15中获得表达,表达蛋白SR-δ1分子质量约为32 kD,与预期大小相符,能与Reo-3阳性血清发生特异性反应,说明SR-δ1有很高的抗原性。
把表达的SR-δ1重组蛋白经初步纯化后用作包被抗原,建立了检测Reo-3血清抗体的间接ELISA方法,并确定了最适抗原包被浓度(0.6μg/mL)、最适血清稀释度(1:100)、血清最适作用时间(150 min)、最适封闭液(8%脱脂奶粉)、最适封闭时间(45min)、酶标抗体最适稀释度(1:8000)、酶标二抗最适作用时间(60min)、底物最适显色时间(10 min)和判定界值(0.292)。包被的重组抗原不与EcT(鼠痘病毒)、SeV(仙台病毒)、MHV(小鼠肝炎病毒)、PVM(小鼠肺炎病毒)阳性血清发生交叉反应,表明建立的ELISA诊断方法具有良好的特异性。批内重复试验和批间重复试验变异系数均小于10%,说明该方法具有很好的重复性。该诊断方法与国家实验动物检测中心建立的ELISA检测试剂盒比较,特异性、敏感性和符合率分别为98.5%、100%和98.7%。上述结果表明该诊断方法具有良好的特异性和敏感性,显示了良好的应用前景。
本研究成功克隆了Reo-3 S1基因,进行了表达。利用表达的SR-δ1蛋白成功建立了快速简便的ELISA诊断方法,为实验动物的质量控制、开发商品化试剂盒提供了技术支持。
Reovirus type 3(Reo-3), a orthoreovirus of the Reoviridae, has a linear double -stranded RNA genome. Reo-3 is the must detection project for the SPF level laboratory animal, such as rat, mouse, Cavia procellus and hamster. Its nature infection host is quite widespread, such as human, rat, mouse, monkey, cattle and so on, and can silent infect animal and produce the antibody in vivo. It brings difficulty for animal test and the external pathogenic factor examination such as blood product, monoclonal antibody, cell culture etc. Thus, it is necessary to establish a quick and accurate diagnostic method.
Serological methods, such as the enzyme-linked immunosorbent assay (ELISA), are commonly used to determine whether laboratory animals are infected with Reo-3. The antigen gain method mainly is the cell culture, but is very difficult to obtain the high titer the virus; Although may multiply in the chicken embryo, but does not have the regularity. In our study ,δ1 is expressed by prokaryotic expression system pQE31 and we establishes new ELISA method using the purified recombinant antigen .The serum antibodies of Reo-3 can be detected easily and quickly.
Based on hydrophilicity and antigenity analysis of the amino acids ofδ1 using biosoftware DNAstar, PCR primers were designed according to gene order of Reo-3 S1(gi:61780). SR gene was amplified by one step reverse transcription polymerase chain reaction (RT-PCR), and was cloned into pMD18-T vector for sequence analysis .The masculine recombinant plasmid was digested by restriction enzymes Sph I+Sal I and then was sub-cloned into prokaryotic expression vector pQE-31, and was induced expression in E.coli M15(pREP-4).The expressed SR-δ1 protein was identified by SDS-PAGE and Western-blot analysis. The results showed that the molecular weight of the expressed protein was 32 kD and the protein could specifically react with antiserum against Reo-3.
The indirect ELISA for detection of antibodies of Reo-3 was established using SR-δ1 as coated antigen with the optimal working parameters, including 0.6μg/mL of the SR-δ1 protein antigen for coating, testing sera dilution at 1:100, second antibody (Sigma) dilution at 1:8000, the standard of determining as positive sample S/P≥0.292 and others. It revealed a negative reaction with the positive sera of EcT, SeV, MHV and PVMT. demonstrating specificity of the diagnosis method is excellent. The variation coefficient of inter- and inner- batch antigen production testing quality control serum which can guarantee the quality of antigen and the accuracy of testing results is below 10 percents, demonstrating that repeatability of the antigen is good. Compared with the Reo-3 ELISA kit by national monitoring center of the quality laboratory animals, the two methods had 98.7% agreement by detecting 75 samples. the speciality of the test is 98.5%, the sensitivity, 100%. The result shows that the ELISA assay has excellent specificity , high sensitivity and excellent reproducibility, and could be used to detect the antibody against Reo-3.
In conclusion, Reo-3 S1 gene was successfully cloned and expressed in E.coli and purified. They lay the foundation for further studies of the protein structure and function ofδ1. The simple and fast diagnosis method using SR-δ1 expressed as diagnosis antigen can be a technique support for further developing the commercial kit of diagnosis and quality control for Laboratory Animals.
引文
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