脑心肌炎病毒间接elisa抗体检测脑心肌炎病毒间接elisa抗体检测方法的建立(附件)【字数：7765】
1
目 录
Abstract 1
Key words 1
引言1
1 材料与方法 2
1.1 材料 2
1.1.1 菌株与载体粒2
1.1.2 主要试剂2
1.1.3 主要仪器2
1.2 方法 2
1.2.1 pET32aVP1重组质粒的构建和鉴定2
1.2.1.1 EMCV VP1基因引物的设计和合成2
1.2.1.2 EMCV VP1基因的扩增和鉴定2
1.2.1.3 pET32aVP1重组质粒的构建2
1.2.1.4 重组质粒的提取和鉴定3
1.2.2 pET32aVP1重组蛋白的原核表达和大量纯化3
1.2.2.1 重组蛋白的原核表达和鉴定3
1.2.2.2 重组蛋白的反应原性鉴定3
1.2.2.3 重组蛋白的大量表达及纯化3
1.2.3 EMCV VP1间接ELISA抗体检测方法的建立和反应条件化4
1.2.3.1 目的蛋白浓度测定4
1.2.3.2 抗原包被和待检血清最佳稀释倍数的选择4
1.2.3.3 血清最佳孵育时间的选择4
1.2.3.4 酶标二抗HRPSPA最佳稀释度的选择4
1.2.3.5 酶标二抗HRPSPA最佳作用时间的选择4
1.2.3.6 最佳显色时间的选择4
1.2.3.7 临界值确定4
1.2.4 特异性检验4
1.2.5重复性检验4
1.2.5.1 批内重复检验4
1.2.6.1 批间重复检验4
2 结果 5
2.1 EMCV VP1目的基因的扩增 5
2.1.1 pET32aVP1重组质粒的的构建与鉴定5
2.1.2 重组质粒pET32aVP1的双酶切鉴定5
2.2 pET32aVP1重组蛋白的原核表达和大量纯化5
2.2.1 重组蛋白的原核表达和鉴定5
2.2.2 重组蛋白的反应原性鉴定6
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.2.3 重组蛋白大量表达及纯化7
2.3 EMCV VP1间接ELISA抗体检测方法的建立和反应条件优化 7
2.3.1 目的蛋白浓度测定7
2.3.2 抗原包被和待检血清最佳稀释倍数的选择7
2.3.3 血清最佳孵育时间的选择8
2.3.4 酶标二抗HRPSPA最佳稀释度的选择 8
2.3.5 酶标二抗HRPSPA最佳作用时间的选择 8
2.3.6 最佳显色时间的选择8
2.3.7 临界值确定9
2.4 特异性检验9
2.5 重复性检验9
2.5.1 批内重复检验9
2.5.2 批间重复检验9
3 讨论 10
致谢 10
参考文献 10 脑心肌炎病毒间接ELISA抗体检测方法的建立
动物药学专业学生 范慧
指导教师 白娟
摘要：脑心肌炎病毒(Encephalomyocarditis virus，EMCV)可引起多种动物及人的以脑炎、心肌炎为主要特征的急性传染病，该病的发生与流行尤其给养猪业都造成了巨大的经济损失。本研究成功构建pET32aVP1重组质粒，并转化大肠杆菌BL21，用IPTG诱导表达，并用包涵体洗液洗涤获得纯化的VP1重组蛋白。以该蛋白为抗原，建立脑心肌炎病毒间接ELISA抗体检测方法，经对各反应条件优化后，当抗原包被浓度为2μg/mL，血清稀释度为1:100，血清孵育时间为1h，酶标二抗HRP
SPA稀释度为1:10000，作用时间为30min，TMB底物显色为8min，此时P/N比值最大，反应条件最佳。当OD450≥0.28，且P/N≥2.1时判断为阳性，OD450≦0.197时为阴性，介于两者之间为可疑。用该检测方法检测PCV2、CSFV、PRV、PRRSV阳性血清，结果均显示为阴性，表明该检测方法具有良好的特异性。
Development of indirect ELISA assay for the identification of antibody to Encephalomyocarditis virus in swine
Student majoring in Veterinary Pharmacy Fan hui
Tutor Bai juan
Abstract：EMCV, which cased acute infectious diseases characterized by encephalitis and myocarditis, had been observed in many kinds of animals and even in humen. The occurrence and epidemic of this disease caused huge economic losses. In this study, the recombinant plasmid pET32aVP1 was constructed and trandformed into BL21, E.Coli competent cell. The recombinant protein VP1 was expressed by inducetion with IPTG, and then purification was performed. In this report, the indirect ELISA based on VP1 protein was developed for detecting antibody to EMCV. Resurts show that the optimal antigen concentration for coating was 2μg/mL, the optimal dilution of serum was 1:100, incubation time of serum was 1h, the dilution of HRPSPA was 1:10000 and the reaction time was 30min, and the reaction time of TMB was 8min.When OD450≥0.28and P/N≥2.1, the rusurt of serum sample was positive, while OD450≦0.197 was negative.Resurts between these could not be confirmed. The method had no cross reaction with other positive serum including PCV2、CSFV、PRV、PRRSV, which shows this method has high specification.
目 录
Abstract 1
Key words 1
引言1
1 材料与方法 2
1.1 材料 2
1.1.1 菌株与载体粒2
1.1.2 主要试剂2
1.1.3 主要仪器2
1.2 方法 2
1.2.1 pET32aVP1重组质粒的构建和鉴定2
1.2.1.1 EMCV VP1基因引物的设计和合成2
1.2.1.2 EMCV VP1基因的扩增和鉴定2
1.2.1.3 pET32aVP1重组质粒的构建2
1.2.1.4 重组质粒的提取和鉴定3
1.2.2 pET32aVP1重组蛋白的原核表达和大量纯化3
1.2.2.1 重组蛋白的原核表达和鉴定3
1.2.2.2 重组蛋白的反应原性鉴定3
1.2.2.3 重组蛋白的大量表达及纯化3
1.2.3 EMCV VP1间接ELISA抗体检测方法的建立和反应条件化4
1.2.3.1 目的蛋白浓度测定4
1.2.3.2 抗原包被和待检血清最佳稀释倍数的选择4
1.2.3.3 血清最佳孵育时间的选择4
1.2.3.4 酶标二抗HRPSPA最佳稀释度的选择4
1.2.3.5 酶标二抗HRPSPA最佳作用时间的选择4
1.2.3.6 最佳显色时间的选择4
1.2.3.7 临界值确定4
1.2.4 特异性检验4
1.2.5重复性检验4
1.2.5.1 批内重复检验4
1.2.6.1 批间重复检验4
2 结果 5
2.1 EMCV VP1目的基因的扩增 5
2.1.1 pET32aVP1重组质粒的的构建与鉴定5
2.1.2 重组质粒pET32aVP1的双酶切鉴定5
2.2 pET32aVP1重组蛋白的原核表达和大量纯化5
2.2.1 重组蛋白的原核表达和鉴定5
2.2.2 重组蛋白的反应原性鉴定6
2 *好棒文|www.hbsrm.com +Q: ￥351916072￥
.2.3 重组蛋白大量表达及纯化7
2.3 EMCV VP1间接ELISA抗体检测方法的建立和反应条件优化 7
2.3.1 目的蛋白浓度测定7
2.3.2 抗原包被和待检血清最佳稀释倍数的选择7
2.3.3 血清最佳孵育时间的选择8
2.3.4 酶标二抗HRPSPA最佳稀释度的选择 8
2.3.5 酶标二抗HRPSPA最佳作用时间的选择 8
2.3.6 最佳显色时间的选择8
2.3.7 临界值确定9
2.4 特异性检验9
2.5 重复性检验9
2.5.1 批内重复检验9
2.5.2 批间重复检验9
3 讨论 10
致谢 10
参考文献 10 脑心肌炎病毒间接ELISA抗体检测方法的建立
动物药学专业学生 范慧
指导教师 白娟
摘要：脑心肌炎病毒(Encephalomyocarditis virus，EMCV)可引起多种动物及人的以脑炎、心肌炎为主要特征的急性传染病，该病的发生与流行尤其给养猪业都造成了巨大的经济损失。本研究成功构建pET32aVP1重组质粒，并转化大肠杆菌BL21，用IPTG诱导表达，并用包涵体洗液洗涤获得纯化的VP1重组蛋白。以该蛋白为抗原，建立脑心肌炎病毒间接ELISA抗体检测方法，经对各反应条件优化后，当抗原包被浓度为2μg/mL，血清稀释度为1:100，血清孵育时间为1h，酶标二抗HRP
SPA稀释度为1:10000，作用时间为30min，TMB底物显色为8min，此时P/N比值最大，反应条件最佳。当OD450≥0.28，且P/N≥2.1时判断为阳性，OD450≦0.197时为阴性，介于两者之间为可疑。用该检测方法检测PCV2、CSFV、PRV、PRRSV阳性血清，结果均显示为阴性，表明该检测方法具有良好的特异性。
Development of indirect ELISA assay for the identification of antibody to Encephalomyocarditis virus in swine
Student majoring in Veterinary Pharmacy Fan hui
Tutor Bai juan
Abstract：EMCV, which cased acute infectious diseases characterized by encephalitis and myocarditis, had been observed in many kinds of animals and even in humen. The occurrence and epidemic of this disease caused huge economic losses. In this study, the recombinant plasmid pET32aVP1 was constructed and trandformed into BL21, E.Coli competent cell. The recombinant protein VP1 was expressed by inducetion with IPTG, and then purification was performed. In this report, the indirect ELISA based on VP1 protein was developed for detecting antibody to EMCV. Resurts show that the optimal antigen concentration for coating was 2μg/mL, the optimal dilution of serum was 1:100, incubation time of serum was 1h, the dilution of HRPSPA was 1:10000 and the reaction time was 30min, and the reaction time of TMB was 8min.When OD450≥0.28and P/N≥2.1, the rusurt of serum sample was positive, while OD450≦0.197 was negative.Resurts between these could not be confirmed. The method had no cross reaction with other positive serum including PCV2、CSFV、PRV、PRRSV, which shows this method has high specification.
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