猪瘟病毒e2蛋白主要抗原区的原核表达猪瘟病毒e2蛋白主要抗原区的原核表达及其单克隆抗体的制备(附件)【字数：10817】
1
目 录
Abstract1
Key words1
引言1
1 材料与方法2
1.1病毒、细胞与动物 2
1.2主要试剂 2
1.2.1 SDSPAGE相关配制2
1.2.2 Western blot相关溶液的配制2
1.2.3 ELISA相关溶液的配制2
1.2.4 培养基的配制3
1.3 E2基因片段的扩增 3
1.3.1 总RNA的制备 3
1.3.2 RTPCR扩增产物 3
1.4表达载体的构建 4
1.5重组蛋白的诱导表达和纯化4
1.6重组蛋白的Westernblot分析5
1.7抗原最佳包被浓度和血清最佳稀释度的选择5
1.8动物免疫6
1.9细胞融合6
1.9.1饲养细胞的制备 6
1.9.2 骨髓瘤细胞的制备 6
1.9.3 脾淋巴细胞的制备 6
1.9.4 细胞融合与杂交瘤细胞的选择性培养 7
1.10杂交瘤细胞筛选8
1.11杂交瘤细胞的克隆化8
1.11.1杂交瘤细胞的冻存8
1.11.2杂交瘤细胞的复苏8
1.12 E2蛋白特异性单克隆抗体的鉴定8
2．结果与分析8
2.1 CSFV E2基因的RTPCR扩增8
2.2 重组质粒的构建9
2.3重组蛋白表达、纯化的鉴定9
2.4杂交瘤细胞的筛选10
2.5单克隆抗体的鉴定10
3. 讨论 11
致谢12
参考文献12
猪瘟病毒E2蛋白主要抗原区的原核表达
及其单克隆抗体的制备
动物医学专业学生 常晓静
指导教师 范红结
摘要：猪瘟病毒（Classical swine fever virus，CSFV）是一种引起猪的高度传染性疾病的病原体，是影响养猪业的重要制约因素，易引起大 *好棒文|www.hbsrm.com +Q: @351916072@
范围流行，造成巨大的经济损失。本研究用RTPCR扩增CSFV E2蛋白主要抗原区的编码基因，通过原核表达的方式进行诱导表达，使E2蛋白获得高效表达。将表达蛋白纯化并作为免疫原，免疫BALB/c小鼠，取小鼠脾细胞与骨髓瘤细胞SP2/0进行细胞融合，以纯化后的蛋白作为包被抗原，用间接ELISA检测方法筛选阳性细胞克隆，经过4次克隆，最终获得1株能够稳定分泌抗E2蛋白特异性单克隆抗体的杂交瘤细胞株E17B，为建立猪瘟检测阻断ELISA方法的建立奠定了基础。
Prokaryotic expression of the major antigenic region of classical swine fever virus E2 protein and preparation of a monoclonal antibody against E2 protein
Student majoring in Veterinary Medicine Chang Xiaojing
Tutor Fan Hongjie
Abstract： Classical swine fever virus (CSFV) is a highly infectious pathogen of pigs and believed to be a major constraint to pig production. Classical swine fever can lead to a wide range of epidemic and result in huge economic losses. A gene encoding the major antigenic domains of E2 protein of classical swine fever virus(CSFV) was amplified by RTPCR. The expression of E2 protein was induced by the way of prokaryotic expression. The purified protein was used as immunogen to immunize BALB/c mice, of which splenocytes were fused with SP2/0 cells. A hybridoma cell line stably secreting a monoclonal antibody (McAb) directed against the E2 protein was screened by ELISA, which established foundation materials for the establishment of classical swine fever to blocking ELISA.
目 录
Abstract1
Key words1
引言1
1 材料与方法2
1.1病毒、细胞与动物 2
1.2主要试剂 2
1.2.1 SDSPAGE相关配制2
1.2.2 Western blot相关溶液的配制2
1.2.3 ELISA相关溶液的配制2
1.2.4 培养基的配制3
1.3 E2基因片段的扩增 3
1.3.1 总RNA的制备 3
1.3.2 RTPCR扩增产物 3
1.4表达载体的构建 4
1.5重组蛋白的诱导表达和纯化4
1.6重组蛋白的Westernblot分析5
1.7抗原最佳包被浓度和血清最佳稀释度的选择5
1.8动物免疫6
1.9细胞融合6
1.9.1饲养细胞的制备 6
1.9.2 骨髓瘤细胞的制备 6
1.9.3 脾淋巴细胞的制备 6
1.9.4 细胞融合与杂交瘤细胞的选择性培养 7
1.10杂交瘤细胞筛选8
1.11杂交瘤细胞的克隆化8
1.11.1杂交瘤细胞的冻存8
1.11.2杂交瘤细胞的复苏8
1.12 E2蛋白特异性单克隆抗体的鉴定8
2．结果与分析8
2.1 CSFV E2基因的RTPCR扩增8
2.2 重组质粒的构建9
2.3重组蛋白表达、纯化的鉴定9
2.4杂交瘤细胞的筛选10
2.5单克隆抗体的鉴定10
3. 讨论 11
致谢12
参考文献12
猪瘟病毒E2蛋白主要抗原区的原核表达
及其单克隆抗体的制备
动物医学专业学生 常晓静
指导教师 范红结
摘要：猪瘟病毒（Classical swine fever virus，CSFV）是一种引起猪的高度传染性疾病的病原体，是影响养猪业的重要制约因素，易引起大 *好棒文|www.hbsrm.com +Q: @351916072@
范围流行，造成巨大的经济损失。本研究用RTPCR扩增CSFV E2蛋白主要抗原区的编码基因，通过原核表达的方式进行诱导表达，使E2蛋白获得高效表达。将表达蛋白纯化并作为免疫原，免疫BALB/c小鼠，取小鼠脾细胞与骨髓瘤细胞SP2/0进行细胞融合，以纯化后的蛋白作为包被抗原，用间接ELISA检测方法筛选阳性细胞克隆，经过4次克隆，最终获得1株能够稳定分泌抗E2蛋白特异性单克隆抗体的杂交瘤细胞株E17B，为建立猪瘟检测阻断ELISA方法的建立奠定了基础。
Prokaryotic expression of the major antigenic region of classical swine fever virus E2 protein and preparation of a monoclonal antibody against E2 protein
Student majoring in Veterinary Medicine Chang Xiaojing
Tutor Fan Hongjie
Abstract： Classical swine fever virus (CSFV) is a highly infectious pathogen of pigs and believed to be a major constraint to pig production. Classical swine fever can lead to a wide range of epidemic and result in huge economic losses. A gene encoding the major antigenic domains of E2 protein of classical swine fever virus(CSFV) was amplified by RTPCR. The expression of E2 protein was induced by the way of prokaryotic expression. The purified protein was used as immunogen to immunize BALB/c mice, of which splenocytes were fused with SP2/0 cells. A hybridoma cell line stably secreting a monoclonal antibody (McAb) directed against the E2 protein was screened by ELISA, which established foundation materials for the establishment of classical swine fever to blocking ELISA.
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