猪链球菌2型菌毛相关基因sbp4的猪链球菌2型菌毛相关基因sbp4的原核表达及免疫原性分析(附件)【字数：10469】
1
目 录
Abstract1
Key words1
引言1
1 材料与方法2
1.1 材料 2
1.1.1 猪链球菌菌株2
1.1.2 表达载体构建相关试剂2
1.1.3 sbp4诱导相关试剂2
1.1.4 主要仪器3
1.2 方法 3
1.2.1 PET28a质粒的增值和提取3
1.2.2 引物3
1.2.3 sbp4基因的PCR扩增3
1.2.4 PCR产物的小量胶回收 4
1.2.5 PETsbp4重组表达载体的构建4
1.2.5.1 目的片段与质粒载体的双酶切与回收 4
1.2.5.2 目的片段与质粒载体的连接 4
1.2.5.3 连接产物的转化 5
1.2.5.4 PCR鉴定重组质粒 5
1.2.5.5 阳性克隆转化 5
1.2.5.6 PCR鉴定阳性重组质粒 5
1.2.6 sbp4的诱导表达5
1.2.6.1 含阳性质粒的Ecoli的诱导表达样品处理5
1.2.6.2 SDSPAGE6
1.2.6.3sbp4表达蛋白的纯化6
1.2.6.4 Westernblot7
2 结果与分析8
2.1 sPET28a质粒提取结果8
2.2 sbp4基因的PCR扩增结果8
2.3 pET28asbp4重组质粒的PCR鉴定8
2.4 pET28asbp4重组质粒的测序鉴定9
2.5 sbp4重组蛋白表达结果9
2.6 sbp4重组蛋白纯化结果10
2.7 Westernblot结果 11
3 讨论11 3.1 本实验的研究意义11 3.2 本实验的创新之处12 3.2.1 质粒提取条件的创新11 3.2.2 Westernblot的创新12 3.3 本实验不足之处12
3.3.1 PET28asbp4重组表达载体的构建12
致谢12< *好棒文|www.hbsrm.com +Q: &351916072&
br /> 参考文献13 猪链球菌2型菌毛相关基因sbp4
的原核表达及免疫原性分析
动物医学专业学生 冉玲
指导教师 张炜
摘要：猪链球菌2型（Streptococcus suis type 2，SS2）是一种重要的人兽共患病原，可感染能够导致人和猪的脑膜炎、心内膜炎、关节炎和败血性休克等。本实验以猪链球菌2型强毒株ZY05719为研究对象，对强毒株特有的5个疑似菌毛结构基因sbp4进行了原核表达和产物免疫原性分析。用PCR方法扩增出sbp4基因后，然后与pET28a质粒进行双酶切，连接后转化入DH5α中构建重组质粒，提取重组质粒再转化入BL21中并IPTG诱导表达。诱导产物通过SDSPAGE检测，约59KD处出现与预期相一致的蛋白条带。Westernblot分析发现，该蛋白可以被SS2高免血清所识别，具有良好的免疫原性。本实验为进一步研究菌毛相关蛋白的致病作用及相关疫苗的制备奠定了基础。
Prokaryotic expression and immunogenicity
analysis of pili related gene sbp4 of Streptococcus suis serotype 2
Student majoring in veterinary medicine RanLing
Tutor Zhang Wei
Abstract：Streptococcus suis serotype 2 is a major pathogen that causes infections and death in pigs and humans. In this study, we investigated the prokaryotic expression and immunogenicity of 5 pili structural genes sbp4, a virulent strain of Streptococcus suis type 2, which was a specific strain of ZY05719. The sbp4 gene was amplified by PCR, then double digested with pET28a plasmid, and then transformed into DH5a. The recombinant plasmid was constructed, then the recombinant plasmid was extracted, then transformed into BL21 and induced by IPTG. The products were detected at 59KD by SDSPAGE, and the protein bands were consistent with the expectation. Westernblot analysis showed that the protein could be identified by SS2 high serum and had good immunogenicity. This study laid the foundation for further study of the pathogenicity of pili associated proteins and the preparation of related vaccines.
目 录
Abstract1
Key words1
引言1
1 材料与方法2
1.1 材料 2
1.1.1 猪链球菌菌株2
1.1.2 表达载体构建相关试剂2
1.1.3 sbp4诱导相关试剂2
1.1.4 主要仪器3
1.2 方法 3
1.2.1 PET28a质粒的增值和提取3
1.2.2 引物3
1.2.3 sbp4基因的PCR扩增3
1.2.4 PCR产物的小量胶回收 4
1.2.5 PETsbp4重组表达载体的构建4
1.2.5.1 目的片段与质粒载体的双酶切与回收 4
1.2.5.2 目的片段与质粒载体的连接 4
1.2.5.3 连接产物的转化 5
1.2.5.4 PCR鉴定重组质粒 5
1.2.5.5 阳性克隆转化 5
1.2.5.6 PCR鉴定阳性重组质粒 5
1.2.6 sbp4的诱导表达5
1.2.6.1 含阳性质粒的Ecoli的诱导表达样品处理5
1.2.6.2 SDSPAGE6
1.2.6.3sbp4表达蛋白的纯化6
1.2.6.4 Westernblot7
2 结果与分析8
2.1 sPET28a质粒提取结果8
2.2 sbp4基因的PCR扩增结果8
2.3 pET28asbp4重组质粒的PCR鉴定8
2.4 pET28asbp4重组质粒的测序鉴定9
2.5 sbp4重组蛋白表达结果9
2.6 sbp4重组蛋白纯化结果10
2.7 Westernblot结果 11
3 讨论11 3.1 本实验的研究意义11 3.2 本实验的创新之处12 3.2.1 质粒提取条件的创新11 3.2.2 Westernblot的创新12 3.3 本实验不足之处12
3.3.1 PET28asbp4重组表达载体的构建12
致谢12< *好棒文|www.hbsrm.com +Q: &351916072&
br /> 参考文献13 猪链球菌2型菌毛相关基因sbp4
的原核表达及免疫原性分析
动物医学专业学生 冉玲
指导教师 张炜
摘要：猪链球菌2型（Streptococcus suis type 2，SS2）是一种重要的人兽共患病原，可感染能够导致人和猪的脑膜炎、心内膜炎、关节炎和败血性休克等。本实验以猪链球菌2型强毒株ZY05719为研究对象，对强毒株特有的5个疑似菌毛结构基因sbp4进行了原核表达和产物免疫原性分析。用PCR方法扩增出sbp4基因后，然后与pET28a质粒进行双酶切，连接后转化入DH5α中构建重组质粒，提取重组质粒再转化入BL21中并IPTG诱导表达。诱导产物通过SDSPAGE检测，约59KD处出现与预期相一致的蛋白条带。Westernblot分析发现，该蛋白可以被SS2高免血清所识别，具有良好的免疫原性。本实验为进一步研究菌毛相关蛋白的致病作用及相关疫苗的制备奠定了基础。
Prokaryotic expression and immunogenicity
analysis of pili related gene sbp4 of Streptococcus suis serotype 2
Student majoring in veterinary medicine RanLing
Tutor Zhang Wei
Abstract：Streptococcus suis serotype 2 is a major pathogen that causes infections and death in pigs and humans. In this study, we investigated the prokaryotic expression and immunogenicity of 5 pili structural genes sbp4, a virulent strain of Streptococcus suis type 2, which was a specific strain of ZY05719. The sbp4 gene was amplified by PCR, then double digested with pET28a plasmid, and then transformed into DH5a. The recombinant plasmid was constructed, then the recombinant plasmid was extracted, then transformed into BL21 and induced by IPTG. The products were detected at 59KD by SDSPAGE, and the protein bands were consistent with the expectation. Westernblot analysis showed that the protein could be identified by SS2 high serum and had good immunogenicity. This study laid the foundation for further study of the pathogenicity of pili associated proteins and the preparation of related vaccines.
版权保护: 本文由 hbsrm.com编辑，转载请保留链接: www.hbsrm.com/yxlw/dwyx/27.html
