鸡朊样蛋白Shadoo、Doppel基因的序列分析及PrP基因的毕赤酵母表达
发布时间：2018-09-03 06:37
【摘要】：【目的】克隆并分析鸡Pr P样蛋白(Shadoo、Doppel)基因(SPRN、PRND),探究它们与Pr PC的关系,为禽源Shadoo(Sho)、Doppel(Dpl)结构与功能、与Pr PC互作关系机制的研究提供理论依据与分子基础;同时,构建鸡Pr P基因(PRNP)的真核表达载体p PIC9K-Ch Pr P(25~248),通过毕赤酵母表达系统获得的重组鸡朊蛋白(Ch Pr P),为朊蛋白在不同生理状态下的折叠形态及PrPC向PrPSc转换机制的研究提供材料。【方法】根据Gen Bank收录的鸡SPRN(Ch SPRN)序列设计特异性引物,以鸡脑组织基因组DNA为模板,扩增鸡脑组织中SPRN基因完整ORF;通过比对多物种Dpl蛋白的氨基酸序列找到PRND基因的保守区,设计简并引物并依据鸡的密码子偏好性对引物进行优化,以逐步降低其简并性。用此特异性引物以聚合酶链式反应(PCR)扩增出鸡PRND(Ch PRND)基因;将两者分别克隆到p MD-18-T载体,测序鉴定后应用生物信息学方法与软件进行分析并将序列上传Gen Bank数据库。结合Sho、Dpl与Pr PC相关研究,分别分析其相互关系。根据毕赤酵母的密码子偏好性、G+C含量等特点对目的基因Pr P(25~248)进行密码子优化与基因合成。同时,扩增优化前的目的序列。之后,将两者均连于p PIC9K、转至GS115。经PCR扩增和接种MM、MD平板鉴定后,诱导表达、SDS-PAGE检测。【结果】Ch SPRN长354bp,在第23位与第56位发生G→A突变,均为C→Y突变。分析氨基酸序列并利用同源法构建Ch Sho与Ch Pr P的三级结构,结果显示它们N-端同源性为44.25%,C-端结构相似性为23.33%;多物种Dpl氨基酸序列比对发现,第11~150位(139个氨基酸残基)为Dpl氨基酸序列保守区,以此区域设计引物并进行PCR扩增,得到约417bp的目的条带,将其提交Gen Bank,登录号为KP140962。综合分析Pr PC与Dpl相关研究发现,尽管Dpl与Pr PC具有相似的翻译后修饰和空间结构,但两者多表现为拮抗作用,尤其是Pr PC N-端包含八肽重复区的第23~88个残基对于保护Purkinje细胞免受Dpl诱导的神经退化作用至关重要;经鉴定,成功构建了p PIC9K-Pr P(25~248),并在GS115中获得表达;与未经优化前相比,优化后蛋白具有更高的表达量。【结论】(1)Ch Sho与Ch Pr P在序列与结构中都表现出惊人相似性;(2)首次成功克隆获得鸡PRND基因;(3)利用毕赤酵母真核表达系统成功获得重组鸡朊蛋白,且通过密码子优化得到了比优化前较高的蛋白表达量。
[Abstract]:[objective] to clone and analyze chicken Pr P-like protein (Shadoo,Doppel) gene (SPRN,PRND) and explore their relationship with Pr PC, so as to provide theoretical basis and molecular basis for the study of the structure and function of Shadoo (Sho) Doppel (Dpl) and the mechanism of interaction with Pr PC. The eukaryotic expression vector p PIC9K-Ch Pr P (25248 of chicken Pr P gene (PRNP) was constructed. The recombinant chicken prion protein (Ch Pr P), was obtained by Pichia pastoris expression system. The folding morphology of (Ch Pr P), as prion protein in different physiological states and the mechanism of PrPC to PrPSc conversion were studied. Donor materials. [methods] specific primers were designed according to the chicken SPRN (Ch SPRN) sequence included in Gen Bank. Using genomic DNA of chicken brain tissue as template, the intact ORF; of SPRN gene was amplified from chicken brain tissue. The conserved region of PRND gene was found by comparing the amino acid sequences of multi-species Dpl protein. Degenerate primers were designed and optimized according to the codon preference of chicken. To gradually reduce its degeneracy. Chicken PRND (Ch PRND) gene was amplified by polymerase chain reaction (PCR) with this specific primer and cloned into p MD-18-T vector respectively. After sequencing and identification, the sequence was analyzed by bioinformatics method and software, and the sequence was uploaded to Gen Bank database. Based on the research of Sho,Dpl and Pr PC, the relationship between them is analyzed. According to the characteristics of codon preference of Pichia pastoris, the codon optimization and gene synthesis of the target gene Pr P (25t248 were carried out. At the same time, the optimized target sequence was amplified. Then connect both to p PIC9K, to GS115. After PCR amplification and MM,MD plate inoculation, the induced expression of Ch SPRN was detected by SDS-PAGE. [results] the length of Ch SPRN was 354bp. The amino acid sequence was analyzed and the tertiary structure of Ch Sho and Ch Pr P was constructed by homology method. The results showed that their N-terminal homology was 44.25 and the similarity of C-terminal structure was 23.33.The amino acid sequence alignment of multiple species Dpl was found. The Dpl amino acid sequence was conserved at position 11150 (139 amino acid residues). Primers were designed and amplified by PCR. The target band of about 417bp was obtained and submitted to Gen Bank, accession number as KP140962.. A comprehensive analysis of Pr PC and Dpl showed that although Dpl and Pr PC had similar posttranslational modifications and spatial structures, both of them showed antagonistic effects. In particular, the 23th-88 residues of the N-terminal of Pr PC containing octapeptide repeats are important to protect Purkinje cells from the neurodegeneration induced by Dpl. It was identified that p PIC9K-Pr P (25t248) was successfully constructed and expressed in GS115. [conclusion] (1) Ch Sho and Ch Pr P showed remarkable similarity in sequence and structure; (2) chicken PRND gene was cloned successfully for the first time; (3) recombinant chicken prion protein was successfully obtained by using Pichia pastoris eukaryotic expression system. A higher protein expression level was obtained by codon optimization than before.
【学位授予单位】：甘肃农业大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S852.23;Q78
本文编号：2219198
[Abstract]:[objective] to clone and analyze chicken Pr P-like protein (Shadoo,Doppel) gene (SPRN,PRND) and explore their relationship with Pr PC, so as to provide theoretical basis and molecular basis for the study of the structure and function of Shadoo (Sho) Doppel (Dpl) and the mechanism of interaction with Pr PC. The eukaryotic expression vector p PIC9K-Ch Pr P (25248 of chicken Pr P gene (PRNP) was constructed. The recombinant chicken prion protein (Ch Pr P), was obtained by Pichia pastoris expression system. The folding morphology of (Ch Pr P), as prion protein in different physiological states and the mechanism of PrPC to PrPSc conversion were studied. Donor materials. [methods] specific primers were designed according to the chicken SPRN (Ch SPRN) sequence included in Gen Bank. Using genomic DNA of chicken brain tissue as template, the intact ORF; of SPRN gene was amplified from chicken brain tissue. The conserved region of PRND gene was found by comparing the amino acid sequences of multi-species Dpl protein. Degenerate primers were designed and optimized according to the codon preference of chicken. To gradually reduce its degeneracy. Chicken PRND (Ch PRND) gene was amplified by polymerase chain reaction (PCR) with this specific primer and cloned into p MD-18-T vector respectively. After sequencing and identification, the sequence was analyzed by bioinformatics method and software, and the sequence was uploaded to Gen Bank database. Based on the research of Sho,Dpl and Pr PC, the relationship between them is analyzed. According to the characteristics of codon preference of Pichia pastoris, the codon optimization and gene synthesis of the target gene Pr P (25t248 were carried out. At the same time, the optimized target sequence was amplified. Then connect both to p PIC9K, to GS115. After PCR amplification and MM,MD plate inoculation, the induced expression of Ch SPRN was detected by SDS-PAGE. [results] the length of Ch SPRN was 354bp. The amino acid sequence was analyzed and the tertiary structure of Ch Sho and Ch Pr P was constructed by homology method. The results showed that their N-terminal homology was 44.25 and the similarity of C-terminal structure was 23.33.The amino acid sequence alignment of multiple species Dpl was found. The Dpl amino acid sequence was conserved at position 11150 (139 amino acid residues). Primers were designed and amplified by PCR. The target band of about 417bp was obtained and submitted to Gen Bank, accession number as KP140962.. A comprehensive analysis of Pr PC and Dpl showed that although Dpl and Pr PC had similar posttranslational modifications and spatial structures, both of them showed antagonistic effects. In particular, the 23th-88 residues of the N-terminal of Pr PC containing octapeptide repeats are important to protect Purkinje cells from the neurodegeneration induced by Dpl. It was identified that p PIC9K-Pr P (25t248) was successfully constructed and expressed in GS115. [conclusion] (1) Ch Sho and Ch Pr P showed remarkable similarity in sequence and structure; (2) chicken PRND gene was cloned successfully for the first time; (3) recombinant chicken prion protein was successfully obtained by using Pichia pastoris eukaryotic expression system. A higher protein expression level was obtained by codon optimization than before.
【学位授予单位】：甘肃农业大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S852.23;Q78
【参考文献】
相关期刊论文 前1条
1 高永军;高晨;陈建明;石琦;郭燕;张宝云;董小平;;人朊蛋白相关蛋白Shadoo的表达、纯化及抗体的制备[J];中国病原生物学杂志;2007年03期
，本文编号：2219198
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