雞朊樣蛋白Shadoo、Doppel基因的序列分析及PrP基因的畢赤酵母表達(dá)
發(fā)布時(shí)間：2018-09-03 06:37
【摘要】：【目的】克隆并分析雞Pr P樣蛋白(Shadoo、Doppel)基因(SPRN、PRND),探究它們與Pr PC的關(guān)系,為禽源Shadoo(Sho)、Doppel(Dpl)結(jié)構(gòu)與功能、與Pr PC互作關(guān)系機(jī)制的研究提供理論依據(jù)與分子基礎(chǔ);同時(shí),構(gòu)建雞Pr P基因(PRNP)的真核表達(dá)載體p PIC9K-Ch Pr P(25~248),通過畢赤酵母表達(dá)系統(tǒng)獲得的重組雞朊蛋白(Ch Pr P),為朊蛋白在不同生理狀態(tài)下的折疊形態(tài)及PrPC向PrPSc轉(zhuǎn)換機(jī)制的研究提供材料�！痉椒ā扛鶕�(jù)Gen Bank收錄的雞SPRN(Ch SPRN)序列設(shè)計(jì)特異性引物,以雞腦組織基因組DNA為模板,擴(kuò)增雞腦組織中SPRN基因完整ORF;通過比對(duì)多物種Dpl蛋白的氨基酸序列找到PRND基因的保守區(qū),設(shè)計(jì)簡(jiǎn)并引物并依據(jù)雞的密碼子偏好性對(duì)引物進(jìn)行優(yōu)化,以逐步降低其簡(jiǎn)并性。用此特異性引物以聚合酶鏈?zhǔn)椒磻?yīng)(PCR)擴(kuò)增出雞PRND(Ch PRND)基因;將兩者分別克隆到p MD-18-T載體,測(cè)序鑒定后應(yīng)用生物信息學(xué)方法與軟件進(jìn)行分析并將序列上傳Gen Bank數(shù)據(jù)庫(kù)。結(jié)合Sho、Dpl與Pr PC相關(guān)研究,分別分析其相互關(guān)系。根據(jù)畢赤酵母的密碼子偏好性、G+C含量等特點(diǎn)對(duì)目的基因Pr P(25~248)進(jìn)行密碼子優(yōu)化與基因合成。同時(shí),擴(kuò)增優(yōu)化前的目的序列。之后,將兩者均連于p PIC9K、轉(zhuǎn)至GS115。經(jīng)PCR擴(kuò)增和接種MM、MD平板鑒定后,誘導(dǎo)表達(dá)、SDS-PAGE檢測(cè)�！窘Y(jié)果】Ch SPRN長(zhǎng)354bp,在第23位與第56位發(fā)生G→A突變,均為C→Y突變。分析氨基酸序列并利用同源法構(gòu)建Ch Sho與Ch Pr P的三級(jí)結(jié)構(gòu),結(jié)果顯示它們N-端同源性為44.25%,C-端結(jié)構(gòu)相似性為23.33%;多物種Dpl氨基酸序列比對(duì)發(fā)現(xiàn),第11~150位(139個(gè)氨基酸殘基)為Dpl氨基酸序列保守區(qū),以此區(qū)域設(shè)計(jì)引物并進(jìn)行PCR擴(kuò)增,得到約417bp的目的條帶,將其提交Gen Bank,登錄號(hào)為KP140962。綜合分析Pr PC與Dpl相關(guān)研究發(fā)現(xiàn),盡管Dpl與Pr PC具有相似的翻譯后修飾和空間結(jié)構(gòu),但兩者多表現(xiàn)為拮抗作用,尤其是Pr PC N-端包含八肽重復(fù)區(qū)的第23~88個(gè)殘基對(duì)于保護(hù)Purkinje細(xì)胞免受Dpl誘導(dǎo)的神經(jīng)退化作用至關(guān)重要;經(jīng)鑒定,成功構(gòu)建了p PIC9K-Pr P(25~248),并在GS115中獲得表達(dá);與未經(jīng)優(yōu)化前相比,優(yōu)化后蛋白具有更高的表達(dá)量。【結(jié)論】(1)Ch Sho與Ch Pr P在序列與結(jié)構(gòu)中都表現(xiàn)出驚人相似性;(2)首次成功克隆獲得雞PRND基因;(3)利用畢赤酵母真核表達(dá)系統(tǒng)成功獲得重組雞朊蛋白,且通過密碼子優(yōu)化得到了比優(yōu)化前較高的蛋白表達(dá)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.
【學(xué)位授予單位】：甘肅農(nóng)業(yè)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類號(hào)】：S852.23;Q78
本文編號(hào)：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.
【學(xué)位授予單位】：甘肅農(nóng)業(yè)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類號(hào)】：S852.23;Q78
【參考文獻(xiàn)】
相關(guān)期刊論文 前1條
1 高永軍;高晨;陳建明;石琦;郭燕;張寶云;董小平;;人朊蛋白相關(guān)蛋白Shadoo的表達(dá)、純化及抗體的制備[J];中國(guó)病原生物學(xué)雜志;2007年03期
，本文編號(hào)：2219198
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