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  本文選題：原麝　切入點：促卵泡激素β基因　出處：《東北林業(yè)大學》2016年碩士論文　論文類型：學位論文

【摘要】：促卵泡激素由α和p亞基組成,激素的生物活性、免疫活性均由p亞基所決定。研究表明,畜牧生產(chǎn)中FSHβ基因已成為衡量動物繁殖性狀的候選基因之一,且該基因亦是調(diào)控原麝繁殖與泌香的重要基因。本研究以原麝為研究對象,采用基因克隆、原核表達研究原麝FSHp基因在大腸桿菌中的表達情況,制備原麝FSHp多克隆抗體,主要結(jié)果如下：本研究參考文獻選取引物,擴增得到了原麝FSHp基因的全序列,基因長為4170bp,編碼區(qū)(CDS)序列長度為390bp。生物信息學分析,結(jié)果表明：原麝FSHβ基因蛋白屬于糖蛋白家族成員,為親水性蛋白。原麝與其它物種的FSHβ基因CDS序列一致性比對,結(jié)果表明：原麝與林麝序列同源性最高(98.7%)；與梅花鹿序列一致性為93.2%；與�？苿游镄蛄幸恢滦栽�95.5%-97.0%之間。以FSHβ亞基基因編碼區(qū)序列、氨基酸序列構(gòu)建的系統(tǒng)進化樹,結(jié)果顯示：原麝與林麝關(guān)系最近。生物合成FSHp基因去信號肽序列,并成功構(gòu)建了去信號肽FSHβ基因的原核表達載體PGEX-6P-1-FSHβ。 IPTG誘導表達、超聲波破碎菌體。SDS-PAGE凝膠電泳檢測：得到大小約為38KDa的融合蛋白,且融合蛋白主要以包涵體的形式存在。Western blot鑒定表明,重組的融合蛋白具有良好的免疫原性。將得到的融合蛋白純化后免疫小鼠,分離小鼠血清,得到FSHp多克隆抗體,對多克隆抗體進行間接Elisa檢測,結(jié)果表明：抗血清最高效價為1：51200。
[Abstract]:Follicle stimulating hormone is composed of 偽 and p subunits, and its biological activity and immune activity are all determined by p subunit. It has been shown that FSH 尾 gene has become one of the candidate genes to measure animal reproductive traits in animal husbandry. The gene is also an important gene to regulate the reproduction and secretion of the original musk deer. In this study, we studied the expression of FSHp gene in Escherichia coli by gene cloning, and prepared the polyclonal antibody to FSHp of promusk deer. The main results are as follows: the whole sequence of FSHp gene of musk deer was amplified from primer selected in the literature. The length of the gene was 4170 BP and the length of coding region was 390 bp. bioinformatics analysis. The results showed that FSH 尾 gene protein was a member of glycoprotein family and was a hydrophilic protein. The CDS sequence of FSH 尾 gene was consistent with that of other species. The results showed that the homology between the original musk deer and the forest musk deer was the highest (98.775), the consistency with the sika deer sequence was 93.22.The sequence consistency with the Bovine fauna was 95.5- 97.0%. The phylogenetic tree was constructed from the coding region of FSH 尾 subunit gene and amino acid sequence. The results showed that the relationship between the procalcide-musk deer and the forest musk deer was close. The FSHp gene was synthesized and the prokaryotic expression vector PGEX-6P-1-FSH 尾. IPTG was successfully constructed to induce the expression of the FSH 尾 gene. Ultrasonic fragmentation of bacteria. SDS-PAGE gel electrophoresis showed that the fusion protein was about 38KDa in size, and the fusion protein mainly existed in the form of inclusion body. Western blot analysis showed that the fusion protein could be identified as inclusion body. The recombinant fusion protein had good immunogenicity. The purified fusion protein was used to immunize mice, the mouse serum was isolated, the polyclonal antibody of FSHp was obtained, and the polyclonal antibody was detected indirectly by Elisa. The results showed that the highest titer of the antiserum was 1: 51200.
【學位授予單位】：東北林業(yè)大學
【學位級別】：碩士
【學位授予年份】：2016
【分類號】：Q953

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