原麝促卵泡激素β基因克隆及原核表达

发布时间：2018-03-16 03:10

本文选题：原麝　切入点：促卵泡激素β基因　出处：《东北林业大学》2016年硕士论文　论文类型：学位论文

【摘要】：促卵泡激素由α和p亚基组成,激素的生物活性、免疫活性均由p亚基所决定。研究表明,畜牧生产中FSHβ基因已成为衡量动物繁殖性状的候选基因之一,且该基因亦是调控原麝繁殖与泌香的重要基因。本研究以原麝为研究对象,采用基因克隆、原核表达研究原麝FSHp基因在大肠杆菌中的表达情况,制备原麝FSHp多克隆抗体,主要结果如下：本研究参考文献选取引物,扩增得到了原麝FSHp基因的全序列,基因长为4170bp,编码区(CDS)序列长度为390bp。生物信息学分析,结果表明：原麝FSHβ基因蛋白属于糖蛋白家族成员,为亲水性蛋白。原麝与其它物种的FSHβ基因CDS序列一致性比对,结果表明：原麝与林麝序列同源性最高(98.7%)；与梅花鹿序列一致性为93.2%；与牛科动物序列一致性在95.5%-97.0%之间。以FSHβ亚基基因编码区序列、氨基酸序列构建的系统进化树,结果显示：原麝与林麝关系最近。生物合成FSHp基因去信号肽序列,并成功构建了去信号肽FSHβ基因的原核表达载体PGEX-6P-1-FSHβ。 IPTG诱导表达、超声波破碎菌体。SDS-PAGE凝胶电泳检测：得到大小约为38KDa的融合蛋白,且融合蛋白主要以包涵体的形式存在。Western blot鉴定表明,重组的融合蛋白具有良好的免疫原性。将得到的融合蛋白纯化后免疫小鼠,分离小鼠血清,得到FSHp多克隆抗体,对多克隆抗体进行间接Elisa检测,结果表明：抗血清最高效价为1：51200。
[Abstract]:Follicle stimulating hormone is composed of 伪 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.
【学位授予单位】：东北林业大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：Q953

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