山羊卵泡刺激素基因的克隆、序列分析及表达载体构建-基础兽医学专业论文.docxVIP

摘要当前畜牧业生产中所使用的卵泡刺激素(FSH)产品都是从牛、羊、猪等家畜的脑 摘要 当前畜牧业生产中所使用的卵泡刺激素(FSH)产品都是从牛、羊、猪等家畜的脑 垂体中直接提取的，由于材料来源有限、纯度差且常有其它激素的污染，影响了FSH的 广泛应用。用基因工程方法可大量生产高纯度的FSH，降低生产费用，这对畜牧业生产 具有重要意义。 本试验是从新屠宰的母山羊脑垂体中提取总RNA，反转录获得cDNA，以此cDNA 为模板，PCR扩增目的基因片段，分别获得长为360bp的山羊FSH-a亚基DNA片段和 长为390bp的山羊FSH．B亚基DNA片段，与预期的目的基因片8L大d,—致。将它们分 了在同一种内，甚至在所有的哺乳动物中FSH．a亚基是极为保守的；而FSH—B亚基尽 管具有种属特异性，但也是相当保守的。将所得的山羊FSH-a、B亚基基因分别用加相 应限制性内切酶酶切位点的引物大量扩增，回收后与pMD 18-T连接，筛选出阳性重组 子，大量双酶切回收获得带粘性末端的山羊FSH．a、B亚基基因，将它们分别与同两个 酶大量双酶切回收获得的带粘性末端的PF质粒进行连接，筛选阳性重组子。依此方法 分别构建了FSH单亚基真核表达载体grSH-2-PF、gFSH-B-PF和FSH双亚基真核表达 载体庐sH-a—B-PF、dFSH—o-gFSH B，PF。户7一 本研究首次进行了山羊FSH的基因克隆、序列分析及表达载体构建方面的探索，填 补了国内外重组山羊FSH基础研究的空白，为今后重组山羊FSH的大量生产应用提供 了理论基础和实验依据。 关键词山恙卵泡刺激素； 基因克隆； 序列分析； 表达载体构建i ／ V AbstractThe Abstract The products of follicle stimulating hormone(FS}n used in animal husbandry afe all exl／ltc把d from the pitui倒es ofbovine．goat,pig and咖other animals．11lack offal and low purity of this kind of FSH limit ils extensive application．ByⅡrtlealls of gene engineering,large quantity and high purity FSH can be produced and the production expenditure alSO c∞be reduced．which is im【portant to animal husbandry． TotalRNAwas extractedfrompituitaryofnewbutcheredfemalegoatforFSH_旺and 13 subuni_t genes．eDNAⅥ雠synlhesized by RT-PCR n塌d由M】s respectively and these eDNA we∞used∞templates in PCR amplifications．．nle PCR products Were 360t,p and 390bp whichjustwerethe samelength ofthe predictedgoatFSH-a and B subunit genes．They were cloned幻pMD 18-T respectively and selected two positive recombinams at rdndorfl to analysis their sequences respectively,and compared their genes and amino acids with some other mamma】s．T1】e results showed that the genes and amino acids ofgoat FSH-a and B sublmits aU have very high homology with other n墙mInals and their homology all e)【c∞d 80％．These results proved thra FSH·n subuni^has high conservation among mammals and FSH-13 subunit is also quite conservative among mammals although it shows species specifieity．Goat FSH-Q and 13 subunit genes、