山羊卵泡刺激素基因的克隆、序列分析及表达载体构建
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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-α亚基DNA片段和长为390bp的山羊FSH-β亚基DNA片段，与预期的目的基因片段大小一致。将它们分别克隆至pMD 18-T，随机挑选几个阳性重组子进行测序。将测序结果与绵羊等多种哺乳动物该基因及相应的氨基酸进行同源性比较、分析。结果显示山羊FSH-α、β亚基基因及其各自对应的氨基酸与其他哺乳动物的同源性都非常高，一般均在80％以上，验证了在同一种内，甚至在所有的哺乳动物中FSH-α亚基是极为保守的；而FSH-β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-α、β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-α、β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gFSH-α-PF、gFSH-β-PF和FSH双亚基真核表达载体gFSH-α-β-PF、dFSH-α-gFSHβ-PF。
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。
The products of follicle stimulating hormone(FSH) used in animal husbandry are all extracted from the pituharies of bovine, goat, pig and some other animals. The lack of material and low purity of this kind of FSH limit its extensive application. By the means of gene engineering, large quantity and high purity FSH can be produced and the production expenditure also can be reduced, which is important to animal husbandry.
Total RNA was extracted from pituitary of new butchered female goat for FSH-a and b subunit genes. cDNA were synthesized by RT-PCR reactions respectively and these cDNA were used as templates in PCR amplifications. The PCR products were 360bp and 390bp which just were the same length of the predicted goat FSH- a and P subunit genes. They were cloned to pMD 18-T respectively and selected two positive recombinants at random to analysis their sequences respectively, and compared their genes and amino acids with some other mammals. The results showed that the genes and amino acids of goat FSH- a and P subunits all have very high homology with other mammals and their homology all exceed 80%. These results proved that FSH- a subunit has high conservation among mammals and FSH- P subunit is also quite conservative among mammals although it shows species specificity. Goat FSH- a and P subunit genes were reamplified respectively by the new primers which had the corresponding restriction enzymes. The PCR products were purified and linked with pMD 18-T vector respectively. The positive recombinants were selected and digested with double restriction enzymes respectively. Goat FSH- a and P subunit genes which had sticky ends were purified and linked respectively with PF which also had the corresbonding sticky ends and were purified. The positive recombinants were selected finally. By this means, eukaryotic expression vectors of FSH single subunit-gFSH-a-PF, gFSH-b -PF and eukaryotic expression vectors of FSH double subunits-gFSH-a-b-PF, dFSH-a -gFSH- P -PF were all constructed successfully.
In this experiment, gene cloning, sequence analysis and construction of expression vector of goat FSH were fist studied. This work initiated the basic research of recombinant goat FSH which supplied the theoretic basis and experiment references to its quantity production and application.
Master: Zhang Li Major: Basic Veterinary Science Supervisor: Prof. Li Qing-zhang
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-α亚基DNA片段和长为390bp的山羊FSH-β亚基DNA片段，与预期的目的基因片段大小一致。将它们分别克隆至pMD 18-T，随机挑选几个阳性重组子进行测序。将测序结果与绵羊等多种哺乳动物该基因及相应的氨基酸进行同源性比较、分析。结果显示山羊FSH-α、β亚基基因及其各自对应的氨基酸与其他哺乳动物的同源性都非常高，一般均在80％以上，验证了在同一种内，甚至在所有的哺乳动物中FSH-α亚基是极为保守的；而FSH-β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-α、β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-α、β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gFSH-α-PF、gFSH-β-PF和FSH双亚基真核表达载体gFSH-α-β-PF、dFSH-α-gFSHβ-PF。
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。
The products of follicle stimulating hormone(FSH) used in animal husbandry are all extracted from the pituharies of bovine, goat, pig and some other animals. The lack of material and low purity of this kind of FSH limit its extensive application. By the means of gene engineering, large quantity and high purity FSH can be produced and the production expenditure also can be reduced, which is important to animal husbandry.
Total RNA was extracted from pituitary of new butchered female goat for FSH-a and b subunit genes. cDNA were synthesized by RT-PCR reactions respectively and these cDNA were used as templates in PCR amplifications. The PCR products were 360bp and 390bp which just were the same length of the predicted goat FSH- a and P subunit genes. They were cloned to pMD 18-T respectively and selected two positive recombinants at random to analysis their sequences respectively, and compared their genes and amino acids with some other mammals. The results showed that the genes and amino acids of goat FSH- a and P subunits all have very high homology with other mammals and their homology all exceed 80%. These results proved that FSH- a subunit has high conservation among mammals and FSH- P subunit is also quite conservative among mammals although it shows species specificity. Goat FSH- a and P subunit genes were reamplified respectively by the new primers which had the corresponding restriction enzymes. The PCR products were purified and linked with pMD 18-T vector respectively. The positive recombinants were selected and digested with double restriction enzymes respectively. Goat FSH- a and P subunit genes which had sticky ends were purified and linked respectively with PF which also had the corresbonding sticky ends and were purified. The positive recombinants were selected finally. By this means, eukaryotic expression vectors of FSH single subunit-gFSH-a-PF, gFSH-b -PF and eukaryotic expression vectors of FSH double subunits-gFSH-a-b-PF, dFSH-a -gFSH- P -PF were all constructed successfully.
In this experiment, gene cloning, sequence analysis and construction of expression vector of goat FSH were fist studied. This work initiated the basic research of recombinant goat FSH which supplied the theoretic basis and experiment references to its quantity production and application.
Master: Zhang Li Major: Basic Veterinary Science Supervisor: Prof. Li Qing-zhang
引文
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76 Montgomery GW, Penty JM, Sise JA, Tou HM. Genes Encoding the Alpha and Beta Chains of Follicle-Stimulating Hormone are not Sites for the Booroola (FecB) Mutation in Sheep. J Reprod Fertil. 1992, 95(3): 895～901
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82 Requel B, Daniel Dl, Milagros A, Adelaida L, Jose CM. Induction of Puberty with Human Chorionic Gonadotopin and Follicle Stimulating Hormone in Hypogonadotropic Hypogonadism. Fertility and Sterilitys. 1999, 71(2): 244～248
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86 Schoot DC, Coelingh BH, Mannaerts BM, Lamberts SW, Bouchard P, Fauser BC. Human Recombinant Follicle-Stimulating Hormone Induces Growth of Preovulatory Follicles without Concomitant Increase in Androgen and Estrogen Biosynthesis in a
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