梅花鹿卵泡刺激素的基因克隆、表达和活性分析
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摘要
卵泡刺激素(follicle stimulating hormone，FSH)广泛用于牛、羊等动物的超排、人工受精等繁殖育种工作中。可以通过几种方法获得FSH，如从绝经期妇女尿中提取，或从猪、牛、羊等动物脑垂体中提取或采用重组DNA技术生产重组FSH。从绝经期妇女尿中及猪、牛、羊等动物脑垂体中提取的FSH存在严重问题，如材料来源有限、提取纯化工作费时、费力、质量差、纯度低、常有其它激素的污染等。采用基因重组技术生产重组FSH就可解决这些问题。例如解决材料来源问题、简化生产工艺、降低生产成本、批次间差异最小、并且没有其它激素污染、无抗体反应。本文采用RT-PCR、SDS-PAGE、Western blot、Radioimmunoassy等方法首次研究了梅花鹿FSH的基因克隆、表达和活性分析，结果如下：
1．梅花鹿FSH-α亚基基因是由363个核苷酸组成，编码120个氨基酸。该序列已提交GenBank被接受，序列号为AY066018。将梅花鹿FSH-α亚基基因核苷酸序列与其它动物该序列相比，结果显示梅花鹿与山羊的同源性最高，达到99％，它们之间只有3个核苷酸不同。梅花鹿与豚鼠、狨的核苷酸同源性最低，为83％～84％，有57～59个核苷酸不同。将梅花鹿FSH-α亚基氨基酸序列与其它动物该序列相比，结果显示梅花鹿与山羊的同源性最高，达到99％。它们之间只有1个氨基酸不同。梅花鹿与牛、绵羊、印度水牛的同源性也高达到97％～98％，有3～4个氨基酸不同。从编码FSH-α亚基基因的核苷酸序列来看，在这些比较的动物中，FSH-α亚基基因核苷酸序列是高度保守的。
2．梅花鹿FSH-β亚基基因由390个核苷酸组成，编码129个氨基酸。该序列已提交GenBank被接受，序列号为AY156688。将梅花鹿FSH-β亚基基因核苷酸序列与其它动物相比，结果显示梅花鹿与绵羊、山羊、牛的同源性最高，达到92％～93％，它们之间有24～30个核苷酸不同。梅花鹿与仓鼠、大鼠、褐鼠的核苷酸同源性最低，为81％～82％，有71～74个核苷酸不同。从FSH-β亚基氨基酸序列来看，梅花鹿与绵羊、山羊、牛的同源性最高，达到92％～93％，它们之间有9～10个氨基酸不同。梅花鹿与朱鹮、鹌鹑的同源性最低，为60％～65％，有45～52个氨基酸不同。
3．重组FSH对促进昆明小鼠卵泡发育有一定效果，可促进次级卵泡向成熟卵泡的发育；对雌性未成年昆明小白鼠卵巢增重效果较明显；对促进小鼠子宫重量的增加有一定效果；重组FSH可提高小鼠血清中E_2含量，但不同个体反应不同。
Follicle stimulating hormone (FSH) had been used widespreadly on superrovulation and artificial fertilization in propagation of cattle,sheep.FSH can be obtained by several techniques.One of them was by purifying urine from menopausal women and extracted FSH from the anterior pituitary of pig,cattle.sheep or FSH was made by using recombinant technique.But the FSH made by urine or the anterior pituitary often had some severities matters^uch as short of primary materials,time-consuming,baborious,copy,low purity and contaminated with other hormones.Those problems can be solved by using recombinant technique.The rFSH which was made by using recombinant technique,which it had some superiorities,for example providing enough primary materials,simplified generative technics, decreased generative cost,not contaminated with other hormones and differences among batches were minimized,no antibody reacrion.The gene cloning,expression and bioactivities of FSH of Cervus nippon was made firstly by RT-PCR,SDS- PAGE,Western blotjladioimmunoassy et al techniques.The results were showed as follows:
1. The nucleotide sequence which encoding Cervus nippon FSH-a subunit was composed of 363 nucleotides.lt was composed of 120 amino acids.lt was directly submitted to GenBank and accepted the number as AY066018. The nucleotide sequence of Cervus nippon FSH-a subunit was compared with other animals.lt had the highest homology with goat,which reached 99%.There were only 3 nucleotides difference. It had lowest homology in nucleotide sequence with guinea pig,Callithrix jacchus, which is 83%-84%. There are 57*59nucleotides difference. It had the highest homology of amino acid sequence of Cervus nippon with goat, which reached 99%. There was only 1 amino acid difference between Cervus nippon and goat It had higher homology among bovine, sheep and buffalo, which reached 92%-93%.There were 3-4 amino acids difference among them. It was found that the nucleotide sequence of the a-subunit in these animals were highly conservative.
2. The nucleotide sequence which encoding Cervus nippon FSH-b subunit was composed of 390 nucleotides. It was composed of 129 amino acids.It was submitted to GenBank and accepted as AY 156688. The nucleotide sequence of Cervus nippon FSH-b subunit was compared withotheranimals.lt had the highest homology among Cervus nippon, sheep, goat and bovine in nucleotide sequence, which reached 92%-93%. There were 24-30 nucleotides difference among them. It had lower homology in nucleotide sequence among Rattus norvegtcusjatfhodopus sungorus,which it was only 81%-82%. There were 71-74 nucleotides difference among them.Its amino acid sequence was compared with some other animals.The result showed that it had the highest homology with sheep,goat,bovine,which reached 92%-93%.There were 9-10 amino acids difference among them. It had lower
homology in amino acid with Coturnix japamca,Nippoma nippon, its homology was 60%-65%.There were 45-52 amino acids difference among them.
3. The recombinant FSH stimulated the growth of Kunming mouse's follicle.It stimulated the secondary follicle to mature follicle.The recombinant FSH stimulated the growth of Kunming mouse's uterus and ovary.The product of recombinant FSH of Cervus nippon raised the content of E2 in mouse's serum, but it had different effect on different body.
Doctor: Guan Hongbin Major Basic Veterinary Science Supervisor: Professor Li Qingzhang
1．梅花鹿FSH-α亚基基因是由363个核苷酸组成，编码120个氨基酸。该序列已提交GenBank被接受，序列号为AY066018。将梅花鹿FSH-α亚基基因核苷酸序列与其它动物该序列相比，结果显示梅花鹿与山羊的同源性最高，达到99％，它们之间只有3个核苷酸不同。梅花鹿与豚鼠、狨的核苷酸同源性最低，为83％～84％，有57～59个核苷酸不同。将梅花鹿FSH-α亚基氨基酸序列与其它动物该序列相比，结果显示梅花鹿与山羊的同源性最高，达到99％。它们之间只有1个氨基酸不同。梅花鹿与牛、绵羊、印度水牛的同源性也高达到97％～98％，有3～4个氨基酸不同。从编码FSH-α亚基基因的核苷酸序列来看，在这些比较的动物中，FSH-α亚基基因核苷酸序列是高度保守的。
2．梅花鹿FSH-β亚基基因由390个核苷酸组成，编码129个氨基酸。该序列已提交GenBank被接受，序列号为AY156688。将梅花鹿FSH-β亚基基因核苷酸序列与其它动物相比，结果显示梅花鹿与绵羊、山羊、牛的同源性最高，达到92％～93％，它们之间有24～30个核苷酸不同。梅花鹿与仓鼠、大鼠、褐鼠的核苷酸同源性最低，为81％～82％，有71～74个核苷酸不同。从FSH-β亚基氨基酸序列来看，梅花鹿与绵羊、山羊、牛的同源性最高，达到92％～93％，它们之间有9～10个氨基酸不同。梅花鹿与朱鹮、鹌鹑的同源性最低，为60％～65％，有45～52个氨基酸不同。
3．重组FSH对促进昆明小鼠卵泡发育有一定效果，可促进次级卵泡向成熟卵泡的发育；对雌性未成年昆明小白鼠卵巢增重效果较明显；对促进小鼠子宫重量的增加有一定效果；重组FSH可提高小鼠血清中E_2含量，但不同个体反应不同。
Follicle stimulating hormone (FSH) had been used widespreadly on superrovulation and artificial fertilization in propagation of cattle,sheep.FSH can be obtained by several techniques.One of them was by purifying urine from menopausal women and extracted FSH from the anterior pituitary of pig,cattle.sheep or FSH was made by using recombinant technique.But the FSH made by urine or the anterior pituitary often had some severities matters^uch as short of primary materials,time-consuming,baborious,copy,low purity and contaminated with other hormones.Those problems can be solved by using recombinant technique.The rFSH which was made by using recombinant technique,which it had some superiorities,for example providing enough primary materials,simplified generative technics, decreased generative cost,not contaminated with other hormones and differences among batches were minimized,no antibody reacrion.The gene cloning,expression and bioactivities of FSH of Cervus nippon was made firstly by RT-PCR,SDS- PAGE,Western blotjladioimmunoassy et al techniques.The results were showed as follows:
1. The nucleotide sequence which encoding Cervus nippon FSH-a subunit was composed of 363 nucleotides.lt was composed of 120 amino acids.lt was directly submitted to GenBank and accepted the number as AY066018. The nucleotide sequence of Cervus nippon FSH-a subunit was compared with other animals.lt had the highest homology with goat,which reached 99%.There were only 3 nucleotides difference. It had lowest homology in nucleotide sequence with guinea pig,Callithrix jacchus, which is 83%-84%. There are 57*59nucleotides difference. It had the highest homology of amino acid sequence of Cervus nippon with goat, which reached 99%. There was only 1 amino acid difference between Cervus nippon and goat It had higher homology among bovine, sheep and buffalo, which reached 92%-93%.There were 3-4 amino acids difference among them. It was found that the nucleotide sequence of the a-subunit in these animals were highly conservative.
2. The nucleotide sequence which encoding Cervus nippon FSH-b subunit was composed of 390 nucleotides. It was composed of 129 amino acids.It was submitted to GenBank and accepted as AY 156688. The nucleotide sequence of Cervus nippon FSH-b subunit was compared withotheranimals.lt had the highest homology among Cervus nippon, sheep, goat and bovine in nucleotide sequence, which reached 92%-93%. There were 24-30 nucleotides difference among them. It had lower homology in nucleotide sequence among Rattus norvegtcusjatfhodopus sungorus,which it was only 81%-82%. There were 71-74 nucleotides difference among them.Its amino acid sequence was compared with some other animals.The result showed that it had the highest homology with sheep,goat,bovine,which reached 92%-93%.There were 9-10 amino acids difference among them. It had lower
homology in amino acid with Coturnix japamca,Nippoma nippon, its homology was 60%-65%.There were 45-52 amino acids difference among them.
3. The recombinant FSH stimulated the growth of Kunming mouse's follicle.It stimulated the secondary follicle to mature follicle.The recombinant FSH stimulated the growth of Kunming mouse's uterus and ovary.The product of recombinant FSH of Cervus nippon raised the content of E2 in mouse's serum, but it had different effect on different body.
Doctor: Guan Hongbin Major Basic Veterinary Science Supervisor: Professor Li Qingzhang
引文
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