绵羊Gfrα1基因的部分cDNA克隆与抗原表位多肽的原核表达

畜牧兽医学报 ›› 2012, Vol. 43 ›› Issue (9): 1377-1384.doi:

绵羊Gfrα1基因的部分cDNA克隆与抗原表位多肽的原核表达

刘燕，罗奋华，包佳婧，刘林洪，单飞彪，吴应积^*

(内蒙古大学哺乳动物生殖生物学及生物技术教育部重点实验室，呼和浩特 010021)

收稿日期:2011-12-29 出版日期:2012-09-25 发布日期:2012-09-25
通讯作者: 吴应积，E-mail：yingji_wu@yahoo.com
作者简介:刘燕（1985-），女，内蒙古包头人，硕士生，主要从事动物生殖与分子生物学研究，E-mail： liuyannmg410@gmail．com
基金资助:
教育部创新团队计划子课题（IRT0833）；高等学校博士学科点博导类基金项目（20101501110001）

Cloning of Gfrα1 Partial cDNA in Sheep and Prokaryotic Expression of Epitope Polypeptide

LIU Yan, LUO Fen-Hua, BAO Jia-Jing, LIU Lin-Hong, DAN Fei-Biao, WU Ying-Ji^*

(The Key Laboratory for Mammalian Reproductive Biology and Biotechnology， Ministry of
Education，Inner Mongolia University，Hohhot 010021, China)

Received:2011-12-29 Online:2012-09-25 Published:2012-09-25

摘要/Abstract

摘要： 旨在克隆绵羊Gfrα1基因序列，深入研究绵羊精原干细胞（SSCs)。本研究通过RTPCR扩增和分子克隆的方法克隆到了绵羊Gfrα1基因的编码区大部分片段。结果，克隆得到的绵羊Gfrα1基因的大部分cDNA序列长1 312 bp，包括1 311 bp的开放阅读框（ORF），编码437个氨基酸，与牛、人、黑猩猩、大鼠和小鼠相应核苷酸序列的同源性分别为98.5%、91.3 %、91.0%、88.9%和88.0%。根据获得的cDNA序列，预测已知片段的抗原表位区，并将抗原表位区序列插入pET-44a（+）载体，经转化得到重组菌，经IPTG诱导表达，并通过亲和柱层析，纯化制备GFRα1抗原表位多肽。Western blot检测发现，经诱导表达的GFRα1抗原表位多肽约为18.2 ku，与预测的大小一致。绵羊Gfrα1基因的克隆和表达研究，为进一步制作该基因的多克隆抗体奠定基础，为绵羊SSCs的分子水平鉴定及功能分析提供了研究条件。

Abstract: In order to clone the DNA sequence of Gfrα1 gene in sheep and deeply research the sheep spermatogonial stem cells (SSCs), in the present study, the partial sheep Gfrα1 cDNA were cloned using RT-PCR and molecular cloning technique. The cloned partial Gfrα1 gene was 1 312 bp in length，including an ORF of 1 311 bp encoding 437 amino acids. The nucleotide sequences of sheep Gfrα1 gene were compared with the counterpart sequences of Bos Taurus，homo sapiens, Pongo abelii, Rattus norvegicus and Mus musculus, and the nucleotide homology was 98.5%, 91.3 %, 91.0%, 88.9% and 88.0%, respectively. According to the cloned Gfrα1 gene sequence, the epitope was forecasted and then the cDNA was inserted into pET-44a（+） vector．Subsequently, the recombinant was induced by IPTG. The recombinant epitope polypeptide was purified according to Affinity column chromatography. Western blot analysis indicated that the molecular weight of the expressed epitope polypeptide was the same as predicted size of approximate 18.2 ku. The data collected provided the important information for further making Gfrα1 gene polyclonal antibody and authenticating on molecular level for spermatogonial stem cells in sheep.

中图分类号:

S826
Q785

刘燕, 罗奋华, 包佳婧, 刘林洪, 单飞彪, 吴应积. 绵羊Gfrα1基因的部分cDNA克隆与抗原表位多肽的原核表达[J]. 畜牧兽医学报, 2012, 43(9): 1377-1384.

LIU Yan, LUO Fen-Hua, BAO Jia-Jing, LIU Lin-Hong, DAN Fei-Biao, WU Ying-Ji. Cloning of Gfrα1 Partial cDNA in Sheep and Prokaryotic Expression of Epitope Polypeptide[J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2012, 43(9): 1377-1384.

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参考文献

［1］HAMRA F K, SCHULTZ N, CHAPMAN K M, et al. Defining the spermatogonial stem cell［J］. Dev Biol, 2004, 269(2): 393-410.

［2］RYU B Y, ORWIG K E, OATLEY J M, et al. Efficient generation of transgenic rats through the male germline using lentiviral transduction and transplantation of spermatogonial stem cells［J］. J Androl, 2007, 28(2): 353-360.

［3］NAGANO M, SHINOHARA T, AVARBOCK M R, et al. Retrovirusmediated gene delivery into male germ line stem cells［J］. FEBS Lett, 2000, 475(1): 7-10.

［4］NAGANO M, BRINSTER C J, ORWIQ K E. Transgenic mice produced by retroviral transduction of male germline stem cells［J］, Proc Natl Acad Sci USA, 2001, 98(23): 13090-13095.

［5］HAMRA F K, GATLIN J, CHAPMAN K M, et al. Production of transgenic rats by lentiviral transduction of male germline stem cells［J］. Proc Natl Acad Sci USA, 2002， 99(23): 14931-14936.

［6］KO K, ARAúZOBRAVO M J, KIM J, et al. Conversion of adult mouse unipotent germline stem cells into pluripotent stem cells［J］. Nat Protoc, 2010， 5(5): 921-928.

［7］KINARM K, NAT ALIA T, WU G M, et al. Induction of Pluripotency in adult unipotent germline stem cells ［J］. Cell Stem Cell, 2009, 5: 87-96.

［8］CONRAD S, RENNINGER M, HENNENLOTTER J, et al. Generation of pluripotent stem cells from adult human testis［J］. Nature, 2008, 456(7220): 344-349.

［9］GUAN K, NAYERNIA K, MAIER L S, et al. Pluripotency of spermatogonial stem cells from adult mouse testis［J］. Nature, 2006, 440: 1199-1203.

［10］KANATSUSHINOH ARA M, INOUE K, LEE J, et al. Generation of pluripotent stem cells from neonatal mouse testis［J］. Cell, 2004, 119: 1001-1012.

［11］TEGELENBOSCH R A, DE ROOIJ D G. A quantitative study of spermatogonial multiplicati -on and stem cell renewal in the C3H/101 F1 hybrid mouse［J］. Mutat Res, 1993, 290(2): 193-200.

［12］KUBOTA H, AVARBOCK M R, BRINSTER R L. Spermatogonial stem cells share some, but not all, phenotypic and functional characteristics with other stem cells［J］. Proc Natl Acad Sci USA, 2003, 100(11): 6487-6492.

［13］SHINOHARA T, ORWIG K E, AVARBOCK M R, et al. Spermatogonial stem cell enrichment by multiparameter selection of mouse testis cells［J］. Proc Natl Acad Sci USA, 2000, 97(15): 8346-8351.

［14］SHINOHARA T, AVARBOCK M R, BRINSTER R L. Beta1-and alpha6-integrin are surface markers on mouse spermatogonial stem cells［J］. Proc Natl Acad Sci USA, 1999, 96(10): 5504-5509.

［15］OATLEY J M, BRINSTER R L. Regulation of spermatogonial stem cell self-renewal in mammals［J］. Annu Rev Cell Dev Biol, 2008, 24: 263-286.

［16］HE Z, KOKKINAKI M, JIANG J, et al. Isolation, characterization, and culture of human spermatogonia［J］. Biol Reprod, 2010, 82(2): 363-372.

［17］TREANOR J J, GOODMAN L, DE SAUVAGE F, et al. Characterization of a multicomponent receptor for GDNF［J］. Nature, 1996, 382: 80-83.

［18］MENG X, LINDAHL M, HYVONEN M E, et al. Regulation of cell fate decision of undifferentiated spermatogonia by GDNF［J］. Science, 2000, 287: 1489-1493.

［19］KUBOTA H, AVARBOCK M R, BRINSTER R L. Growth factors essential for selfrenewal and expansion of mouse spermatogonial stem cells［J］. Proc Natl Acad Sci USA, 2004, 101: 16489-16494.

［20］HE Z, JIANG J, HOFMANN M C, et al. Gfra1 silencing in mouse spermatogonial stem cells results in their differentiation via the inactivation of RET tyrosine kinase［J］. Biol Reprod, 2007, 77(4): 723-733.

［21］侯越, 吴应积, 罗奋华，等. 绒山羊Scp3 基因的克隆及睾丸中第一轮减数分裂的发生［J］ . 动物学研究, 2009, 30(3): 341-344.

［22］白音巴图, 罗奋华, 胡甜园，等. 绵羊tnp2基因的克隆及其在体外共培养系统中圆形精子细胞的转录分析［J］. 动物学研究, 2009, 30(3): 262-266.

［23］刘海超, 任俊明, 吴应积. 胶质细胞源性神经营养因子引发精原干细胞自我更新的信号通路［J］. 生物物理学报, 2012, 28(1): 29-36.

［24］JING S, WEN D, YU Y, et al. GDNFinduced activation of the Ret protein tyrosine kinase is mediated by GDNFRα, a novel receptor for GDNF［J］. Cell, 1996, 85(7): 1113-1124.

［25］NAKAGAWA T, SHARMA M, NABESHIMA Y, et al. Functional hierarchy and reversibility within the murine spermatogenic stem cell compartment［J］. Science, 2010, 328: 62-67.

［26］RYU B Y, KUBOTA H, AVARBOCK M R, et al. Conservation of spermatogonial stem cell self-renewal signaling between mouse and rat［J］. Proc Natl Acad Sci USA, 2005, 102: 14302-14307.

［27］KUBOTA H, BRINSTER R L. Culture of rodent spermatogonial stem cells, male germline stem cells of the postnatal animal［J］. Methods Cell Biol, 2008, 86: 59-84.

［28］HERMANN B P, SUKHWANI M, SIMORANGKIR D R, et al. Molecular dissection of the male germ cell lineage identifies putative spermatogonial stem cells in rhesus macaques ［J］. Hum Reprod, 2009, 24: 1704-1716.

［29］陈雪红. 胶质细胞源性神经营养因子受体alphal结构与功能关系的探讨［D］. 青岛: 青岛大学, 2003.

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