绒山羊Izumo基因的克隆及组织表达
摘要
哺乳动物的受精过程,是一个精密而有序的细胞之间的相互作用的过程,其最终的结果就是精卵间通过质膜融合作用,成为受精卵。目前,科学家们虽然通过基因组学理论和蛋白质组学技术在精子表面上发现了数量可观的与受精相关的分子,但是精卵融合过程是在顶体反应发生之后才进行的,其中精子和卵细胞的质膜必须通过相匹配的受体结合,而这个引发精卵融合的分子机制究竟是怎样运作目前还不清楚。在这些已经获得的与精卵融合过程相关的分子中,只有近期在精子上发现的IGSF(免疫球蛋白超家族)新成员Izumo基因,经过基因敲除的小鼠实验,证明它是精卵融合所必需的。
内蒙古白绒山羊作为一种内蒙古地区培育多年的家畜,是羊毛和羊绒的主要来源之一。由于Izumo基因的发现对于了解绒山羊的受精机制有重要的指导意义,为了研究其在内蒙古白绒山羊精卵融合中的分子机制,本实验根据GenBank中已发表的牛的Izumo核苷酸序列,设计并合成了扩增绒山羊Izumo基因的1对引物,实验采用RT-PCR(逆转录聚合酶链式反应)扩增获得了Izumo基因部分序列,并对Izumo基因进行了克隆和序列分析。在此基础之上,以得到的绒山羊Izumo部分序列为模板,采用RACE(快速扩增cDNA末端)技术,扩增了雄性成年内蒙古白绒山羊睾丸组织中Izumo基因的全长序列,并钓取了该基因的CDS(编码区)。另外本实验还制备了地高辛标记的绒山羊Izumo cDNA探针,用利用RNA Northern印记技术,对内蒙古白绒山羊Izumo RNA进行分析,从而测定总RNA中编码Izumo基因的mRNA分子的大小和表达丰度。
1、通过RT-PCR技术,获得484 bp的Izumo基因cDNA序列,与牛的Izumo基因同源性高达98%。
2、通过RACE技术,获得1536 bp的全长序列,钓取到1035 bp的Izumo基因编码区序列,该编码区编码344个氨基酸。
3、通过对绒山羊睾丸和附睾的头、体、尾部组织Izumo基因mRNA的RT-PCR组织表达鉴定,以及对该基因mRNA的Northern印迹鉴定,结果睾丸和附睾的头部、体部、尾部组织均显示阳性条带。
4、本研究为进一步进行绒山羊Izumo基因在精卵融合过程中的功能研究奠定了基础。
Mammalian fertilization process is a precise and orderly process of interaction between cells, the end result is that sperm-egg membrane fusion between the adoption of the role of a fertilized egg. At present, the scientists although the theory and through genomics proteomics technology found in the sperm surface fertilization associated with a significant number of molecules, but the sperm-egg fusion process is after the acrosome reaction carried out, in which sperm and egg plasma membrane must match the receptor binding, and this lead to what the molecular mechanism of sperm-egg fusion is unclear how it will operate. Has been obtained in these processes associated with sperm-egg fusion molecules, only recently discovered in the sperm of IGSF (immunoglobulin superfamily) new members of Izumo gene knockout mice after experiments prove that it is sperm-egg fusion necessary.
Inner Mongolia White Cashmere goats for many years as a foster livestock in Inner Mongolia, is the main source of wool and cashmere, one of the research on the Izumo gene reported in this paper are first. Since the discovery of Izumo gene for understanding the mechanism of fertilization and goats have important guiding significance, in order to study in Inner Mongolia White Cashmere goats in the molecular mechanism of sperm-egg fusion, this study has been published under the GenBank, the nucleotide sequence of bovine Izumo, designed and synthesized goats Izumo gene amplification primers 1, experiment using RT-PCR (reverse transcription polymerase chain reaction) amplification of the Izumo gene sequences, and Izumo gene was cloned and sequenced. In this basis, to get some goats Izumo sequence as a template, using RACE (rapid amplification of cDNA ends) technology, the first expansion of the adult male testis Inner Mongolia Cashmere goats in Izumo gene sequences, and fishing took the gene CDS (coding region). Also this experiment was prepared digoxigenin labeled goat Izumo cDNA probe technology with the use of RNA Northern imprint on the Inner Mongolia White Cashmere goats Izumo RNA analysis, and thus the determination of total RNA, mRNA encoding Izumo gene expression of molecular size and abundance.
1. By RT-PCR technique, the Izumo gene was 484 bp cDNA sequence, and the cattle Izumo gene homology as high as 98%.
2. By RACE technology to obtain 1536 bp full-length sequence, fishing access to the 1035 bp of the Izumo gene coding sequence, the coding region encoding 344 amino acids.
3. Through the goat testis and epididymis head, body, tail tissue Izumo gene mRNA in the RT-PCR identification of tissue expression, and mRNA of the gene in the Northern blotting results of testis and epididymis of the head, body, tail tissue show positive bands.
4. This study further goats Izumo sperm-egg fusion in the process of gene function in the foundation.
内蒙古白绒山羊作为一种内蒙古地区培育多年的家畜,是羊毛和羊绒的主要来源之一。由于Izumo基因的发现对于了解绒山羊的受精机制有重要的指导意义,为了研究其在内蒙古白绒山羊精卵融合中的分子机制,本实验根据GenBank中已发表的牛的Izumo核苷酸序列,设计并合成了扩增绒山羊Izumo基因的1对引物,实验采用RT-PCR(逆转录聚合酶链式反应)扩增获得了Izumo基因部分序列,并对Izumo基因进行了克隆和序列分析。在此基础之上,以得到的绒山羊Izumo部分序列为模板,采用RACE(快速扩增cDNA末端)技术,扩增了雄性成年内蒙古白绒山羊睾丸组织中Izumo基因的全长序列,并钓取了该基因的CDS(编码区)。另外本实验还制备了地高辛标记的绒山羊Izumo cDNA探针,用利用RNA Northern印记技术,对内蒙古白绒山羊Izumo RNA进行分析,从而测定总RNA中编码Izumo基因的mRNA分子的大小和表达丰度。
1、通过RT-PCR技术,获得484 bp的Izumo基因cDNA序列,与牛的Izumo基因同源性高达98%。
2、通过RACE技术,获得1536 bp的全长序列,钓取到1035 bp的Izumo基因编码区序列,该编码区编码344个氨基酸。
3、通过对绒山羊睾丸和附睾的头、体、尾部组织Izumo基因mRNA的RT-PCR组织表达鉴定,以及对该基因mRNA的Northern印迹鉴定,结果睾丸和附睾的头部、体部、尾部组织均显示阳性条带。
4、本研究为进一步进行绒山羊Izumo基因在精卵融合过程中的功能研究奠定了基础。
Mammalian fertilization process is a precise and orderly process of interaction between cells, the end result is that sperm-egg membrane fusion between the adoption of the role of a fertilized egg. At present, the scientists although the theory and through genomics proteomics technology found in the sperm surface fertilization associated with a significant number of molecules, but the sperm-egg fusion process is after the acrosome reaction carried out, in which sperm and egg plasma membrane must match the receptor binding, and this lead to what the molecular mechanism of sperm-egg fusion is unclear how it will operate. Has been obtained in these processes associated with sperm-egg fusion molecules, only recently discovered in the sperm of IGSF (immunoglobulin superfamily) new members of Izumo gene knockout mice after experiments prove that it is sperm-egg fusion necessary.
Inner Mongolia White Cashmere goats for many years as a foster livestock in Inner Mongolia, is the main source of wool and cashmere, one of the research on the Izumo gene reported in this paper are first. Since the discovery of Izumo gene for understanding the mechanism of fertilization and goats have important guiding significance, in order to study in Inner Mongolia White Cashmere goats in the molecular mechanism of sperm-egg fusion, this study has been published under the GenBank, the nucleotide sequence of bovine Izumo, designed and synthesized goats Izumo gene amplification primers 1, experiment using RT-PCR (reverse transcription polymerase chain reaction) amplification of the Izumo gene sequences, and Izumo gene was cloned and sequenced. In this basis, to get some goats Izumo sequence as a template, using RACE (rapid amplification of cDNA ends) technology, the first expansion of the adult male testis Inner Mongolia Cashmere goats in Izumo gene sequences, and fishing took the gene CDS (coding region). Also this experiment was prepared digoxigenin labeled goat Izumo cDNA probe technology with the use of RNA Northern imprint on the Inner Mongolia White Cashmere goats Izumo RNA analysis, and thus the determination of total RNA, mRNA encoding Izumo gene expression of molecular size and abundance.
1. By RT-PCR technique, the Izumo gene was 484 bp cDNA sequence, and the cattle Izumo gene homology as high as 98%.
2. By RACE technology to obtain 1536 bp full-length sequence, fishing access to the 1035 bp of the Izumo gene coding sequence, the coding region encoding 344 amino acids.
3. Through the goat testis and epididymis head, body, tail tissue Izumo gene mRNA in the RT-PCR identification of tissue expression, and mRNA of the gene in the Northern blotting results of testis and epididymis of the head, body, tail tissue show positive bands.
4. This study further goats Izumo sperm-egg fusion in the process of gene function in the foundation.
引文
1 Inoue N, Ikawa M, Isotani A,et al. The immunoglobulin superfamily protein Izumo is required for sperm to fuse with eggs[J]. Nature, 2005, 434:(7030) 234-238.
2王德刚;黄天华;谢庆东;安刚.重组小鼠Izumo的表达及其特异性抗体对小鼠体外精卵融合的影响[J].癌变.畸变.突变, 2007, (03): 68-72.
3安刚;黄天华;谢庆东;王德刚. pCXN2-mIZUMO对C57BL/6小鼠生育能力的影响[J].癌变.畸变.突变, 2007, (04): 60-63.
4 Turano C,Coppari S,Altieri F,Ferraro A. Proteins of the PDI family: unpredicted non-ER locations and functions. J Cell physiol. 2002,193(2): 154-163.
5 Ellerman DA,Myles DG,Primakoff P. A role for sperm surface protein disulfide isomerase activity in gamete fusion: evidenee for the participation of ERP57. Dev Cell. 2006,10: 831-837.
6 Schultz R,Williams C. Developmental biology: sperm-egg fusion unscrambled. Nature. 2005,434(7030):152-153.
7 Talwar GP. Vaccines and passive immunological approaches for fertility and hormone-dependent cancer. Immunol review, 1991, 171: 173-179.
8 Naz RK, Gupta SK, Gupta JC, Vyas HK and Talwar GP. Recent advances in contraceptive vaccine development. Hum Reprod, 2005, 20: 3271-3283.
9 Gupta S. Reproductive immunology. India: Narosa Publishing House, 1999, 303-308.
10 Aitaken JR. Immunocontraceptive vaccine for human use. J Reprod lmmunol, 2002, 57: 273-287.
11 Kirkpatrick JF and Turner J. Reversibility of action and safety during pregnancy of immunization against zona pellucid in wild mares. Reprod Suppl, 2002, 60: 197-202.
12 Talwar GP, Singh O, Pal R, Chatterjee N, Sahal P, Dhall K, Kaul J, Das SK, Suri S, Buckshee K, Saraya L and Saxena BN. A vaccine that prevents pregnancy in woman. Proc Natl Acad Sci U S A, 1994, 91: 2532-2536.
13 Ohl D and Naz RK. Infertility due to antibodies. J Uro1, 1995, 46: 591-602.
14 Bronson RG, Cooper G and Rosenfeld D. Sperm antibodies: their role in infertility. Fertil Steril, 1984, 42: 171-182.
15 Liskin L, Pile JM, Quillan WF. Vasectomy safe and simple. Popul Rep, 1983, 4: 61-100.
16 Silber SJ and Grotjan HE. Microscopic vasectomy reversal 30 years later: asummary of 4010 cases by the same surgeon. J Andro1, 2004, 25: 845-859.
17 Baskin MJ. Temporary sterilization by injection of human spermatozoa: a preliminary report. Am J Obstet Gynecol, 1932, 24: 892-897.
18 Menge AC. Immunizations and infertility. J Reprod Fertil Suppl, 1970, 10: 171-185.
19 Allardyce RA. Effect of ingested sperm on fecundity in the rat. J Exp Med, 1984, 159: 1548-1553.
20 Naz RK. Search for Peptide sequences involved in human antisperm antibody-mediated male immunoinfertility by using phage display technology. Mo1 Reprod Dev, 2005, 72: 25-30.
21 Goldberg E. Sperm specific lactate dehydrogenase and development of a contraceptive vaccine. In Clark DA and Croy BA. Reproduetive Immunology. New York: Elsevier Press 1986. 137-142.
22 Naz RK and Zhu X. Reeombinant fertilization antigen-1 causes a contraceptive effect in actively immunized mice. Biol Reprod, 1998, 59: 1095-1100.
23 Naz RK. Fertilization-related sperm antigens and their immunocontraceptive potentials. Am J Reprod Immunol, 2000, 44: 41-46.
24 Naz RK and Zhu X. Molecular cloning and sequencing of a novel cDNA encoding for a protein involved in human sperm function. Biochem Biophys Res Commun, 2001, 284: 911-917.
25 Naz RK and Chauhan SC. Human sperm-specific Peptide vaccine that causes long-term reversible contraception. Biol Reprod, 2002, 67: 674-680.
26 Trivedi RN and Naz RK. Testis-specific antigen(TSA-1) is expressed in murine sperm and its antibodies inhibit fertilization. Am J Reprod lmmunol, 2002, 47: 38-45.
27 Deng X, Meyers SA, Tollner TL, Yudin Al, Primakoff PD, He DN and Overstreet JW. Immunological response of female macaques to the PH-20 sperm protein following injection of recombinant proteins or synthesized peptides. J Reprod lmmunol, 2002, 54: 93-115.
28 Gaudreault C, Montfort L and Sullivan R. Effect of immunization of hamsters against recommbinent P26h on fertility rates. Reproduction, 2002, 123: 307-313.
29 Dube E, Legare C, Gaudreault C and Sullivan R. Contraceptive responses of female hamsters immunized with recombinant sperm protein P26h. Contraception, 2005, 72: 459-467.
30 Jagadish N, Rana R, Mishra D, Garg M, Chaurasiya D, Hasegawa A, Koyama K andSuri A. Immunogenicity and contraceptive potential of recombinant human sperm associated antigen(SPAG9). J Reprod Immunol, 2005, 67: 69-76.
31 O’Rand MG, Widgren EE, Sivashanmugam P, Richardson RT, Hall SH, French FS, VandeVoort CA, Ramachandra SG, Ramesh V and Rao AJ. Reversible immunocontraception in male monkeys immunized with Eppin. Science, 2004, 306: 1189-1190.
32周密,王健,施惠娟.精卵结合分子机制的研究进展[J].中国男科学杂志, 2007, (12): 79-81.
33刘利敏,陈德宇,黄天华.精卵融合过程中关键精子膜蛋白的研究进展[J].中华男科学杂志, 2009, (03): 70-73.
34毕聪明,彭树英,曹俊伟,王珅.哺乳动物精卵融合机制[J].中国生物工程杂志, 2005, (S1): 45-48.
35刘志春,江一平.精卵融合机制研究进展[J].生殖与避孕, 2008, (08): 38-42.
36周思畅,倪崖,石其贤.四次跨膜蛋白CD9与精卵融合[J].国外医学(计划生育分册), 2005, (01): 16-18.
37郭晓强,杨彩卿.精卵细胞融合分子机制[J].细胞生物学杂志, 2007, (03): 58-61.
38冯伯森,闫一琳.哺乳动物精子与卵子质膜粘附和融合的分子基础[J].生理科学进展, 2005, (01): 78-80.
39娜仁花;旭日干.精子黏附分子-1 cDNA的克隆及表达[J].黑龙江畜牧兽医, 2007, (04): 11-13.
40娜仁花;旭日干.精子受精抗原-1(FA-1) mRNA在绵羊睾丸和附睾中的表达[J].畜牧与兽医, 2006, (11): 4-6.
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2王德刚;黄天华;谢庆东;安刚.重组小鼠Izumo的表达及其特异性抗体对小鼠体外精卵融合的影响[J].癌变.畸变.突变, 2007, (03): 68-72.
3安刚;黄天华;谢庆东;王德刚. pCXN2-mIZUMO对C57BL/6小鼠生育能力的影响[J].癌变.畸变.突变, 2007, (04): 60-63.
4 Turano C,Coppari S,Altieri F,Ferraro A. Proteins of the PDI family: unpredicted non-ER locations and functions. J Cell physiol. 2002,193(2): 154-163.
5 Ellerman DA,Myles DG,Primakoff P. A role for sperm surface protein disulfide isomerase activity in gamete fusion: evidenee for the participation of ERP57. Dev Cell. 2006,10: 831-837.
6 Schultz R,Williams C. Developmental biology: sperm-egg fusion unscrambled. Nature. 2005,434(7030):152-153.
7 Talwar GP. Vaccines and passive immunological approaches for fertility and hormone-dependent cancer. Immunol review, 1991, 171: 173-179.
8 Naz RK, Gupta SK, Gupta JC, Vyas HK and Talwar GP. Recent advances in contraceptive vaccine development. Hum Reprod, 2005, 20: 3271-3283.
9 Gupta S. Reproductive immunology. India: Narosa Publishing House, 1999, 303-308.
10 Aitaken JR. Immunocontraceptive vaccine for human use. J Reprod lmmunol, 2002, 57: 273-287.
11 Kirkpatrick JF and Turner J. Reversibility of action and safety during pregnancy of immunization against zona pellucid in wild mares. Reprod Suppl, 2002, 60: 197-202.
12 Talwar GP, Singh O, Pal R, Chatterjee N, Sahal P, Dhall K, Kaul J, Das SK, Suri S, Buckshee K, Saraya L and Saxena BN. A vaccine that prevents pregnancy in woman. Proc Natl Acad Sci U S A, 1994, 91: 2532-2536.
13 Ohl D and Naz RK. Infertility due to antibodies. J Uro1, 1995, 46: 591-602.
14 Bronson RG, Cooper G and Rosenfeld D. Sperm antibodies: their role in infertility. Fertil Steril, 1984, 42: 171-182.
15 Liskin L, Pile JM, Quillan WF. Vasectomy safe and simple. Popul Rep, 1983, 4: 61-100.
16 Silber SJ and Grotjan HE. Microscopic vasectomy reversal 30 years later: asummary of 4010 cases by the same surgeon. J Andro1, 2004, 25: 845-859.
17 Baskin MJ. Temporary sterilization by injection of human spermatozoa: a preliminary report. Am J Obstet Gynecol, 1932, 24: 892-897.
18 Menge AC. Immunizations and infertility. J Reprod Fertil Suppl, 1970, 10: 171-185.
19 Allardyce RA. Effect of ingested sperm on fecundity in the rat. J Exp Med, 1984, 159: 1548-1553.
20 Naz RK. Search for Peptide sequences involved in human antisperm antibody-mediated male immunoinfertility by using phage display technology. Mo1 Reprod Dev, 2005, 72: 25-30.
21 Goldberg E. Sperm specific lactate dehydrogenase and development of a contraceptive vaccine. In Clark DA and Croy BA. Reproduetive Immunology. New York: Elsevier Press 1986. 137-142.
22 Naz RK and Zhu X. Reeombinant fertilization antigen-1 causes a contraceptive effect in actively immunized mice. Biol Reprod, 1998, 59: 1095-1100.
23 Naz RK. Fertilization-related sperm antigens and their immunocontraceptive potentials. Am J Reprod Immunol, 2000, 44: 41-46.
24 Naz RK and Zhu X. Molecular cloning and sequencing of a novel cDNA encoding for a protein involved in human sperm function. Biochem Biophys Res Commun, 2001, 284: 911-917.
25 Naz RK and Chauhan SC. Human sperm-specific Peptide vaccine that causes long-term reversible contraception. Biol Reprod, 2002, 67: 674-680.
26 Trivedi RN and Naz RK. Testis-specific antigen(TSA-1) is expressed in murine sperm and its antibodies inhibit fertilization. Am J Reprod lmmunol, 2002, 47: 38-45.
27 Deng X, Meyers SA, Tollner TL, Yudin Al, Primakoff PD, He DN and Overstreet JW. Immunological response of female macaques to the PH-20 sperm protein following injection of recombinant proteins or synthesized peptides. J Reprod lmmunol, 2002, 54: 93-115.
28 Gaudreault C, Montfort L and Sullivan R. Effect of immunization of hamsters against recommbinent P26h on fertility rates. Reproduction, 2002, 123: 307-313.
29 Dube E, Legare C, Gaudreault C and Sullivan R. Contraceptive responses of female hamsters immunized with recombinant sperm protein P26h. Contraception, 2005, 72: 459-467.
30 Jagadish N, Rana R, Mishra D, Garg M, Chaurasiya D, Hasegawa A, Koyama K andSuri A. Immunogenicity and contraceptive potential of recombinant human sperm associated antigen(SPAG9). J Reprod Immunol, 2005, 67: 69-76.
31 O’Rand MG, Widgren EE, Sivashanmugam P, Richardson RT, Hall SH, French FS, VandeVoort CA, Ramachandra SG, Ramesh V and Rao AJ. Reversible immunocontraception in male monkeys immunized with Eppin. Science, 2004, 306: 1189-1190.
32周密,王健,施惠娟.精卵结合分子机制的研究进展[J].中国男科学杂志, 2007, (12): 79-81.
33刘利敏,陈德宇,黄天华.精卵融合过程中关键精子膜蛋白的研究进展[J].中华男科学杂志, 2009, (03): 70-73.
34毕聪明,彭树英,曹俊伟,王珅.哺乳动物精卵融合机制[J].中国生物工程杂志, 2005, (S1): 45-48.
35刘志春,江一平.精卵融合机制研究进展[J].生殖与避孕, 2008, (08): 38-42.
36周思畅,倪崖,石其贤.四次跨膜蛋白CD9与精卵融合[J].国外医学(计划生育分册), 2005, (01): 16-18.
37郭晓强,杨彩卿.精卵细胞融合分子机制[J].细胞生物学杂志, 2007, (03): 58-61.
38冯伯森,闫一琳.哺乳动物精子与卵子质膜粘附和融合的分子基础[J].生理科学进展, 2005, (01): 78-80.
39娜仁花;旭日干.精子黏附分子-1 cDNA的克隆及表达[J].黑龙江畜牧兽医, 2007, (04): 11-13.
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