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绵羊精子赤道膜蛋白片段1(SPESP1)的克隆、原核表达及纯化

DOI: 10.13560/j.cnki.biotech.bull.1985.2015.07.023, PP. 155-160

Keywords: 绵羊,SPESP1,cDNA克隆,原核表达,蛋白纯化

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Abstract:

旨在克隆绵羊Spesp1cDNA并表达,得到纯化的GST-SPESP1融合蛋白。以绵羊睾丸组织总cDNA为模板,根据GenBank公布的绵羊基因组序列设计引物,PCR扩增得到Spesp1cDNA,构建原核表达重组载体pGEX-Spesp1,将其转化到E.coliBL21中表达,并优化其表达条件,利用SDS-PAGE切胶纯化法得到纯化的融合蛋白,用Westernblot鉴定所得融合蛋白。结果表明,经测序,克隆得到的Spesp1cDNA序列与GenBank中预测的cDNA序列对比有两个碱基不同,并造成一个氨基酸的差异。在E.coliBL21中成功表达重组融合蛋白,其最优表达条件为:37℃、4h、0.005%IPTG终浓度,SDS-PAGE和Westernblotting中,融合蛋白位于约64kD处并可以被抗GST和抗羊SPESP1的抗体识别,与预计相符,表明融合蛋白成功表达。成功纯化得到GST-SPESP1融合蛋白,为研究SPESP1在精卵细胞膜融合中的功能奠定了基础。

References

[1]  Yamashita M, Yamagata K, Tsumura K, et al. Acrosome reaction of mouse epididymal sperm on oocyte zona pellucid[J]. J Reprod Dev, 2007, 53(2):255-262.
[2]  Inoue N, Ikawa M, Isotani A, et al. The immunoglobulin superfamily protein Izumo is required for sperm to fuse with eggs[J]. Nature Publishing Group, 2005, 434(7030):234-238.
[3]  Le Naour F, Rubinstein E, Jasmin C, et al. Severely reduced female fertility in CD9-deficient mice[J]. Science, 2000, 287(5451):319-321.
[4]  Kaji K, Oda S, Shikano T, et al. The gamete fusion process is defective in eggs of Cd9-deficient mice[J]. Nat Genet, 2000, 24(3):279-282.
[5]  Chalbi M, Barraud-Lange V, Ravaux B, et al. Binding of sperm protein Izumo1 and its egg receptor Juno drives Cd9 accumulation in the intercellular contact area prior to fusion during mammalian fertilization[J]. Development, 2014, 141(19):3732-3739.
[6]  Yanagimachi R. Mammalian fertilization[M]. New York:Raven Press, 1994.
[7]  Sebkova N, Ded L, Vesela K, et al. Progress of sperm IZUMO1 relocation during spontaneous acrosome reaction[J]. Reproduction, 2014, 147(2):231-240.
[8]  Satouh Y, Inoue N, Ikawa M, et al. Visualization of the moment of mouse sperm-egg fusion and dynamic localization of IZUMO1[J]. J Cell Sci, 2012, 125(21):4985-4990.
[9]  Wolkowicz MJ, Digilio L, Klptz K, et al. Equatorial segment protein(ESP)is a human alloantigen involved in sperm-egg binding and fusion[J]. J Androl, 2008, 29(3):272-282.
[10]  Lv ZM, Wang M, Xu C. Antifertility characteristics of the N-terminal region of mouse equatorial segment protein[J]. Anat Rec(Hoboken), 2010, 293(1):171-181.
[11]  Fujihara Y, Murakami M, Inoue N, et al. Sperm equatorial segment protein 1, SPESP1, is required for fully fertile sperm in mouse[J]. Cell Science, 2010, 123(9):1531-1536.
[12]  Miyado K, Yamada G, Yamada S, et al. Requirement of CD9 on the egg plasma membrane for fertilization[J]. Science, 2000, 287(5451):321-324.
[13]  Bianchi E, Doe B, Goulding D, et al. Juno is the egg Izumo receptor and is essential for mammalian fertilization[J]. Nature, 2014, 508(7497):483-487.
[14]  Kawai Y, Hama T, Mayumi T, et al. Flow cytometric analysis of mouse sperm using monoclonal anti-sperm antibody OBF13[J]. Journal of Reproductive Immunology, 1989, 16(1):71-82.
[15]  Wolkowicz MJ, Shetty J, Westbrook A, et al. Equatorial segment protein defines a discrete acrosomal subcompartment persisting throughout acrosomal biogenesis1[J]. Biology of Reproduction, 2003, 69(3):735-745.
[16]  Liu ZD, Xing WJ, Wang LQ, et al. Prokaryotic expression, ascitic polyclonal antibody preparation and identification of cashmere goat izumol[J]. Agriculture Science in China, 2010, 9(4):605-613.

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