| 长牡蛎精子膜蛋白的提取及其部分生化性质的研究 |
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| 引用本文： | 刘丽燕,杨爱国,王清印,周丽青,刘志鸿.长牡蛎精子膜蛋白的提取及其部分生化性质的研究[J].中国水产科学,2007,14(6):889-895. |
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| 作者姓名： | 刘丽燕 杨爱国 王清印 周丽青 刘志鸿 |
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| 作者单位： | 1. 农业部海洋渔业资源可持续利用重点开放实验室,中国水产科学研究院,黄海水产研究所,山东,青岛,266071;上海水产大学,生命学院,上海,200090;湛江恒兴南方海洋科技有限公司,广东,湛江,524001
2. 农业部海洋渔业资源可持续利用重点开放实验室,中国水产科学研究院,黄海水产研究所,山东,青岛,266071 |
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| 摘 要： | 从提取时间和TritonX-100浓度两个方面对长牡蛎(Crassosstrea gigas)精子膜蛋白提取条件进行优化,确定最佳提取条件:将精液150 g离心去除精原细胞,经PBS缓冲液和Tris-HCl缓冲液清洗后,3 000 g离心10 min(4 ℃)得精子悬液;然后加入3倍体积含1% TritonX-100的膜蛋白提取液,冰浴振摇1 h;50 000 g离心15 min(4 ℃),收集上清液即为精子膜蛋白溶液.SDS-PAGE电泳后,考马斯亮蓝R-250染色检测到21种膜蛋白,分子量在26～156 kD之间,PAS染色检测到糖蛋白有14种,苏丹黑B染色检测到脂蛋白有17种,其中有13种膜蛋白既是糖蛋白又是脂蛋白.实验结果还发现1种分子量为38 kD的r16膜蛋白既为糖蛋白又为脂蛋白,且高碘酸-Shiff试剂和苏丹黑B染色最深,条带最清晰,蛋白丰度最高,与此相对的分子量为55 kD的r12膜蛋白,考马斯亮蓝R-250染色也较深,条带清晰,但其糖蛋白和脂蛋白染色相对r16膜蛋白较浅,说明r16和r12两种膜蛋白其蛋白丰度相差不大,其糖基化程度及脂化程度可能相差较大,此两种膜蛋白有待于进一步分离纯化.此研究结果可为长牡蛎精子膜蛋白在精子发生和受精过程中作用的研究提供基础资料.
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| 关 键 词： | 长牡蛎 精子膜蛋白 糖蛋白 脂蛋白 |
| 文章编号： | 1005-8737-(2007)06-0889-07 |
| 修稿时间： | 2006年11月13 |
Extraction and biochemical analysis of sperm membrane protein from pacific oyster ( Crassosstrea gigas ) |
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| LIU Li-yan,YANG Ai-guo,WANG Qing-yin,ZHOU Li-qing,LIU Zhi-hong.Extraction and biochemical analysis of sperm membrane protein from pacific oyster ( Crassosstrea gigas )[J].Journal of Fishery Sciences of China,2007,14(6):889-895. |
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| Authors: | LIU Li-yan YANG Ai-guo WANG Qing-yin ZHOU Li-qing LIU Zhi-hong |
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| Abstract: | The extraction condition of sperm membrane proteins from Pacific oyster(Crassostrea gigas) were optimized in two aspects:the extraction time and the concentration of TritonX-100.The final optimized extraction conditions:sperms were centrifuged by 150 g to remove spermatogonia,cleaned twice in PBS buffer and tris-HCl buffer respectively,and centrifuged for 10 min at 3 000 g to abtain sperm suspension.Then the sperm suspension was put into triple volumes of extraction buffer including 1% TritonX-100,cooled in ice for 1 h,centrifuged for 15 min at 50 000 g,and the sperm membrane solution was finally collected.By SDS-PAGE gels electrophoresis,21 kinds of sperm membrane proteins were dyed ranging from 26 kD to 156 kD by coomassie brilliant blue R-250,14 kinds of glycoprotein were detected by periodic acid-schiff reagent(PAS),17 kinds of lipoproteins were detected by Sudan Black B,and 13 kinds of membrane proteins were identified as either glycoprotein or lipoprotein.The results show that the r16 membrane protein was not only glycoprotein but also lipoprotein,whose molecular weight was 38 kD.This r16 menbrane protein was dyed clearest by coomassie brilliant blue R-250,periodic acid-schiff reagent(PAS) and Sudan Black B.R12 membrane protein was dyed as clear as r16 membrane protein,whose molecular weight was 55 kD,but r12 membrane protein wasn't dyed clearer than r16 membrane protein.It proved that r16 membrane protein and r12 membrane protein were similar in protein abundance,hower they were diffierent in glycosylation and lipid.It is necessary to separate and purify these two membrane proteins.These results would provide useful information for separation and purification of sperm membrane protein in Crassostrea gigas and established further foundation for spermatogenesis and fertilization research. |
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| Keywords: | Crassostrea gigas sperm membrane protein glycoprotein lipoprotein |
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