摘要
目的:先前研究发现促性腺激素释放激素(GnRH)激动剂对大鼠睾丸间质细胞中膜联蛋白5(annex-in5)的翻译和转录水平都有一定的影响,推测annexin5可能是睾酮分泌调节中的一个信号分子。为研究annexin5在雄性生殖调节中的作用,本研究拟获得具有活性的大鼠annexin5重组蛋白,并观察其对人精子运动功能的影响。方法:化学合成法合成大鼠annexin5的编码基因序列,将该基因插入组氨酸标签(HIS)融合表达载体pET28a中,在T7启动子控制下,通过异丙基-β-D-硫代半乳糖苷(IPTG)诱导表达HIS融合蛋白;以亲和层析法纯化表达融合蛋白并用活化部分凝血激酶时间(APTT)交叉试验法验证该蛋白的抗凝血活性。15例供精者的精液标本一式2份,1份加重组蛋白annexin5(终浓度为10-8mol/L),1份做对照,分别处理20和60min,用计算机辅助精液分析系统(CASA)分析精子活力、活动率;处理20min后,做精子爬高试验。结果:合成的目的基因产物长度为974bp,插入序列与annexin5的序列完全一致。在IPTG诱导下,BL21(DE3)重组菌高效表达出相对分子质量为36000的融合蛋白,经纯化获得95%纯度的融合蛋白,纯化蛋白具有很强的抗凝血活性。Annexin5处理20min后精子活动率提高了21%(P<0.05),活力提高了40%(P<0.01),而处理60min后精子活力、活动率与对照组比较均无显著性差异;精子爬高高度与对照组比较具有极显著差异[(37.84±6.35)mmvs(49.5±12.27)mm,P<0.01]。结论:成功构建了大鼠annexin5重组表达载体并在大肠埃希菌中高效表达annexin5蛋白,且该蛋白体外处理精子20min时提高精子的运动能力。
Objective:Gonadotropin releasing hormones (GnRH) regulate the expression of annexin 5 in Leydig cells,and annexin 5 is supposed to be a signal molecule in regulating testosterone secretion.This study aimed to investigate the function of annexin 5 in male reproduction by observing its effect on human sperm motility in vitro.Methods:The encoding sequence of rat annexin 5 was chemically synthesized and inserted into the HIS fusion expression vector pET28a.The expression of the fusion protein HIS-annexin 5 was induced by isopropy-β-D-thiogalactoside (IPTG) under the control of the T7 promoter,and the products were purified by affinity chromatography.The anticoagulant activity of annexin 5 was determined by the modified activated partial thromboplastin time (APTT) test.Semen samples from 15 donors were assigned to a control and an annexin 5 group,the latter treated with recombinant annexin 5 at the concentration of 10-8 mol/L.Sperm motility and the percentage of grade a+b sperm were measured by computer-assisted semen analysis (CASA) after 20 and 60 min exposure,and the sperm ascending experiment was done after 20 min treatment.Results:The product of the synthesized target gene was 947 bp in length,and the inserted sequence corresponded to the published encoding sequence of rat annexin 5.The plasmid pET28a-annexin 5 was transformed into E.coli BL21(DE3) and IPTG induced a fusion protein with a relative molecular weight of about 36 000,a purity of 95% and a high anticoagulant activity.Compared with the control group,sperm motility and the percentage of grade a+b sperm were increased by 40% (P0.01) and 21% (P0.01),respectively,after 20 min treatment with annexin 5,but neither showed any significant improvement after 60 min.The sperm ascending altitude was remarkably elevated after annexin 5 treatment,with extremely significant difference from the control group (37.84±6.35 vs 49.5±12.27,P0.01).Conclusion:An annexin 5 recombinant expression vector was successfully constructed.The protein annexin 5 can be efficiently expressed in E.coli and effectively improve human sperm motility in vitro.
出处
《中华男科学杂志》
CAS
CSCD
北大核心
2010年第5期400-404,共5页
National Journal of Andrology
基金
国家自然科学基金(30770801)~~
关键词
膜联蛋白5
精子
表达
活力
活动率
爬高试验
annexin 5
sperm
expression
motility
grade a+b sperm
sperm ascending experiment