大鼠膜联蛋白5的重组表达及其对人精子体外运动功能的影响

Expression of rat annexin 5 and its effect on human sperm motility in vitro

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.