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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: Previous studies have found that gonadotropin-releasing hormone (GnRH) agonist exerts a certain influence on the translation and transcription of Annex-5 in rat testicular stromal cells. It is speculated that annexin 5 may be a regulator of testosterone secretion In a signal molecule. In order to study the role of annexin5 in male reproductive regulation, the present study aimed to obtain an active recombinant rat annexin5 protein and observe its effect on human sperm motor function. METHODS: The coding sequence of annexin5 was synthesized by chemical synthesis. The gene was inserted into histidine tag (HIS) fusion expression vector pET28a. The recombinant plasmid was transfected by isopropyl-β-D- The fusion protein was induced by lactose glycoside (IPTG). The fusion protein was purified by affinity chromatography and the anticoagulant activity of the protein was verified by the cross-test of activated partial thromboplastin time (APTT). The sperm samples from 15 donors were divided into two groups. One group was given annexin5 (final concentration was 10-8 mol / L) and the other group was treated as control. The samples were processed for 20 and 60 min respectively and analyzed by computer assisted semen analysis system (CASA) Sperm motility, activity rate; deal with 20min, do sperm climbing test. Results: The length of the target gene was 974bp. The inserted sequence was identical to that of annexin5. Under the induction of IPTG, BL21 (DE3) recombinant strain highly expressed 36000 molecular weight of the fusion protein, purified to obtain 95% purity of the fusion protein, the purified protein has a strong anticoagulant activity. The sperm motility increased by 21% (P <0.05) and the vitality increased by 40% (P <0.01) after 20min treatment with Annexin5, but there was no significant difference between the control and sperm motility after 60min treatment There was a significant difference between the two groups ([(37.84 ± 6.35) mm vs (49.5 ± 12.27) mm, P <0.01]. Conclusion: The annexin5 recombinant plasmid was successfully constructed and the annexin5 protein was highly expressed in Escherichia coli, and the sperm motility of sperm was enhanced after 20min treatment of sperm in vitro.