重组人卵透明带蛋白(rhZP3)的生物活性研究

为了研究毕赤酵母表达的重组人卵透明带蛋白(rhZP3)的生物活性,分别用空白培养液,含孕酮或rhZP3的培养液对人精子进行顶体诱发实验,用考马斯亮蓝染色法对顶体状态进行评价;用不同浓度的rhZP3以及空白培养液分别处理精子,然后再与卵子进行结合实验,观察经过不同处理的精子在精卵结合中的情况;用抗rhZP3抗血清与阴性血清分别处理卵子,再与精子进行结合实验,观察经过不同处理后的卵子在精卵结合中的情况。rhZP3诱发顶体反应实验结果显示,rhZP3处理组与空白对照组之间差异显著(P<0.01);精卵结合实验结果显示,各实验组和对照组之间存在显著性差异(P<0.01),rhZP3、抗rhZP3抗体均能抑制精卵结合。实验结果表明,rhZP3具有天然人卵透明带蛋白相似的活性。

Studies of the Biological Activities of the Recombinant Human Zona Pellucida Protein 3 (rhZP3)

Human zona pellucida(hZP),which is a complex matrix surrounding oocytes, is comprised of three glycoproteins (hZP1,hZP2 and hZP3).The native hZP3 possesses the sperm-receptor activity and the function of inducing acrosome reaction, so it has long been used as a candidate antigen to develop immunocontraceptive vaccines and as a native agent to detect sperm function. For investigating the activities of purified recombinant human zona pellucida protein 3(rhZP3)expressed in Pichia pastoris , the rhZP3-induced acrosome reaction(AR) was determined and the effects of rhZP3 and anti-rhZP3 serum on sperm-egg interaction were evaluated. The sperm were incubated with BWW medium, progesterone and rhZP3 respectively, then the acrosome status of sperm was evaluated by Coomassie brilliant blue (CBB) staining. The rhZP3-exposed and nonexposed mouse sperm were incubated with zona-intact mouse oocytes and the mouse oocytes pretreated with anti-rhZP3 serum and negative serum were co-incubated with mouse sperm, then the number of sperm bound per ZP and the fertilization rate (FR) were compared. The rhZP3-exposed and nonexposed human sperm were incubated with zona-intact human oocytes and the human oocytes pretreated with anti-rhZP3 serum and negative serum were co-incubated with human sperm, then the number of sperm bound per ZP were counted. After inducing acrosome reaction of human sperm, the sperm showed a significant differences between rhZP3 and control group (P<0.01). Statistical differences between rhZP3-exposed and nonexposed sperm groups, anti-rhZP3-treated and control oocytes groups were observed not only in human but also in mouse(P<0.01). The results showed that both rhZP3 and anti-rhZP3 antibody could strongly interfere with sperm-ZP. Results of the present study show that the biological activities of rhZP3 are similar to native human zona pellucida protein. rhZP3 has the possibilities for developing the contraceptive vaccine and the reagents to detect sperm function and the antibodies to zona pellucida.