【作者】 张巧玉；

【导师】 史常旭；

【作者基本信息】 第三军医大学 ， 泌尿外， 2004， 博士

【摘要】 随着基因重组技术的发展和人们对生殖分子生物学的深入认识,免疫性避孕疫苗的研究是将来避孕节育研究的重要方向,免疫性避孕疫苗可能成为控制人口过度增长的理想措施。针对精子/睾丸抗原的免疫性避孕疫苗因可能适用于两性,而一直受到研究者的青睐。目前在利用精子抗原开发避孕疫苗的研究中,虽然对某些特异性精子蛋白抗原及表位多肽进行了免疫避孕效应研究,但尚未取得满意的结果。主要可能有以下两个方面的原因造成:1、精子/睾丸抗原为自身蛋白,抗原性弱;2、生殖道粘膜免疫应答水平低下,未能产生足够有效的特异抗体滴度。因此,增强生殖道特异抗体反应是提高精子抗原避孕效应的一个新的切入点。而生殖道粘膜诱发抗精子蛋白特异抗体的关键则是寻找合适的靶抗原、免疫途径和免疫佐剂。 精子蛋白抗原必须具备睾丸/精子特异性并参与精卵相互作用的特性。其中精子表面蛋白Sp17在人和各种哺乳动物如兔、小鼠及灵长类动物中的精母细胞、精细胞和睾丸/精子中表达,是一种高度保守的哺乳动物蛋白。鼠、兔和人的Sp17有61%的同源性,而人与狒狒有97%的同源性。在受精过程中,sp17通过与卵细胞透明带表面的糖蛋白、乙酰肝素和葡聚糖等结合参与顶体反应来促进受精。因此,Sp17是受精过程的关键分子之一,是开发避孕疫苗理想的侯选抗原。 要使合适的抗原产生理想特异的有效粘膜免疫应答必须选择合适的免疫途径和免疫佐剂,其中粘膜免疫是近来有关免疫途径研究的一个热点。一个粘膜部位的免疫反应可以诱发远隔部位粘膜的免疫反应,是粘膜疫苗发挥作用的基础。在生殖道性传播疾病的免疫研究中,鼻内免疫产生的阴道特异IgA和血清特异IgG抗体反应强于阴道或口服接种途径。鼻粘膜免疫不仅诱导出多个粘膜部位尤其是生殖道的抗体反应,且能诱导系统免疫反应,并可显著降低免疫原用量,说明鼻内免疫对抗原较为敏感,尤其是蛋白类抗原。多数疫苗经肌肉、皮下、腹腔或真皮注射,主要诱导系统免疫,诱导粘膜免疫反应的作用弱。因此,对于避孕疫苗来说,鼻内免疫可能是一种比较合适的途径。但大多数可溶性蛋白抗原在未给予佐剂的条件下经鼻内免疫后诱导的免疫反应水平较低,粘膜免疫中要求使用粘膜免疫佐剂来增强精子抗原的免疫原性和诱导所更多还原

【Abstract】 Being applicable to both sexes, immunological contraceptive vaccine, whose target is spermAestis antigens, has been always drawing immense attention from researchers. In current research on developing contraceptive vaccine using sperm antigens, major focuses are as follow: 1.To screen, identify, isolate, clone and sequence fertility- or infertility-related specific sperm/testis antigen genes and to determine the key active epitopes using various methods. 2. To obtain needed proteins by analytic methods, synthesis or DNA recombination techniques, and 3.To investigate the immunological contraceptive effects of certain specific sperm protein antigens and epitope peptides. However, no satisfactory contraceptive effects have been found yet, and the case is also true with the researches on sperm protein Sp17. The main causes involved are: 1.Being autologous proteins, sperm/testis antigens have weak antigenicity; 2. There are inadequate and ineffective specific antibody titers produced due to low levels of immune response in mucosa of the reproductive tract. Since the titers of specific antibodies in the reproductive tract are closely related to fertility, the design of contraceptive vaccines should aim at effectively triggering mucosal immune responses in the reproductive tract. Therefore, it will be a new direction to enhance contraceptive effects of sperm antigens by reinforcing specific antibody responses in the reproductive tract. The key factors of inducing specific anti-sperm protein antibodies in mucosa of the reproductive tract include appropriate target antigens, immunization routes and immunological adjuvants.A contraception vaccine based on sperm antigens must provided with being expressed specially in the testis/sperm,moreover,taking part in interaction of sperm and ovum。 Several sperm specific antigens identified as prospective candidates for immunocontraception are of testicular origin.Sp17 is a key molecul during fertilization . It was originally found to be expressed exclusively in the spermatocyte,sperm cell and testis of humans and a variety of mammals such as rabbit, mouse and primates ,and specificity binds with theglycoprotein and glucosan of pellucid zone. Sp17 is a highly conserved mammalian protein, Human and mouse and rabbit Spl7 were shown to share 61% homology, Human and macaque Spl7 were shown to share a very high degree of identity (97%).During fertilization,Spl7 takes part in acrosome response by binding with the heparitin of pellucid zone of ovum and enhances the ablity of fertilization. In addition, Spl7 can affect acrosome respones and motoricity of sperm by binding after interaction sit of it’s N tip and cAMP-dependence protein kinase Ila regulatory subit binds with protein kinase A anchoring protein.Therefore,Spl7 is a suitable antigen of contraceptive vaccine.Mucosal immunity has become a research highlight over recent years. It stimulates local and remote mucosal and systemic immune responses. Intranasal immunity not only stimulates antibody response in multiple mucosal sites, particulary in the reproductive tract, but also stimulates systemic immune response, thus drawing particular attention from researchers. Administered intramuscularly, subcutaneously, intraperitoneally or intradermally, injections of most vaccines mainly stimulate systemic immunity, rarely stimulating mucosal immune response. Therefore, intranasal immunity will be a prosperous immunization strategy for contraceptive vaccines. However, immunological adjuvants must be used to enhance the immunogenicity of sperm antigens and to stimulate needed types of immune responses in mucosal immunity. Most soluble protein antigens immunized intranasally stimulate low levels of immune responses if not being used with adjuvants. So, the use of immunological adjuvant plays a significant role in mucosal immunity. With regard to antigens, immunization routes and specificity of anti-infection, properties of specific adjuvants may vary essentially. There has been no research report on the intranasal immunization route of sperm antigens and the application of immunological adjuvants at home or abroad.This study for the first time used cytokine IL-5 as the immunological adjuvant for intranasal immunization of sperm protein Spl7. Sperm protein Spl7 was cloned and prokaryotically expressed using genetic recombination technique. cDNA sequence encoding IL-5 protein in mature mice was inserted into prokaryotic expression vector pET/SP17 to construct recombinant expression vector of IL-5-Spl7 fusion gene, and then the IL-5-Spl7 fusion protein was prokaryotically expressed efficiently and purified. Male mice were intranasally immunized with resultant recombinant protein. Then the following data were observed: 1 The titer changes of anti-SP17antibody in serum and the reproductivetract, 2 The effects on fertility, 3 Pathologic changes of vital organs such as the liver, kidney, spleen, lung, brain, testis and ovary, and 4 Changes in the rates of teratism and micronucleus of sperms in filial generation.The main results and conclusion of the study are as follow:1. cDNA sequence encoding sperm protein Spl7 was amplified from mice testis using RT-PCR. Recombinant plasmid pET/Spl7 was constructed by inserting the gene in the expression vector pET-28a(+) using oriented cloning. Full-length mice IL-5 gene was amplified with plasmid pGEM-l-IL-5 as the template and cloned into pET/Spl7 via the restriction sites of BamH I and Hind HI and linked to the upstream of Spl7 gene via the base sequence encoding the linker YPQD, thus successfully constructing recombinant plasmid pET/IL-5-Spl7. The fusion gene of IL-5-Spl7 was confirmed to meet the design requirements using double enzymatic excision and sequencing.2. The recombinant plasmids pET/Spl7 and pET/IL-5-Spl7 were transfected into E. Coli BL21(DE3). IPTG was used to stimulate gene expression. SDS-PAGE analysis revealed that both Spl7 and IL-5-Spl7 were expressed, and the molecular weight of the proteins were approximately 25kD and 49kD respectively, being approximate to the theoretical values of Spl7 and IL-5-Spl7 recombinant proteins. The purity of recombinant proteins Spl7 and IL-5-Spl7 was 88.9% and 91% respectively after being purified using Ni2+-ANT affinity column. Western blotting confirmed that the expressed proteins were Spl7 and IL-5-Spl7 respectively using anti-his mAb.3. The mice were immunized intranasally with the recombinant proteins and the titers of specific antibodies IgG and IgA in serum and the reproductive tract were determined. At 2 weeks of the primary immunization, Spl7-specific antibody IgG response was present in the mice of the experimental groups. Following immunization, antibody titers increased as the time elapsed and reached a peak at 6 weeks of the primary immunization. The highest levels of induced anti-Spl7 antibody were observed in the IL-5-Spl7 group, reaching 1:12800, which sustained for about 4 weeks before slight decrease. At 2 weeks of the primary immunization, specific antibody IgA was present in mice vaginal irrigation fluid of each experimental group and the levels increased by degrees, reaching peak values at 6 weeks of the primary immunization. The highest levels of antibody were observed in the IL-5-Spl7 group, reaching 1:12800. No Spl7-specific IgG or IgA antibody was detected in the negative control group. Rank sum testing showed that from 4 weeks after the primaryimmunization, the titers of specific IgG and IgA antibodies in both the IL-5-Spl7 group and the Spl7+IL-5 group were significantly higher than that in the Spl7 group at the same stage(p<0.05), and that there was no significant difference between the !L-5-Spl7 group and the Spl7+IL-5 group.4. At 10 days of immunization, detection of the proliferation rate of mice splenic lymphocyte and IL-4 and INF- Y secreted by lymphocytes showed that, following immunization of IL-5-Spl7, the proliferation rate of mice splenic lymphocyte increased, so did the IL-4 and INF- Y in supernatant of lymphocyte culture. Moreover, the increase in IL-4 was much more obvious. Analysis of mice serum subtypes revealed that, following IL-5-Spl7 immunization, the ratio of IgGl:IgG2a increased. The above findings suggested that intranasal immunization with IL-5-Spl7 stimulated strong mucosal immunity in the reproductive tract and systemic immune response in mice, which mainly stimulated Tl^-type immune response, helping humoral immune response.5. Following intranasal immunization, the pregnancy rates of the groups IL-5-Spl7, IL5+Spl7, Spl7 decreased to 15.4%, 28.6%, $10% respectively. The number of newborn per birth in each group decreased too, significantly lower than that of the Spl7 group. These findings demonstrated that sperm antigen Spl7 with or without the use of adjuvant stimulated immunological contraceptive effects to certain degrees, but cannot totally block mice reproduction.6. Histologic studies following immunization revealed: 1 There was no apparent pathologic changes in the liver and spleen and no signs of lymphocyte infiltration, 2 There were perivascular spaces in brain, 3 There was no apparent pathologic changes in the kidney and lung, 4 The follicles were morphologically normal and no atrophy occurred, 5 The diameters of testis seminiferous tubules were normal with complete basal lamina, and there were abundant sperms and spermatoblasts of various stages. Micronucleus tests of bone marrow obtained from immunized mice and their offspring showed that there was no significant difference in PCE / NCE and micronucleus rate between the experimental and normal groups.更多还原