【作者】 孙黎黎；

【导师】 梁志清；

【作者基本信息】 第三军医大学 ， 妇产科学， 2010， 博士

【摘要】 免疫性避孕一直是男性避孕节育研究的重要领域,具有巨大的临床应用前景和良好的社会经济价值。然而迄今为止没有理想的避孕疫苗问世,究其原因最大的可能在于没有理想的靶抗原。作为避孕疫苗应用的的靶抗原应当至少满足两个条件,即抗原特异表达于生殖系统且参与生殖过程中的重要环节。近年来,附睾蛋白酶抑制蛋白(Epidydimal protease inhibitor,Eppin)因在非人灵长类动物实验中显示出可靠的避孕效应而备受关注。它特异表达于睾丸和附睾,在精子成熟和生殖过程中具有重要作用。利用重组Eppin蛋白免疫雄猴获得了78%抗生育效果,停止免疫后,71%的受试雄猴恢复了生育力。体外实验发现来自不育雄猴血清中的抗Eppin抗体可直接破坏人Eppin-精囊凝固蛋白(Semenogelin,Sg)复合体,干扰Eppin调控前列腺特异性抗原PSA对Sg的水解作用,影响人精子活动力,进而抑制生育力。因此,Eppin可能成为理想的避孕疫苗靶抗原。虽然对Eppin的研究有了一定的进展,但仍存在如下问题:(1)蛋白抗原分子量较大,难以实现质量控制;(2)未对抗原中可能导致远期毒副反应的不良表位做剔除或加工。(3)抗体产生不稳定;(4)隐匿的保护性表位功能未能表达,所引发的保护性免疫不够强大,免疫避孕效果不理想。因此,为了提高Eppin抗原的保护性,就不得不从天然抗原整个分子水平向表位水平过渡:摒除不良表位,保留、改善保护性表位以得到更理想的疫苗分子。表位是蛋白质抗原性的基础,正确而详细地绘制蛋白质表位图谱对设计疫苗分子结构及免疫干预治疗等具有重要意义。本研究首先通过RT-PCR法从人新鲜睾丸组织扩增获得Eppin cDNA,构建入原核表达载体,诱导表达并经纯化获得重组人Eppin蛋白(第一部分)。同时,利用生物信息学方法分析Eppin蛋白的二级结构及抗原性、亲水性、柔韧性、表面可及性等理化特性,预测Eppin蛋白的主要功能区域,根据该区域抗原趋势指数分析结果,采用错位重叠套式方法设计该区域的肽段。利用人工合成肽技术固相合成多肽,并与载体蛋白钥孔血蓝蛋白(KLH)、牛血清蛋白(BSA)偶联,用KLH偶联肽与弗氏完全佐剂、不完全佐剂等体积混合,充分乳化后足垫皮下注射免疫小鼠,通过间接ELISA方法观察各个表位肽刺激抗体的产生,通过体外、体内抗生育实验观察表位肽的抗生育效应,以此筛选出其优势中和B细胞表位,并以rhEppin免疫为阳性对照,分析其抗生育潜能及安全性(第二部分)。最后,设计并制备基于Eppin优势B细胞表位的2分支MAP抗原肽,以弗氏佐剂为佐剂,腹部皮下免疫雄性恒河猴,ELISA方法监测免疫后被免疫雄猴血清抗体滴度,CBA方法检测Th1/Th2细胞因子的改变,睾丸活检术取睾丸标本电镜观察免疫后睾丸超微结构的变化,并与发情期雌猴合笼2个周期,观察Eppin的MAP抗原肽对恒河猴的抗生育效果(第三部分)。结果显示:①成功构建了pET-32a-Eppin重组质粒,并获得了具有抗原特异性的可溶性Eppin蛋白。②通过分析Eppin蛋白二级结构及表面特性,初步预测出Eppin蛋白的89～133位氨基酸为主要功能区域,设计合成8条代表该氨基酸区域的肽段,并与载体蛋白KLH偶联,命名为F1-F8。③采用偶联肽足垫皮下注射免疫小鼠,发现8个候选表位肽都能激发高滴度的抗体,但仅F5肽所获得的抗血清能与人精子天然Eppin蛋白结合,并能显著抑制精卵结合,使雄鼠的生育力受到很大限制,从而获得Eppin优势中和B细胞表位为F5:FVYGGCQGNN(105～114区段)。④Eppin的MAP抗原肽免疫的10只雄猴中有2只血清IgG抗体滴度始终无明显提高,淘汰,剩余8只抗体滴度可达1:400~1600,但需2~3周加强免疫一次。合笼后发现对照组5只有4只受孕,实验组8只仅3只受孕,显示了较肯定的避孕效果。电镜结果未发现免疫后睾丸组织超微结构的损害,CBA细胞因子检测未见Th1/Th2极化改变,各细胞因子免疫前后无明显差异。提示合成肽对T细胞功能的影响甚微,今后的剂型改造可以考虑增强T细胞功能,在佐剂、递呈方式上做优化。本研究结果表明生物信息学方法表位预测并合成偶联肽免疫策略是筛选蛋白优势中和B细胞表位的有效方法;Eppin蛋白的第105～114区段是Eppin优势中和B细胞表位区域;Eppin表位肽用于免疫避孕具有较好的安全性和有效性;基于Eppin优势表位的避孕疫苗尚需要在表位水平进一步修饰改造才能满足人用避孕疫苗的要求。更多还原

【Abstract】 The world’s population is growing at a tremendous rate and despite of the availability of various contraceptive modalities there is an urgent requirement for a better contraception method, which is reversible, safe and highly effective, easily obtained, inexpensive and suitable for all stages of reproductive life. Contraceptive vaccines can fulfill most of the properties of an ideal contraceptive. Up to now, there has been no indication of an ideal contraceptive vaccine being developed probably due to the lack of an ideal target antigen. The target antigen applied in contraceptive vaccine must meet at least two conditions. First, it should be expressed specifically in the reproductive system; second, it should act as an important component in the reproductive process.Recently, epididymal protease inhibitor (Eppin), a sperm specific antigen, was identified as an ideal and promising target for the development of a series of safer and more effective contraceptive vaccines. Human-derived Eppin is specifically expressed in epididymis and testis. Eppin was detected in Sertoli cells in 12 day-old mice and afterwards expresses during the maturation process of spermatids. When used for active immunization of adult male Macaca monkeys, seven out of nine males (78%) developed high titers to recombinant Eppin, and all these high titer monkeys were infertile. Five out of seven (71%) high anti-Eppin titer males recovered fertility when immunization was stopped. This study revealed that Eppin is a promising target for male contraception.However, previous studies showed that the contraceptive effect of the full-length protein was not sufficient to satisfy clinical demand. This may ascribe to: Eppin protein not only has B-cell and T cell epitopes providing immune recognition, but also has toxic or inhibitory epitopes, non-neutralized dominant epitopes, pathological epitopes and cross-reactive epitopes with auto-antigen. Therefore, in order to induce an immune response with stronger specificity and an increased immune effect, we must effectively avoide the former and modify the latter, allowing more ideal vaccine molecules to be developed. Epitope vaccine, synthesized with antigen epitope, is a newly developed vaccine technique with the advantages of a simple manufacturing processes, relatively low cost, stable chemical property and strong immune specificity with minimal toxicity or side-effects. Screening as well as identification of epitopes is the primary and most important task during preparation of an epitope vaccine.Eppin cDNA was obtained by RT-PCR from fresh human testis, and then cloned into expression vector pET-32a, the recombinant vector was transformed into BL21 (DE3) and induced by IPTG. The recombinant protein was purified by Ni2+-NTA-column.The antigenic activity of purified recombinant protein was assessed with Western blot (Part I).The epitope prediction was performed according to the correlation of B-cell epitope with physico- chemical property or secondary structures. After reviewing references, analyzing the homology and rejecting the crossing part of Eppin,8 sequential overlapping decapeptides (six amino acids overlap) targeting the Kunitz domain were designed and synthesized by solid-phase synthesis using a synthesis peptide technique.The dominant cellular epitopes were screened and identified by further experiments in vitro and in vivo. It was theoretically confirmed that the antigens had strong antigenicity and prevented potentially adverse immune reactions caused by pathological antigen and cross-reactive epitopes (Part II).Finally, the two branches of multiple antigen peptide (MAP) was designed and synthesized by solid-phase synthesis using a synthesis peptide technique. The sexually mature male rhesus monkeys were immunized with MAP by abdominal subcutaneous injection in Freund’s adjuvant. The presence and reactivity of antibody (IgG) against peptides in the serum were analyzed using ELISA. The levels of Th1/Th2 cytokine in serum were detected by non-human primate Th1/Th2 cytokine kit (BDTM cytometric bead array) (Part III).The results showed that:①Enzyme digestion analysis and sequencing showed that the target fragment was successfully inserted into the vector pET-32a (+).Soluble Eppin protein possessing specific antigenicity was expressed and purified successfully.②B-cell epitope prediction of the human testis and epididymis specific sequence of Eppin was made based on the characteristics of hydrophilicity, flexibility/mobility, accessibility, polarity, exposed surface and turns, using DNASTAR’S Lasegene software (DNAStar Inc.). Eight B-cell epitopes corresponding to 89-133 sites amino acids domain were selected, synthesized and coupled with keyhole limpet hemocyanin (KLH), or bovine serum albumin (BSA) by Xi’an Huachen Bio-technology Co., Ltd, China. Homology analysis using the BLAST function of NCBI didn’t show any peptide sequences which share significant homology with other proteins with the exception of Eppin protein itself. The purity of each peptide was above 90% as analyzed by reverse phase high-performance liquid chromatography (HPLC).③All eight peptides can elicit similar specific antibody titers, however,only the antisera from F5 showed the capacity to combined with natural Eppin in semen,and significantly decreased the rate of pregnancy in mice. Therefore the immunodominant neutralizing B-cell epitope of Eppin is F5: FVYGGCQGNN (Eppin 105-114).④High–anti-Eppin titers of 1:400 ~ 1600 were detected in eight of the ten monkeys immunized with Eppin MAP; two monkeys were low responders with titers <1:400 on postimmunization day 120. Because the purpose of this study was to test the efficacy of antibodies to Eppin MAP on fertility, the two low-titer monkeys were dropped from the study group and their fertility was not tested. Each male monkey in the immune and control groups were subjected to fertility testing by cohabiting with a proven fertile female during her two ovulatory cycles. Three out of eight males in the immune group were infertile and one out of five males in the control group was infertile. This revealed the MAP of Eppin had an affirmative contraceptive effect. The electron microscope examination of testis biopsy also showed no obvious abnomality. But in the detecting of Th1/Th2 cytokines, there was no significant difference in the males of immune and control groups.The results of this research indicates that epitope prediction by bioinformatics method combinated with synthetic peptide coupling carrier protein immunization strategy is an an effective way and a beneficial alternative to analysis B-cell epitope.The immunodominant neutralizing B-cell epitope of Eppin located at the 105-114 amnio acids.Epitope peptide of Eppin is an effective and safe modality for male contraception.However contraceptive vaccine based on Eppin dominant epitope needs further modification on the level of epitope to meet the requirements of contraceptive vaccine for human use.更多还原