【作者】 阎萍；

【导师】 梁志清；

【作者基本信息】 第三军医大学 ， 妇产科学， 2009， 博士

【摘要】 世界人口的持续快速增长,男性愿意共同承担计划生育的责任,因此开发新型的男性避孕方法是当务之急。避孕疫苗(contraceptive vaccine, CV)具有高效、安全、经济、可逆等优点,是目前避孕研究中极具前景的研究热点之一。避孕疫苗根据其作用的靶点不同,分为靶向配子的产生、配子的功能和配子的结局三类。男性避孕疫苗主要是通过干扰激素调控的精子发生、抑制精子活动力或是精卵相互作用过程中的任何一步达到抑制生育的作用。卵泡刺激素(follicle stimulating hormone, FSH)通过与其特异性表达于睾丸支持细胞(Sertoli cell, SC)的受体(FSHR)结合来启动和维持男性的精子发生。因而FSHR可作为男性避孕疫苗的靶抗原之一。研究发现,FSHR的胞外区(extracellular domain, ECD)存在多个参与激素受体结合或信号传导的不连续区域。既往的研究采用单抗或多抗探针、噬菌体展示技术、肽竞争结合或表位作图的方法来筛选激素受体结合的位点,但是筛选出的位点用于抗生育力研究的较少,或是抑制生育力效果欠佳。附睾蛋白酶抑制剂(epididymis protease inhibitor, Eppin)表达于精子顶体后区、睾丸的支持细胞、间质细胞和圆形精子以及附睾上皮的主细胞,可能与精子的成熟密切相关。射精后覆盖于精子表面的Eppin与精液凝固蛋白(semenogelin, Sg)饱和性结合,为精子提供抗菌作用。研究显示重组Eppin蛋白免疫雄猴能显著抑制雄猴的生育力,停止免疫后71%雄猴能恢复生育力,因此Eppin是开发避孕疫苗的候选抗原。因此本研究的目的在于筛选FSHR的优势中和性B细胞表位,观察多肽疫苗抗生育的有效性和安全性。本研究将hFSHR ECD按蛋白质氨基酸序列分为1-140aa和120-350aa两段,通过基因扩增和构建原核表达载体,从而诱导蛋白的表达并纯化。通过ECD二级结构的分析、在线B细胞表位预测和分子对接技术最后选定了4个候选肽,联合泛DR辅助T细胞表位(pan-DR epitope, PADRE)合成融合肽段。采用蛋白初次免疫-候选肽强化免疫的策略免疫成年雄性Balb/C小鼠,观察各表位肽激发抗体的产生及对小鼠生育力的影响。由于单一针对FSHR所获得的避孕效应只能中等程度的抑制小鼠生育力,因此联合Eppin免疫,以观察多价疫苗对生育力的抑制效应。以重组蛋白疫苗为对照,分析表位肽疫苗的抗生育潜能和安全性。方法:1. hFSHR ECD和Eppin原核蛋白的诱导表达hFSHR ECD的1-140aa段和120-350aa段基因分别采用桥连拼接PCR扩增,Eppin基因序列以人睾丸cDNA为模板,经PCR扩增获得。目的基因连入pET 32a载体,1mM IPTG诱导原核蛋白表达,并对表达的蛋白进行纯化和鉴定。纯化后的重组hFSHR蛋白按所含的氨基酸分别记为F140和F240。2. FSHR ECD B细胞表位的分析预测采用DNAstar protein软件,从二级结构、亲水性、可及性、可塑性和抗原指数等几方面对FSHR ECD进行分析;利用B细胞表位在线预测,从亲水性、可及性、转角、极性和抗原性等参数对胞外区进行在线分析;以hFSH和FSHR相互作用复合体的晶体结构为初始模版(PDB ID:1xwd),利用InsightⅡ分子模拟软件包完成FSHR与FSH的分子对接。3. FSHR B细胞表位融合肽的合成为增强免疫原性,FSHR B细胞表位前接泛DR Th表位肽(PADRE),融合肽送上海吉尔生化有限公司合成。4. B细胞表位肽疫苗的免疫效应采用重组hFSHR蛋白质初次免疫、B细胞表位肽强化的免疫策略,与弗氏佐剂混合后免疫成年雄性Balb/C小鼠。初免后2周进行强化免疫,每周强化一次,共3次。采用ELISA法检测动态监测小鼠血清特异性IgG滴度变化。Western blot法检测小鼠血清与人睾丸蛋白的结合。在此基础上,联合Eppin优势B细胞表位免疫小鼠,ELISA法动态监测小鼠血清特异性IgG滴度变化。5. B细胞表位肽疫苗对生育力的抑制作用末次免疫结束后2周和6周,分别将雄鼠与健康有生育力的雌鼠按1: 3混合喂养2周,观察雌鼠受孕率和每胎产仔数。6.免疫小鼠的生殖内分泌改变及机制研究末次合笼实验结束后2周,随机处死小鼠,采集小鼠血清、睾丸、附睾。从精子计数、精子活力和精子尾部低渗膨胀实验(HOS)等方面对附睾精子进行分析;从睾丸重量和睾丸精子2方面对睾丸进行观察;放免法测定小鼠血清睾酮(T)水平;联合Eppin免疫组还观察小鼠血清对正常人精子的抑制作用和检测小鼠附睾冲洗液中IgA滴度。7.免疫小鼠睾丸组织学检查处死小鼠分离睾丸组织固定,切片后行HE染色,观察睾丸的组织学改变。8.生育力恢复实验小鼠初次免疫后40周(末次接种后36周),ELISA检测血清特异性抗体滴度降至1: 5000以下,雄鼠与雌鼠按1: 3混合喂养2周,观察雌鼠受孕率和每胎产仔数。结果:1. hFSHR ECD和Eppin原核蛋白的诱导表达通过PCR成功扩增FSHR ECD 1-140aa段、120-350aa段和Eppin的基因序列,分别构建pET 32a原核表达载体。诱导表达的原核蛋白质为包涵体,纯化复性后Bradford法测定蛋白质含量:F140为1.26 mg/ml,F240为1.19 mg/ml,Eppin为5.5mg/ml,并检测蛋白质的纯度>95%,重组蛋白免疫小鼠获得的抗血清能识别人睾丸蛋白中Mr约为75kD或34kD左右的蛋白质,具有较好的生物学特性。2. hFSHR ECD B细胞表位的分析预测综合hFSHR ECD二级结构分析、在线B细胞表位预测和分子对接分析结果,最后选择4个候选B细胞表位,分别位于ECD的29~38、32~44、269~280和285~300氨基酸区域内。3. FSHR B细胞表位融合肽的合成含有PADRE和B细胞表位的融合肽由上海吉尔生化有限公司合成,多肽纯度达到85%以上。4. B细胞表位肽的免疫效应基于FSHR的抗原与弗氏佐剂混合后免疫Balb/C雄性成年小鼠(8周龄),ELISA法在血清中可检测到特异性IgG,最高效价可达1: 400000以上,小鼠血清可识别人睾丸蛋白中分子量为75kD左右的蛋白质。联合Eppin的优势B细胞表位肽C与FSHR的肽2-4免疫Balb/C雄性成年小鼠,血清中特异性IgG的最高效价超过1: 400000。说明该免疫策略能有效的诱导机体产生高滴度的特异性抗体。5. B细胞表位肽疫苗对生育力的抑制作用重组hFSHR蛋白初免、B细胞表位肽强化免疫小鼠后,P2、P3和P4组小鼠生育力明显下降,以P2组最为显著,雌鼠妊娠率平均降至为26.67%,每胎产仔数降至4.19只,仅略高于蛋白全程免疫组。联合Eppin免疫后,雌鼠妊娠率和每胎产仔数更低,以F2EP2C组最为显著,分别降至6.67%和2.5只,稍高于FSHR和Eppin蛋白联合免疫4次组。说明肽2可能是FSHR的一个优势B细胞表位,基于此的表位肽疫苗具有免疫性避孕效应,联合FSHR和Eppin优势B细胞表位肽的多价疫苗能够显著地抑制小鼠生育力,效果优于单一使用。6.免疫小鼠的生殖内分泌改变及机制研究随机处死雄性小鼠后,在FSHR免疫后生育力下降组的小鼠观察到:附睾精子计数和活动力等均下降;睾丸缩小,睾丸精子减少;血睾酮水平下降。联合Eppin免疫后,生殖道特异性IgA滴度较高,在精子减少的基础上,精子的活力亦明显降低,与小鼠抗血清能显著抑制正常人精子活力的结果一致。7.免疫小鼠睾丸组织学检查通过小鼠睾丸组织学检查发现,FSHR免疫后生育力下降的雄鼠显示睾丸组织有不同程度的改变,主要表现为曲细精管管径缩小、腔内生精细胞排列层次紊乱,管腔内精子数目减少,部分管腔内精子缺如。联合Eppin免疫后,睾丸组织的病理改变并未加重。8.生育力恢复实验由于小鼠睾丸有病理组织学改变,在初次免疫40周(末次免疫后36周)后,生育力下降组小鼠的生育力稍有升高但是未能恢复至正常。结论:1、通过PCR获得了hFSHR ECD和hEppin的DNA序列,成功地构建了含有目的基因的原核表达载体并诱导蛋白表达。经纯化和复性后,重组蛋白的纯度大于95%,具有较好的生物学特性。2、利用DNAstar对hFSHR ECD二级结构进行分析,预测潜在的B细胞表位,结合B细胞表位在线预测和分子对接技术以提高预测的准确性,综合分析结果,最后选择了4个候选B细胞表位肽。3、为了增强B细胞表位肽的免疫原性,联合候选肽与PADRE合成融合肽,合成的肽纯度均>85%。4、采用FSHR蛋白初免、B细胞表位肽强化免疫的策略能有效诱导机体产生高滴度的特异性抗体。FSHR的B细胞表位肽疫苗能通过减少精子数量而降低小鼠的生育力,但是伴有睾丸缩小、血睾酮降低和睾丸组织学改变的副作用;5、联合Eppin与FSHR免疫,在精子数量减少的同时,精子活力下降,导致小鼠的生育力进一步下降,但是的副反应并没有进一步加重。以后还需在疫苗剂型上深入研究以增强表位肽的免疫原性而降低甚至避免副作用。本研究为B细胞表位肽疫苗和多价避孕疫苗的研究提供了实验基础。更多还原

【Abstract】 As the world’s population continues to soar and men are willingness to participate in family planning, it is urgent to develop new contraceptive methods. The most properties of an ideal contraceptive should be effective, safe, inexpensive and reversible. The contraceptive vaccine (CV) can fulfill most of the properties and is an exciting proposition. Based on various targets, the CVs fall into three categories: vaccines inhibiting gamete production, gamete function and gamete outcome. The CVs for male focus on interfering spermatogenesis, inhibiting sperm motility or any process involved in sperm-egg interaction which resulted in fertility suppression.Follicle stimulating hormone is a glycoprotein hormone secreted by the pituitary gland which is essential for the initiation and maintenance of spermatogenesis in males. It exerts its biological actions through binding to its receptor (FSHR) expressed exclusively on Sertoli cells. For its importance on spermatogenesis, it is a potential target for male contraceptive. Literature showed that various discrete, discontinuous regions of the extracellular domain (ECD) are crucial for either FSH binding or subsequent signal transduction or both. In the past several decades, apart from monoclonal or polyclonal antibodies employed as probes, other approaches have been explored to understand structure-function relationship between FSH and FSHR, such as filamentous phage display, peptide challenge experiment and epitope mapping. However, the sites assessed in previous studies neither provide ideal suppressive fertility in animals nor go further to fertility test.Eppin (epididymis protease inhibitor) is a ~133 amino acid protein that specifically expressed on posterior part of perforatorium, Sertoli cells, Leydig cells, round spermatids and principal cell in epididymal epithelium. It is likely to be important for sperm maturation. Eppin presents on the surface of ejaculated sperm and becomes saturated with semenogelin (Sg), which is the major component of semen cogulum. Thus the Eppin-Sg complex provided sperm the antimicrobial activity while through the female reproductive tract. While the Macaca radiate receiced active immunization with recombinant Eppin, seven out of nine males showed inhibited fertility, and the fertilizing capacities of five out of seven males were restored when stopping immunization. For this reason, Eppin is a candidate molecular for CV.Therefore, our researches aimed at identify immunodominant neutralizing B-cell epitopes of FSHR and investigate its efficacy and safty as a CV for males. In this study, we divided the extracellular of hFSHR into two fragments on the basis of the amino acids it contained, including 1-140 amino acids and 120-350 amino acids, respectively. The gene sequences were inserted in plasmid pET 32a and the prokaryotic proteins were induced to express and purified. Four candidate peptides were selected after analysis of secondary structure of ECD, online prediction of B cell epitope and molecular docking. The four B cell peptides were in tandem with Pan DR epitope (PADRE) to enhance their immunogenicity. We employed the protein prime-peptide boost strategy to explore their antifertility efficacy on adult male mice. As vaccines based on FSHR produced only moderate fertility inhibition, we applied Eppin for combination immunization and observed its effect.Methods:1. The inductive expression of prokaryotic proteins for ECD of hFSHR and hEppinThe gene sequence of 1-140 amino acids and 120-350 amino acids of ECD were obtained by GeneSOEing PCR. The gene sequence of Eppin was amplificated by PCR. These genes were inserted in plasmid pET 32a and the prokaryotic proteins were expressed under induction of 1 mM IPTG. The recombinant proteins were purified and identified. The purified FSHR proteins were signed as F140 and F240, respectively.2. Prediction of B cell epitope of ECD of hFSHRThe ECD of hFSHR was analyzed with DNAstar software for secondary structure, hydrophilicity, accessibility, plasticity and antigen index. At meantime, the potential B cell epitope was predicted online. The molecular docking of hFSHR and hFSH was performed using the InsightⅡsoftware package. The InsightⅡ/Homology module was used to build the initial FSHR model with crystal structure of complex of human FSH and FSHR (PDB ID: 1xwd) as template.3. Synthesis of confusion peptide of hFSHR The candidate peptides were conjugated with PADRE to enhance their immunogenic- city and synthesized by GL Biochem (Shanghai) Ltd.4. The immunological effect of B cell epitopeThe sexually mature Balb/C male mice received active immunization with protein prime-peptide boost regimen. The immunogen was emulsified with same volume of Freund’s adjuvant. The first boost vaccination was given at 2 weeks after the prime immunization and repeated for three times at one week interval. The specific antibody IgG was monitored using ELISA. The Western blot analysis was employed to testify whether the antisera from vaccinated mice could bind with the natural FSHR protein or not. Also, the specific antibody IgG of mice received combination immunization with FSHR and Eppin was assessed at certain times.5. Antifertility effectThe productive capacities of immunized male mice were investigated by mating one male with three randomly selected females of proven fertility for 2 weeks. The number of pregnant females and litter sizes were recorded. Two successive mating trials were carried out at 2 weeks and 6 weeks respectively after the final immunization.6. Studies on the effects of inoculation to the reproductive and endocrine system of male mice and its mechanismsTwo weeks after the last fertility assay, male mice were sacrificed, blood was collected, and testis and epididymis were dissected. Sperm were collected from cauda epididymidis and assessed for sperm count, motility and hypo-osmotic swelling test (HOS). The weights of testis were taken notes and the spermatids in testis were calculated. The serum testosterone level was detected by radioimmunoassay (RIA). In addition, the inhibition effects of antiserum on normal human sperm and the IgA in epididymis lavage fluid were evaluated for animals vaccinated with combination immunization.7. Histological examination of testisAfter male mice were sacrificed, testis were removed, fixed and embedded. The slices were stained by hematoxylin-eosin (HE) and observed under light microscope.8. Fertility recovery assayForty weeks after primary immunization, one male was cohabited with three female for 2 weeks. The number of contraceptive mice and progeny size were counted. Results:1. The inductive expression of prokaryotic proteins for ECD of hFSHR and hEppinThe gene sequences of hFSHR and hEppin were amplificated and inserted in plasmid pET 32a. The prokaryotic proteins were existed as inclusion body. After purified and renaturated, the protein content was assessed by Bradford method. It was 1.26 mg/ml for F140, 1.19 mg/ml for F240 and 5.5 mg/ml for Eppin and their purity were over 95%. The Western blot analysis showed that the antiserum induced by recombinant protein could bind with 75kD or 34kD protein, which was existed in the extracted protein from human testis tissue.2. Prediction of B cell epitope of ECD of hFSHRTaken all analytic results into consideration, we selected four potential epitopes, whose location in ECD were 29~38, 32~44, 269~280 and 285~300, respectively.3. Synthesis of confusion peptide of hFSHRThe purity of confusion peptides were above 85%.4. The immunological effect of B cell epitopeAfter vaccinated with antigen based on FSHR, specific IgG antibody can be detected in serum with the highest titre above 1: 400000. The Western blot analysis displayed a band about 75kD when extracted protein from human testis tissue was probed with antisera from immunized animals. Similar antibody titres were observed in males with combination immunization. It suggested that it could elicit high specific antibody with the protein prime-peptide boost strategy.5. Antifertility effectThe fertility assay displayed that primed with hFSHR protein and boosted with conjugated peptide, partial contraceptive effects were induced in mice of group P2, P3 and P4. Especially the group P2 showed a significantly reduced pregnant rate of 26.67% and the mean litter sizes were 4.19, a litter high than that of mice immunized four times with recombinant protein. When Eppin were applied together, the inhibition effect was more obviously, mice primed with FSHR and Eppin and boosted with peptide 2 plus peptide C showed a similar higher fertility with that of combination protein prime-boost mice. In detail, the fertility rate significantly reduced to 6.67% and progeny sizes to 2.5. Therefore, peptide 2 might be a dominant B cell epitope of FSHR to produce fertility inhibition and combination vaccination was superior to single application.6. Studies on the effects of inoculation to the reproductive and endocrine system of male mice and its mechanismsFurther investigation for the mechanism of reduced fertility in vaccinated males revealed lower sperm count and motility of cauda epididymidis, smaller testis and less spermatid in testis and lower serum testosterone level. Inoculated with Eppin together, higher IgA titre in epididymis lavage was observed and the motility of sperm depressed significantly as well, this was consistent with inhibition effect of antisera on normal human sperm.7. Histological examination of testisIn fertility suppressed male mice induced by immunization with FSHR, histological examination manifested diameter of seminiferous tubule were decreased along with lack of sufficient sperm in some seminiferous tubules. In light microscope, it revealed a distorted tubular architecture in spermatogenic epithelium. The pathological change in testis did not aggravate after vaccination adding Eppin.8. Fertility recovery assayFor the pathological change existed in testis, the fertility decreased male mice showed a little high fertility rate but can not recover completely at forty weeks after primary immunization when ELISA showed antibody titres were less than 1: 5000.Conclusions:1. We amplificated the gene sequence of human FSHR and Eppin and constructed prokaryotic expression vector successfully. After inductive expression, purification and renaturation, the purity of combinant proteins were over 95%.2. The ECD of hFSHR were analyzed for secondary structure, predicted B cell epitope online and molecular docking. Taken all these results into consideration, four candidate B cell epitope peptides were selected.3. PADRE was in tandem with B cell epitope and the purity of fusion peptide was above 85%.4. The protein prime–peptide boost modality could elicit high titre of specific antibody. Peptide vaccine of FSHR could induce fertility inhibition through reduce quantity of sperm, but with side effects such as smaller testis, lower serum testosterone level and pathological change in testis.5. Combination immunization with FSHR and Eppin could induce significant decreased sperm motility accompanied with less sperm, while the side effects did not aggravated. How to enhance the immunogenicity of B cell epitope but alleviate or avoid side effects should be investigated further. But our study provided experimental ground- work for development of multivalent contraceptive vaccine.更多还原