基于卵泡雌激素受体-FSHR多肽避孕疫苗的优化设计及其抗生育效应的实验研究

发布时间：2018-03-19 04:31

本文选题：疫苗　切入点：免疫　出处：《第三军医大学》2011年博士论文　论文类型：学位论文

【摘要】：FSH是垂体分泌的糖蛋白激素,对雄性精子的产生有至关重要的作用。实验表明阻断FSH或受体活性可以引起少精或无精。人们利用FSH这种生物学功能结合免疫学的理论和方法研究开发FSH/FSHR避孕疫苗。就FSHR避孕疫苗而言,其是通过受体抗体结合间接封闭FSH的生物学功能,抑制精子的生成达到避孕的目的。FSHR是G蛋白偶联受体,有669氨基酸组成,其胞外区是与FSH结合的关键部位。为避免与其它糖蛋白家族发生交叉反应而干扰其它激素的功能,因此对FSHR活性的表位筛选具有重要意义。研究发现通过克隆技术表达的FSHR N端多肽(18～27、25～34、29～38氨基酸)可以避孕,而且生育力在停止免疫后逐渐恢复。以上研究无疑为避孕疫苗的研究带来了曙光。但也仍存在一些问题,如所应用的研究对象数目少,不具有代表性,生育力恢复实验仅为33.4%,其对生殖健康的远期影响尚需进一步的验证等。本实验室前期工作中用人FSHR重组蛋白免疫雄性小鼠后,导致小鼠受孕率明显下降。但一个严重的问题是带来生殖器管的损伤及睾酮的下降。考虑到FSHR蛋白免疫动物造成生殖器官损伤主要是因为蛋白表面含有多种表位,除中和表位外,其它表位如毒性表位、优势非中和性表位等可能引起免疫偏移造成免疫损伤,因此为了减少其副作用,我们利用计算机联合预测结合人工合成的方法获得关于FSHR蛋白胞外区由13氨基酸组成的多肽,并采用不同剂量多肽初免、加强策略免疫雄鼠,观察其避孕效应及对生殖机能的影响,主要内容包括:1.利用各种生物信息学方法对FSHR胞外区蛋白进行二维结构分析、对B细胞表位筛选以及通过分子对接等综合预测,获得优势中和表位肽段,并对获得的肽段进行优化。并通过肽结合实验进一步验证筛选肽段的免疫原性,获得最终优化后B细胞表位多肽。2.使用不同剂量的FHSR多肽免疫雄性小鼠,在不同的免疫时期与正常受孕的雌鼠合笼(比例1:2)观察雌鼠受孕率及产仔率。3.在观察不同剂量FSHR肽段对小鼠避孕效果同时,检测被免疫小鼠的生殖器官有无病理损害、了解被免疫小鼠的精子功能,以及性激素水平。主要采用的实验方法如下:1.采用DNAprotein软件对FSHR蛋白胞外区二级结构分析,抗原性方案、亲水性方案、可及性方案等对B细胞表位进行筛选以及通过分子对接等手段进行综合分析。经与前期获得的B细胞表位进行比较及优化后,获得最佳抗原表位肽段。2.用ELISA方法检测筛选肽段的免疫原性。2.根据预测结果,人工合成FSH受体胞外区肽段,与泛T细胞表位PDR偶联成融合蛋白,免疫小鼠。具体分组是:FSHR肽20ug及80ug两个剂量及对照组PBS、蛋白组共4组。采取肽初免、加强策略,初免与完全佐剂混合、加强免疫与不完全佐剂混合注射于成年雄性小鼠后肢足垫。3.ELISA方法观察免疫前及免疫后0、2、3、4、6、8、10、12、14、16、20周抗体滴度。4.受孕实验:根据抗体滴度,分别于免疫后6周(抗体滴度大于1:500)及10周(抗体滴度大于1:1000)按1:2与雌鼠合笼观察雌鼠受孕率及产仔率,进行统计学分析。5.初免结束后11周每组各取4只小鼠,检测附睾精子数、活力及畸形率。6.通过体外精卵结合实验了解精子功能。7.HE染色在光镜下观察有无生殖器官病理变化。电镜下观察血睾屏障。8.放免法测定被免疫小鼠血睾酮情况。所获得的实验结果:1.通过筛选、优化获得B细胞优势中和表位的肽段---IELRFVLTKLRVI。将获得的FSHR优势表位肽与泛T细胞表位---aKXVAAWTLKAAaZ偶联,获得FSHR融合肽。2.抗体滴度于初免后2周逐渐上升,肽组(80μg)在免疫后10周达到高峰,后逐渐下降。3.肽(20ug)小鼠雌鼠受孕率及产仔率与PBS对照组无区别;而肽(80ug)组及蛋白组小鼠在免疫10周雌鼠受孕率及产仔率下降,与PBS对照组相比有显著差异。4.在初免后11周在光镜及透射电镜下肽组生殖器官无病理损害,血睾屏障无破坏,血睾酮水平无变化,而蛋白组光镜下即有明显精原细胞水肿或点状坏死,睾酮下降。5.不同剂量FSHR肽及蛋白组免疫小鼠后均导致精子数减少,活力降低,精卵融合受抑制.肽(80ug)组及蛋白组的精子畸形率明显升高。根据以上结果,我们可以得知:不同剂量FSHR肽免疫小鼠后结果不同,肽(20ug)组雌小鼠受孕率及产仔率无改变,而肽(80ug)组雌小鼠受孕率及产仔率均下降,与蛋白组相似。虽然不同剂量FSHR肽均导致精子功能下降,但只有肽(80ug)组受孕率下降,因此说明精子功能只有下降到一定程度后才达到避孕效果。而且通过对免疫后生殖器官的观察,肽组即无对生殖器官病理损害,也无对睾酮产生影响。因此就FSHR多肽疫苗免疫小鼠后即可以避孕又对生殖器官无损伤及对性激素无影响而言,其具有成为潜在的理想的避孕疫苗的可能。
[Abstract]:FSH is a glycoprotein hormone secreted by the pituitary, plays a vital role in producing male sperm. The experiment showed that blocking FSH receptor activity or can cause little or no sperm. People of the use of FSH biological function combined with immune theory and method of the research and development of FSH/FSHR contraceptive vaccine. FSHR contraceptive vaccine, which is the combination of the indirect closed receptor antibody the biological function of FSH, inhibit the generation of sperm to achieve the purpose of contraception is.FSHR G protein coupled receptors, 669 amino acids, the extracellular domain is a key part in combination with FSH. In order to avoid sugar and other protein family cross reaction and interference of other hormones, so the activity of FSHR epitope screening has important significance. Research found that FSHR N terminal polypeptide through cloning expression (18 ~ 27,25 ~ 34,29 ~ 38 amino acids) can stop contraception, and fertility in immunized by Recovered. The above research will undoubtedly bring dawn on contraceptive vaccine. But there still exist some problems, such as the number of research objects used less, not representative, fertility recovery experiment is only 33.4%, its impact on long-term reproductive health needs to be further verified. Immunize male mice with human FSHR recombinant our previous work in protein, resulting in pregnancy rate decreased significantly. But a serious problem is to bring down the damage and testosterone genital management. Considering the FSHR protein immune animal causes genital injury is mainly because the egg white surface contains a variety of epitopes, except neutralizing epitope, other epitopes such as toxicity epitope dominant and non neutralizing epitopes may induce immune deviation caused by immune injury, so in order to reduce the side effect, we use a computer to predict with a synthetic method to obtain FSHR The extracellular polypeptide protein consists of 13 amino acids, and peptides with different doses of primary immunization, strengthening immunity strategy of male rats, observe the contraceptive effect and influence on reproductive function, the main contents include: 1. the use of a variety of biological information on the extracellular domain of FSHR protein by two-dimensional structure analysis method, the B cell epitope screening and through molecular docking comprehensive prediction, to gain the advantage of neutralizing epitope peptide and the peptide was optimized. And the peptide binding experiments to further verify the immunogenicity of screening peptide, FHSR peptide immunization of male mice finally optimized B cell epitope peptide.2. with different doses and in different immune period with the normal pregnant female rats were mated (ratio 1:2) to observe the female pregnancy rate and farrowing rate of.3. in the observation of different doses of FSHR peptide of contraceptive effect in mice, detected by mice reproductive organs have no pathological Damage, understanding the sperm function of the immune mice, and the level of sex hormones.
The main experimental methods are as follows: 1. by using analysis of FSHR protein extracellular level two antigenic structure DNAprotein software scheme, hydrophilic scheme and scheme of screening and molecular docking by means of B cell epitopes were analyzed. With the pre - B cell epitopes were compared and optimized.2., obtained immunogenic epitope peptide of.2. detected by ELISA screening peptides according to the prediction results, the synthetic peptide FSH receptor extracellular domain, and the pan T cell epitope PDR conjugates of a fusion protein to immunize mice. The specific group is: FSHR peptide 20ug and two doses of 80ug and control group PBS, protein group altogether 4 groups. Take peptide priming, strengthen strategy, beginning from mixed with Freund's complete adjuvant, strengthen immunity and incomplete adjuvant injection in adult male mice hind footpad of.3.ELISA were observed before immunization and after 0,2,3,4,6,8,1 0,12,14,16,20 week pregnancy tests: according to the antibody titer of.4. antibody titers, respectively at 6 weeks after immunization (antibody titer of more than 1:500) and 10 weeks (the antibody titers greater than 1:1000) according to 1:2 and female rats were mated female rats to observe the pregnancy rate and farrowing rate were analyzed at the beginning of.5. free 11 weeks after the end of each of the 4 in mice, detection of epididymal sperm number, motility and deformity rate of.6. by in vitro sperm egg binding experiments to understand sperm function.7.HE staining was used for reproductive organ pathological changes under light microscope. Under electron microscope the blood testis barrier.8. radioimmunoassay in immunized mice serum testosterone conditions.
The experimental results obtained: 1. through screening, optimization of B cell dominant neutralizing epitope peptide ---IELRFVLTKLRVI. will get the FSHR epitope and pan T cell epitope ---aKXVAAWTLKAAaZ coupling, FSHR fusion peptide.2. antibody titer in the early 2 weeks after immunization increased peptide group (80 g) in the peak at 10 weeks after immunization, decreased gradually after.3. peptide (20ug) in female pregnancy rate and farrowing rate and PBS control group had no difference; and (80ug) peptide and proteomics in mice decreased by 10 weeks female pregnancy rate and litter, compared with PBS control group has significant difference in early.4. free after 11 weeks of light microscopy and transmission electron microscopy peptides of the reproductive organs without pathological damage to the blood testis barrier, no damage, no change in the level of serum testosterone, and protein group under light microscope that there were spermatogonia edema or necrosis, testosterone decreased.5. of different doses of FSHR peptide and protein immune mice after Guide To reduce the number of sperm, motility, sperm egg fusion inhibition. Peptide (80ug) and proteomics of sperm abnormality rate was significantly increased. According to the above results, we can know that different doses of FSHR peptide in mice immunized with peptide (20ug) results in different groups of female mice pregnancy rate and farrowing rate unchanged, and peptide (80ug) group, the pregnancy rate and farrowing rate of female mice was decreased, and the similar protein group. Although different doses of FSHR peptide resulted in sperm function decline, but only the peptide (80ug) group decreased pregnancy rates, so that sperm function only after falling to a certain extent to achieve contraceptive effect. And through the observation of the reproductive organs of the immune after that no peptide group on reproductive organ pathological damage, no influence on testosterone. Therefore immune FSHR polypeptide vaccine mice after contraception and reproductive organs without injury and had no effect on sex hormone, which has become a potential ideal. The possibility of a pregnancy vaccine.

【学位授予单位】：第三军医大学
【学位级别】：博士
【学位授予年份】：2011
【分类号】：R392

【引证文献】