本文選題：疫苗　切入點：免疫　出處：《第三軍醫(yī)大學(xué)》2011年博士論文　論文類型：學(xué)位論文

【摘要】：FSH是垂體分泌的糖蛋白激素,對雄性精子的產(chǎn)生有至關(guān)重要的作用。實驗表明阻斷FSH或受體活性可以引起少精或無精。人們利用FSH這種生物學(xué)功能結(jié)合免疫學(xué)的理論和方法研究開發(fā)FSH/FSHR避孕疫苗。就FSHR避孕疫苗而言,其是通過受體抗體結(jié)合間接封閉FSH的生物學(xué)功能,抑制精子的生成達到避孕的目的。FSHR是G蛋白偶聯(lián)受體,有669氨基酸組成,其胞外區(qū)是與FSH結(jié)合的關(guān)鍵部位。為避免與其它糖蛋白家族發(fā)生交叉反應(yīng)而干擾其它激素的功能,因此對FSHR活性的表位篩選具有重要意義。研究發(fā)現(xiàn)通過克隆技術(shù)表達的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漸恢復(fù)。以上研究無疑為避孕疫苗的研究帶來了曙光。但也仍存在一些問題,如所應(yīng)用的研究對象數(shù)目少,不具有代表性,生育力恢復(fù)實驗僅為33.4%,其對生殖健康的遠期影響尚需進一步的驗證等。本實驗室前期工作中用人FSHR重組蛋白免疫雄性小鼠后,導(dǎo)致小鼠受孕率明顯下降。但一個嚴重的問題是帶來生殖器管的損傷及睪酮的下降。考慮到FSHR蛋白免疫動物造成生殖器官損傷主要是因為蛋白表面含有多種表位,除中和表位外,其它表位如毒性表位、優(yōu)勢非中和性表位等可能引起免疫偏移造成免疫損傷,因此為了減少其副作用,我們利用計算機聯(lián)合預(yù)測結(jié)合人工合成的方法獲得關(guān)于FSHR蛋白胞外區(qū)由13氨基酸組成的多肽,并采用不同劑量多肽初免、加強策略免疫雄鼠,觀察其避孕效應(yīng)及對生殖機能的影響,主要內(nèi)容包括:1.利用各種生物信息學(xué)方法對FSHR胞外區(qū)蛋白進行二維結(jié)構(gòu)分析、對B細胞表位篩選以及通過分子對接等綜合預(yù)測,獲得優(yōu)勢中和表位肽段,并對獲得的肽段進行優(yōu)化。并通過肽結(jié)合實驗進一步驗證篩選肽段的免疫原性,獲得最終優(yōu)化后B細胞表位多肽。2.使用不同劑量的FHSR多肽免疫雄性小鼠,在不同的免疫時期與正常受孕的雌鼠合籠(比例1:2)觀察雌鼠受孕率及產(chǎn)仔率。3.在觀察不同劑量FSHR肽段對小鼠避孕效果同時,檢測被免疫小鼠的生殖器官有無病理損害、了解被免疫小鼠的精子功能,以及性激素水平。 主要采用的實驗方法如下:1.采用DNAprotein軟件對FSHR蛋白胞外區(qū)二級結(jié)構(gòu)分析,抗原性方案、親水性方案、可及性方案等對B細胞表位進行篩選以及通過分子對接等手段進行綜合分析。經(jīng)與前期獲得的B細胞表位進行比較及優(yōu)化后,獲得最佳抗原表位肽段。2.用ELISA方法檢測篩選肽段的免疫原性。2.根據(jù)預(yù)測結(jié)果,人工合成FSH受體胞外區(qū)肽段,與泛T細胞表位PDR偶聯(lián)成融合蛋白,免疫小鼠。具體分組是:FSHR肽20ug及80ug兩個劑量及對照組PBS、蛋白組共4組。采取肽初免、加強策略,初免與完全佐劑混合、加強免疫與不完全佐劑混合注射于成年雄性小鼠后肢足墊。3.ELISA方法觀察免疫前及免疫后0、2、3、4、6、8、10、12、14、16、20周抗體滴度。4.受孕實驗:根據(jù)抗體滴度,分別于免疫后6周(抗體滴度大于1:500)及10周(抗體滴度大于1:1000)按1:2與雌鼠合籠觀察雌鼠受孕率及產(chǎn)仔率,進行統(tǒng)計學(xué)分析。5.初免結(jié)束后11周每組各取4只小鼠,檢測附睪精子數(shù)、活力及畸形率。6.通過體外精卵結(jié)合實驗了解精子功能。7.HE染色在光鏡下觀察有無生殖器官病理變化。電鏡下觀察血睪屏障。8.放免法測定被免疫小鼠血睪酮情況。 所獲得的實驗結(jié)果:1.通過篩選、優(yōu)化獲得B細胞優(yōu)勢中和表位的肽段---IELRFVLTKLRVI。將獲得的FSHR優(yōu)勢表位肽與泛T細胞表位---aKXVAAWTLKAAaZ偶聯(lián),獲得FSHR融合肽。2.抗體滴度于初免后2周逐漸上升,肽組(80μg)在免疫后10周達到高峰,后逐漸下降。3.肽(20ug)小鼠雌鼠受孕率及產(chǎn)仔率與PBS對照組無區(qū)別;而肽(80ug)組及蛋白組小鼠在免疫10周雌鼠受孕率及產(chǎn)仔率下降,與PBS對照組相比有顯著差異。4.在初免后11周在光鏡及透射電鏡下肽組生殖器官無病理損害,血睪屏障無破壞,血睪酮水平無變化,而蛋白組光鏡下即有明顯精原細胞水腫或點狀壞死,睪酮下降。5.不同劑量FSHR肽及蛋白組免疫小鼠后均導(dǎo)致精子數(shù)減少,活力降低,精卵融合受抑制.肽(80ug)組及蛋白組的精子畸形率明顯升高。根據(jù)以上結(jié)果,我們可以得知:不同劑量FSHR肽免疫小鼠后結(jié)果不同,肽(20ug)組雌小鼠受孕率及產(chǎn)仔率無改變,而肽(80ug)組雌小鼠受孕率及產(chǎn)仔率均下降,與蛋白組相似。雖然不同劑量FSHR肽均導(dǎo)致精子功能下降,但只有肽(80ug)組受孕率下降,因此說明精子功能只有下降到一定程度后才達到避孕效果。而且通過對免疫后生殖器官的觀察,肽組即無對生殖器官病理損害,也無對睪酮產(chǎn)生影響。因此就FSHR多肽疫苗免疫小鼠后即可以避孕又對生殖器官無損傷及對性激素?zé)o影響而言,其具有成為潛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.

【學(xué)位授予單位】：第三軍醫(yī)大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2011
【分類號】：R392

【引證文獻】

相關(guān)博士學(xué)位論文 前1條

1 唐帥;Eppin優(yōu)勢表位自組裝納米避孕疫苗的構(gòu)建及實驗研究[D];第三軍醫(yī)大學(xué);2013年

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本文編號：1632866