本文關(guān)鍵詞：SP17-EPPIN融合蛋白避孕疫苗的構(gòu)建及其避孕效應(yīng)的初步研究　出處：《重慶醫(yī)科大學》2011年博士論文　論文類型：學位論文

  更多相關(guān)文章： 精子膜蛋白 Sp17 Eppin 融合蛋白 免疫避孕

【摘要】：避孕疫苗是一種有效、長期、可逆的避孕方法,是目前避孕領(lǐng)域研究的熱點。精子膜蛋白是精子發(fā)生成熟過程中的重要標志分子,與受精和胚胎的早期發(fā)育有密切聯(lián)系,具體涉及精子獲能、精卵識別、頂體反應(yīng)、穿過卵透明帶、精卵融合和受精卵的分裂等方面,其中某些精子膜蛋白能引起機體產(chǎn)生相應(yīng)的抗體,導致免疫性不孕�；诖瞬捎镁幽さ鞍籽兄聘咝А⒖赡�、無副作用的免疫避孕疫苗成為可能。 精子頂體膜蛋白17(Sp17)分布于精子頂體赤道區(qū)及頂體內(nèi)膜。Sp17在精子頂體反應(yīng)后大量表達,其作用可能是與透明帶上的糖基結(jié)合而促進受精作用。附睪蛋白激酶抑制劑(Eppin)是新發(fā)現(xiàn)的一種睪丸和附睪特異性分泌蛋白,射精后的人類精子頭部和尾部表面覆蓋著豐富的Eppin蛋白。當精子進入附睪的輸出管,與附睪頭和尾部所分泌的Eppin通過特異性位點結(jié)合于精子表面。目前認為Eppin蛋白參與調(diào)控精液液化和精子運動。Sp17和Eppin均為潛在的避孕疫苗靶點。目前雖然單一精子膜蛋白疫苗具有避孕效應(yīng),但還不能令人滿意,聯(lián)合不同作用機制的靶抗原構(gòu)建復合避孕疫苗可能是解決該問題的有效嘗試。有鑒于Sp17和Eppin位于精子表面的不同位置,作用機制不同,因此推測Sp17-Eppin融合蛋白疫苗可以功能互補、增效,能夠減少疫苗用量,降低毒副作用,利于生殖功能恢復。第一部分pPIC9K-Sp17-Eppin酵母表達載體的構(gòu)建 目的獲得人Sp17和Eppin基因,將兩者連接成融合基因,構(gòu)建該融合基因的酵母表達載體,同時構(gòu)建Sp17酵母表達載體。 方法提取人睪丸組織總RNA,經(jīng)過反轉(zhuǎn)錄得到總cDNA,根據(jù)Genebank公布的Sp17和Eppin序列設(shè)計引物,PCR擴增得到Sp17和Eppin編碼區(qū)序列,通過linker連接兩個基因,克隆至pPIC9K質(zhì)粒中,構(gòu)建pPIC9K-Sp17-Eppin酵母表達載體。并構(gòu)建pPIC9K-Sp17酵母表達載體。酶切及測序鑒定質(zhì)粒。 結(jié)果用SnaB I和Not I雙酶切pPIC9K-Sp17-Eppin質(zhì)粒,切下876bp的目的條帶,測序未發(fā)生基因突變,堿基序列正確。并酶切pPIC9K-Sp17質(zhì)粒,切下456bp,測序正確。 結(jié)論pPIC9K-Sp17-Eppin及pPIC9K-Sp17酵母表達載體成功構(gòu)建,為獲得Sp17-Eppin融合蛋白及Sp17重組蛋白奠定了基礎(chǔ)。第二部分Sp17-Eppin融合蛋白的表達、純化及鑒定 目的獲得Sp17-Eppin融合蛋白及Sp17重組蛋白,制備疫苗。 方法以單交換的整合方式電穿孔法分別將pPIC9K-Sp17-Eppin和pPIC9K-Sp17表達質(zhì)粒轉(zhuǎn)染酵母菌GSll5,依次進行G418篩選、PCR篩選鑒定、Mut篩選及小量搖瓶培養(yǎng)實驗,篩選高拷貝菌株,確定蛋白表達的最佳收獲時段,篩選結(jié)束后大量搖瓶培養(yǎng),于最佳時段收獲上清液,經(jīng)Ni2+-NTA柱純化后將所收集樣品冷凍抽干備用。純化蛋白用SDS-PAGE、Western-blot鑒定。 結(jié)果成功篩選出Sp17-Eppin融合蛋白和Sp17重組蛋白高效表達菌株,經(jīng)Ni2+-NTA柱純化蛋白純度達95%。用SDS-PAGE、Western-blot鑒定證實獲得了Sp17-Eppin融合蛋白及Sp17重組蛋白。 結(jié)論Sp17-Eppin融合蛋白和Sp17重組蛋白高效、正確的酵母表達系統(tǒng)成功建立,并獲得了高純度蛋白,為進一步的動物實驗奠定了基礎(chǔ)。第三部分Sp17-Eppin融合蛋白疫苗對小鼠的抗生育效應(yīng)研究 目的探討Sp17-Eppin融合蛋白對雄性小鼠的避孕效應(yīng)。 方法設(shè)Sp17-Eppin融合蛋白組(融合蛋白組)、Sp17重組蛋白組(重組蛋白組)及對照PBS組。采用初免加兩次強化的方案,分別將Sp17-Eppin融合蛋白50ug、Sp17重組蛋白50ug及等量佐劑皮下注射Balb/c雄性小鼠,對照組注射PBS及等量佐劑。取初免后各時期小鼠血清,應(yīng)用酶聯(lián)免疫法監(jiān)測血清抗體滴度的變化,測睪酮水平,初免后10周合籠檢測其避孕效應(yīng),12周隨機處死6只小鼠取睪丸、附睪等組織臟器,評估附睪精子計數(shù)、活動率及活率情況,用光鏡和電鏡觀察睪丸組織的病理變化,進行體外小鼠血清對人精子抑制試驗及熒光實驗,21周后再次合籠觀察小鼠生育能力恢復情況。 結(jié)果免疫后融合蛋白組及重組蛋白組小鼠血清出現(xiàn)特異性IgG抗體,抗體滴度在初免后10周達最高峰,峰值分別為12806.67±999.14和6770.67±1088.13,融合蛋白組1:10000的血清抗體滴度可持續(xù)6周。各組血清睪酮水平無明顯差異。合籠試驗融合蛋白組受孕率為21.43%,重組蛋白組受孕率37.50%,PBS組受孕率82.35%,融合蛋白組相比其他兩組受孕率有顯著性降低(P0.01)。融合蛋白組附睪精子計數(shù)、活動率及活率分別為(5.03±1.02)×106/ml、25.50±5.36%、32.00±5.73%,相比其他兩組均顯著性下降(P0.01)。融合蛋白組睪丸重量為75.00±4.65mg,相比其他兩組有明顯減輕(P0.05)。體外實驗中小鼠血清孵育的人精子前向運動百分率60min為32.15±3.43%,3h為22.77±2.65%,相比孵育前有顯著降低(P0.01),熒光實驗可見抗Sp17-Eppin融合蛋白抗體結(jié)合位點在精子頭部和尾部。光鏡下觀察融合蛋白組小鼠睪丸曲細精管管腔內(nèi)的精子數(shù)明顯減少,生精細胞層數(shù)減少。電鏡下可見融合蛋白組生精細胞內(nèi)線粒體腫脹,內(nèi)質(zhì)網(wǎng)擴張。初免21周后再行合籠試驗,融合蛋白組受孕率恢復至57.14%。 結(jié)論Sp17-Eppin融合蛋白疫苗在動物實驗中顯示了良好的抗生育效應(yīng),相比Sp17重組蛋白疫苗具有更好的避孕效應(yīng),其避孕效應(yīng)具有可逆性,但其具有睪丸縮小的副作用,對睪丸的生精功能有一定程度的影響。 本研究在借鑒國內(nèi)外有關(guān)免疫疫苗的理論和技術(shù)的基礎(chǔ)上,以人Sp17和Eppin為靶抗原,構(gòu)建了Sp17-Eppin融合蛋白酵母表達系統(tǒng),獲得了高純度融合蛋白,在動物實驗中發(fā)現(xiàn)Sp17+Eppin融合蛋白疫苗避孕效應(yīng)明顯強于單一Sp17重組蛋白疫苗,達到了避孕效應(yīng)增加、功能互補的目的。本課題是對構(gòu)建融合蛋白避孕疫苗的有益嘗試,對男性免疫避孕疫苗新途徑進行了有效探索,為開發(fā)安全、高效、可逆的理想避孕疫苗打下了堅實的實驗基礎(chǔ)。
[Abstract]:Contraceptive vaccine is an effective, long-term, reversible contraceptive methods, is currently a hot research field. The contraceptive sperm membrane protein is an important marker of spermatogenesis in the mature process, closely related with fertilization and early embryo development, in particular the sperm capacitation, sperm egg recognition, acrosome reaction, through the zona with sperm egg fusion and zygote etc., some sperm membrane proteins can cause the body to produce antibodies, leading to immune infertility. The sperm membrane protein by reversible development of high efficiency, based on no side effects of contraceptive vaccine is possible.
The acrosomal membrane protein 17 (Sp17) located in the equatorial region of the acrosome and acrosomal membrane.Sp17 in sperm acrosome reaction after expression, the effect may be combined to promote fertilization with carbohydrate ZP. Epididymal protein kinase inhibitor (Eppin) is a newly discovered testis and epididymis specific secretory protein of human sperm after ejaculation, the head and tail covered with rich Eppin protein. When the output sperm in the epididymis tube, and secreted by the head and tail of the epididymis by Eppin specific binding sites on the sperm surface. Now that the Eppin protein involved in regulation of sperm motility and semen liquefaction were.Sp17 and Eppin contraceptive vaccine potential targets. Although a single sperm membrane protein vaccine with the contraceptive effect, but it is not satisfactory, the target antigen with different effect mechanism of compound contraceptive vaccine is likely to solve the problem Effective attempt. In view of different positions Sp17 and Eppin located on the sperm surface, the mechanism is different, so we speculated that Sp17-Eppin fusion protein vaccine can be complementary functions, efficiency, can reduce the dosage of the vaccine, reduce the adverse effect to the reproductive function. The first part constructs pPIC9K-Sp17-Eppin yeast expression vector
Objective to obtain human Sp17 and Eppin genes, connect the two into fusion gene, construct the yeast expression vector of the fusion gene, and construct the expression vector of Sp17 yeast.
Methods total RNA was extracted from human testicular tissue after reverse transcription of total cDNA, Genebank and Eppin according to the published Sp17 primers, amplified by PCR sequence Sp17 and Eppin encoding region, connexin two gene by linker, cloned into pPIC9K plasmid, to construct pPIC9K-Sp17-Eppin yeast expression vector. Then we construct the yeast expression vector pPIC9K-Sp17 enzyme. Digestion and sequencing of plasmid.
Results pPIC9K-Sp17-Eppin plasmid was digested with SnaB I and Not I. The target bands of 876bp were cut down. No gene mutation was found in sequencing. The nucleotide sequence was correct. The pPIC9K-Sp17 plasmid was cut down and the 456bp was cut down, and the sequence was correct.
Conclusion the expression vector of pPIC9K-Sp17-Eppin and pPIC9K-Sp17 yeast was successfully constructed, which laid the foundation for obtaining Sp17-Eppin fusion protein and Sp17 recombinant protein. The second part is the expression, purification and identification of Sp17-Eppin fusion protein.
Objective to obtain the Sp17-Eppin fusion protein and the recombinant protein of Sp17 to prepare the vaccine.
Methods to integrate single exchange electroporation respectively pPIC9K-Sp17-Eppin and pPIC9K-Sp17 expression plasmid was transfected into yeast GSll5, followed by G418 screening, screening and identification of PCR, Mut screening and small flask experiments, screening of high copy strain, to determine the optimal harvest time of protein expression, screening after the end of a large number of flask culture, the best time to harvest the supernatant by Ni2+-NTA, purified the collected samples were frozen dry spare. By SDS-PAGE, purified protein Western-blot identification.
Results the Sp17-Eppin fusion protein and Sp17 recombinant protein were successfully screened out. The purity of the purified protein was 95%. by Ni2+-NTA column. The Sp17-Eppin fusion protein and Sp17 recombinant protein were identified by SDS-PAGE and Western-blot.
Conclusion the Sp17-Eppin fusion protein and Sp17 recombinant protein were successfully established, and the high-purity protein was successfully established, which laid the foundation for further animal experiments. The third part is the anti fertility effect of Sp17-Eppin fusion vaccine.
Objective to investigate the contraceptive effect of Sp17-Eppin fusion protein on male mice.
Methods the Sp17-Eppin fusion protein group (fusion protein group), Sp17 group (recombinant protein recombinant protein group) and control group PBS. The initial free plus two intensive programs, respectively, Sp17-Eppin 50ug fusion protein Sp17 recombinant protein 50ug and the same amount of adjuvant subcutaneous injection of Balb/c male mice in control group were injected with PBS and the same amount of adjuvant at the beginning of each period. The mice immunized serum changes using enzyme linked immunosorbent assay monitoring serum antibody titers, measured testosterone levels, beginning from 10 weeks after the cage to detect the contraceptive effect, 12 weeks, 6 mice were killed from the testis, epididymis and other organs, evaluate the epididymal sperm count, activity rate and survival rate of the situation and with the pathological changes under light microscope and electron microscope in mouse testicular tissue, serum inhibition test and fluorescence experiments on human sperm, 21 weeks after cage mice fertility restoration.
Results after immunization with fusion of specific IgG antibody in serum protein group and recombinant antibody titer in mice, the peak of up to 10 weeks after immunization, the peak were 12806.67 + 999.14 and 6770.67 + 1088.13, 1:10000 fusion protein group the serum antibody titer lasted 6 weeks. There was no significant difference in serum testosterone levels. Cage protein the pregnancy rate of group fusion test was 21.43%. The recombinant protein group the pregnancy rate was 37.50%, pregnancy rate is 82.35% PBS, the fusion protein group compared to the other two groups pregnancy rate was significantly decreased (P0.01). The fusion protein group, epididymal sperm count, activity rate and survival rate were (5.03 + 1.02) + 5.36% * 106/ml, 25.50. 32 + 5.73%, compared with the other two groups were significantly decreased (P0.01). The fusion protein group testicular weight was 75 + 4.65mg, compared to the other two groups had significantly reduced (P0.05). In vitro experiments in mice with human serum for sub motility 60mi N is 32.15 + 3.43%, 22.77 + 2.65% 3h, compared to before incubation decreased significantly (P0.01), fluorescence visible anti Sp17-Eppin fusion protein antibody binding sites in the sperm head and tail. Under the light microscope fusion protein group seminiferous tube number of sperm in the lumen was significantly reduced, spermatogenic cells reduce the number of electron microscope. The fusion protein group spermatogenic cells mitochondria swelling, endoplasmic reticulum expansion. 21 weeks after the primary immunization cage test fusion protein: the pregnancy rate recovered to 57.14%.
Conclusion Sp17-Eppin fusion protein vaccine in animal experiments showed that the anti fertility effect of good contraceptive effect, compared with Sp17 recombinant protein vaccine is better, the contraceptive effect is reversible, but it has the side effect of testicular size, have a certain degree of influence on testicular spermatogenic function.
In this study, based on immune theory and technology at home and abroad, with Sp17 and Eppin as target antigen, Sp17-Eppin fusion protein was constructed in yeast expression system, purified fusion protein, found that Sp17+Eppin fusion protein vaccine contraceptive effect was stronger than the single Sp17 recombinant protein vaccine in animal experiments, to contraceptive effect increases, functional complementarity. This research is to construct the beneficial attempt fusion protein contraceptive vaccine, the effective way to explore new male contraceptive vaccine, for the development of safe, efficient, ideal contraceptive vaccine can reverse lay a solid experimental basis.

【學位授予單位】：重慶醫(yī)科大學
【學位級別】：博士
【學位授予年份】：2011
【分類號】：R392

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