SP17-EPPIN融合蛋白避孕疫苗的构建及其避孕效应的初步研究

发布时间：2018-01-16 05:15

本文关键词：SP17-EPPIN融合蛋白避孕疫苗的构建及其避孕效应的初步研究　出处：《重庆医科大学》2011年博士论文　论文类型：学位论文

【摘要】：避孕疫苗是一种有效、长期、可逆的避孕方法,是目前避孕领域研究的热点。精子膜蛋白是精子发生成熟过程中的重要标志分子,与受精和胚胎的早期发育有密切联系,具体涉及精子获能、精卵识别、顶体反应、穿过卵透明带、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机体产生相应的抗体,导致免疫性不孕。基于此采用精子膜蛋白研制高效、可逆、无副作用的免疫避孕疫苗成为可能。精子顶体膜蛋白17(Sp17)分布于精子顶体赤道区及顶体内膜。Sp17在精子顶体反应后大量表达,其作用可能是与透明带上的糖基结合而促进受精作用。附睾蛋白激酶抑制剂(Eppin)是新发现的一种睾丸和附睾特异性分泌蛋白,射精后的人类精子头部和尾部表面覆盖着丰富的Eppin蛋白。当精子进入附睾的输出管,与附睾头和尾部所分泌的Eppin通过特异性位点结合于精子表面。目前认为Eppin蛋白参与调控精液液化和精子运动。Sp17和Eppin均为潜在的避孕疫苗靶点。目前虽然单一精子膜蛋白疫苗具有避孕效应,但还不能令人满意,联合不同作用机制的靶抗原构建复合避孕疫苗可能是解决该问题的有效尝试。有鉴于Sp17和Eppin位于精子表面的不同位置,作用机制不同,因此推测Sp17-Eppin融合蛋白疫苗可以功能互补、增效,能够减少疫苗用量,降低毒副作用,利于生殖功能恢复。第一部分pPIC9K-Sp17-Eppin酵母表达载体的构建目的获得人Sp17和Eppin基因,将两者连接成融合基因,构建该融合基因的酵母表达载体,同时构建Sp17酵母表达载体。方法提取人睾丸组织总RNA,经过反转录得到总cDNA,根据Genebank公布的Sp17和Eppin序列设计引物,PCR扩增得到Sp17和Eppin编码区序列,通过linker连接两个基因,克隆至pPIC9K质粒中,构建pPIC9K-Sp17-Eppin酵母表达载体。并构建pPIC9K-Sp17酵母表达载体。酶切及测序鉴定质粒。结果用SnaB I和Not I双酶切pPIC9K-Sp17-Eppin质粒,切下876bp的目的条带,测序未发生基因突变,碱基序列正确。并酶切pPIC9K-Sp17质粒,切下456bp,测序正确。结论pPIC9K-Sp17-Eppin及pPIC9K-Sp17酵母表达载体成功构建,为获得Sp17-Eppin融合蛋白及Sp17重组蛋白奠定了基础。第二部分Sp17-Eppin融合蛋白的表达、纯化及鉴定目的获得Sp17-Eppin融合蛋白及Sp17重组蛋白,制备疫苗。方法以单交换的整合方式电穿孔法分别将pPIC9K-Sp17-Eppin和pPIC9K-Sp17表达质粒转染酵母菌GSll5,依次进行G418筛选、PCR筛选鉴定、Mut筛选及小量摇瓶培养实验,筛选高拷贝菌株,确定蛋白表达的最佳收获时段,筛选结束后大量摇瓶培养,于最佳时段收获上清液,经Ni2+-NTA柱纯化后将所收集样品冷冻抽干备用。纯化蛋白用SDS-PAGE、Western-blot鉴定。结果成功筛选出Sp17-Eppin融合蛋白和Sp17重组蛋白高效表达菌株,经Ni2+-NTA柱纯化蛋白纯度达95%。用SDS-PAGE、Western-blot鉴定证实获得了Sp17-Eppin融合蛋白及Sp17重组蛋白。结论Sp17-Eppin融合蛋白和Sp17重组蛋白高效、正确的酵母表达系统成功建立,并获得了高纯度蛋白,为进一步的动物实验奠定了基础。第三部分Sp17-Eppin融合蛋白疫苗对小鼠的抗生育效应研究目的探讨Sp17-Eppin融合蛋白对雄性小鼠的避孕效应。方法设Sp17-Eppin融合蛋白组(融合蛋白组)、Sp17重组蛋白组(重组蛋白组)及对照PBS组。采用初免加两次强化的方案,分别将Sp17-Eppin融合蛋白50ug、Sp17重组蛋白50ug及等量佐剂皮下注射Balb/c雄性小鼠,对照组注射PBS及等量佐剂。取初免后各时期小鼠血清,应用酶联免疫法监测血清抗体滴度的变化,测睾酮水平,初免后10周合笼检测其避孕效应,12周随机处死6只小鼠取睾丸、附睾等组织脏器,评估附睾精子计数、活动率及活率情况,用光镜和电镜观察睾丸组织的病理变化,进行体外小鼠血清对人精子抑制试验及荧光实验,21周后再次合笼观察小鼠生育能力恢复情况。结果免疫后融合蛋白组及重组蛋白组小鼠血清出现特异性IgG抗体,抗体滴度在初免后10周达最高峰,峰值分别为12806.67±999.14和6770.67±1088.13,融合蛋白组1:10000的血清抗体滴度可持续6周。各组血清睾酮水平无明显差异。合笼试验融合蛋白组受孕率为21.43%,重组蛋白组受孕率37.50%,PBS组受孕率82.35%,融合蛋白组相比其他两组受孕率有显著性降低(P0.01)。融合蛋白组附睾精子计数、活动率及活率分别为(5.03±1.02)×106/ml、25.50±5.36%、32.00±5.73%,相比其他两组均显著性下降(P0.01)。融合蛋白组睾丸重量为75.00±4.65mg,相比其他两组有明显减轻(P0.05)。体外实验中小鼠血清孵育的人精子前向运动百分率60min为32.15±3.43%,3h为22.77±2.65%,相比孵育前有显著降低(P0.01),荧光实验可见抗Sp17-Eppin融合蛋白抗体结合位点在精子头部和尾部。光镜下观察融合蛋白组小鼠睾丸曲细精管管腔内的精子数明显减少,生精细胞层数减少。电镜下可见融合蛋白组生精细胞内线粒体肿胀,内质网扩张。初免21周后再行合笼试验,融合蛋白组受孕率恢复至57.14%。结论Sp17-Eppin融合蛋白疫苗在动物实验中显示了良好的抗生育效应,相比Sp17重组蛋白疫苗具有更好的避孕效应,其避孕效应具有可逆性,但其具有睾丸缩小的副作用,对睾丸的生精功能有一定程度的影响。本研究在借鉴国内外有关免疫疫苗的理论和技术的基础上,以人Sp17和Eppin为靶抗原,构建了Sp17-Eppin融合蛋白酵母表达系统,获得了高纯度融合蛋白,在动物实验中发现Sp17+Eppin融合蛋白疫苗避孕效应明显强于单一Sp17重组蛋白疫苗,达到了避孕效应增加、功能互补的目的。本课题是对构建融合蛋白避孕疫苗的有益尝试,对男性免疫避孕疫苗新途径进行了有效探索,为开发安全、高效、可逆的理想避孕疫苗打下了坚实的实验基础。
[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.

【学位授予单位】：重庆医科大学
【学位级别】：博士
【学位授予年份】：2011
【分类号】：R392

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