重组小鼠精子蛋白Izumo的制备及其免疫避孕潜力的研究

发布时间：2018-10-26 21:04

【摘要】： 【背景与目的】在众多的免疫避孕候选抗原中，精子抗原被认为是目前最有希望和最具应用前景的避孕疫苗靶抗原。2005年，一种新的精子特异表达的精卵融合关键蛋白Izumo的发现可能为避孕疫苗的研究提供了新思路和新材料。本课题旨在研究原核表达的重组小鼠Izumo(mlzumo)蛋白的免疫避孕潜力，为重组mlzumo蛋白是否能作为避孕疫苗候选靶抗原提供实验数据和科学依据。【材料与方法】①构建pET28a(+)-mlzumo原核表达质粒；②在大肠杆菌中表达带有6His纯化标签的重组蛋白6His-mlzumo；③固化Ni~(2+)亲和层析纯化重组蛋白6His-mIzumo，，并透析复性和Bradford法定量；④通过腹腔注射用6His-mIzumo免疫雌雄性C57BL／6小鼠；⑤Western blot检测免疫小鼠血清抗6His-mlzumo IgG抗体的特异性；⑥ELISA检测血清特异性抗体滴度随免疫时间变化的规律；⑦精卵融合试验观察免疫血清对小鼠体外精卵融合的影响；⑧双荧光染色法对抗6His-mlzumo IgG抗体在精子上的识别部位进行定位；⑨免疫小鼠在末次免疫后第2周与正常小鼠合笼，评价其生育力。【结果】①成功构建了pET28a(+)-mIzumo原核表达质粒；②成功表达和得到了纯化的重组蛋白6His-mlzumo；③雌性和雄性免疫小鼠体内都能产生血清特异性抗6His-mIzumoIgG抗体，特异性抗体与天然mlzumo存在交叉反应；④血清特异性抗6His-mIzumo IgG抗体开始出现在第一次加强免疫后第2周，末次免疫后第2周抗体滴度达到最高并至少持续到末次免疫后第6周；⑤6His-mlzumo免疫血清完全阻断小鼠体外精卵融合的发生；⑥抗6His-mlzumo IgG抗体特异地识别的小鼠AR精子(发生了顶体反应的精子)头部顶体区；⑦雌性和雄性免疫小鼠的生育力与PBS对照组相比没有明显降低。【结论】通过pET原核表达系统成功制备带有6His纯化标签的重组蛋白6His-mlzumo：6His-mlzumo对于雌性和雄性小鼠都具有免疫原性；虽然免疫重组mlzumo蛋白能够使小鼠产生特异识别小鼠AR精子头部顶体区并能阻断体外精卵融合的血清特异性IgG抗体，但对雌性和雄性免疫小鼠生育力的影响不明显。因此，原核表达的重组mlzumo蛋白可能对免疫小鼠没有明显的避孕作用，Izumo是否能作为避孕疫苗研制的靶抗原还需要进一步的研究和探索。
[Abstract]:[background & objective] among the many candidate antigens for immune contraception, sperm antigens are considered to be the most promising and promising target antigens for contraceptive vaccine. The discovery of a new spermatozoa specifically expressed spermatozoa fusion key protein Izumo may provide new ideas and materials for the study of contraceptive vaccine. The purpose of this study was to study the immunological potential of recombinant mouse Izumo (mlzumo) protein expressed in prokaryotic cells, and to provide experimental data and scientific basis for whether recombinant mlzumo protein can be used as candidate antigen for contraceptive vaccine. [materials and methods] 1 to construct pET28a ()-mlzumo prokaryotic expression plasmid, 2 to express the recombinant protein 6His-mlzumo with 6His purified label in Escherichia coli. 3Recombinant protein 6His-mIzumo was purified by immobilized Ni~ (2) affinity chromatography, and refolded by dialysis and measured by Bradford assay. (4) female and male C57BL/6 mice were immunized by intraperitoneal injection of 6His-mIzumo, and the specificity of anti-6His-mlzumo IgG antibody in serum of immunized mice was detected by 5Western blot. The changes of serum specific antibody titer with immune time were detected by 6ELISA, and the effect of immunized serum on mouse spermatozoa fusion in vitro was observed by sperm-egg fusion test. (8) double fluorescent staining was used to locate the recognition site of 6His-mlzumo IgG antibody on sperm, and 9 mice were incubated with normal mice at the 2nd week after the last immunization to evaluate their fertility. [results] 1 the prokaryotic expression plasmid of pET28a ()-mIzumo was successfully constructed, 2 the recombinant protein 6His-mlzumo was successfully expressed and purified. (3) in both female and male immunized mice, serum specific anti 6His-mIzumoIgG antibodies were produced, and there was a cross reaction between the specific antibodies and natural mlzumo. (4) the serum specific anti 6His-mIzumo IgG antibody began to appear at the second week after the first immunization, and the titer of the antibody reached the highest level at the second week after the last immunization and lasted until at least 6 weeks after the last immunization. 56His-mlzumo immune serum completely blocked the development of mouse spermatozoa fusion in vitro. 6) the acrosome region of mouse AR sperm (sperm with acrosome reaction) was specifically recognized by anti 6His-mlzumo IgG antibody. The fertility of female and male immunized mice was not significantly lower than that of PBS control group. [conclusion] the recombinant protein 6His-mlzumo:6His-mlzumo with 6His purified label was successfully prepared by pET prokaryotic expression system for both female and male mice. Although the recombinant mlzumo protein can make mice produce specific serum-specific IgG antibodies that specifically recognize the acrosome region of mouse AR sperm head and block the fusion of spermatozoa in vitro, the effect on fertility of female and male immunized mice is not obvious. Therefore, the recombinant mlzumo protein expressed in prokaryotic cells may not have significant contraceptive effect on immunized mice. Whether Izumo can be used as the target antigen of contraceptive vaccine should be further studied and explored.
【学位授予单位】：汕头大学
【学位级别】：硕士
【学位授予年份】：2007
【分类号】：R392

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