重组人卵透明带ZP3蛋白在毕赤酵母中的表达

利用P.pastoris表达人卵透明带ZP3蛋白。设计特定引物从全长hZP3 cDNA上扩增含跨膜区序列的人卵透明带ZP3基因片段,并在N末端接上串联组氨酸编码序列的重组基因序列;扩增片段插入表达载体pPIC9K中;线性化后的重组质粒转入P.pastoris中,用高浓度G418筛选高拷贝菌株,然后甲醇诱导目的蛋白表达。用SDS-PAGE和Western blot分析表达产物。结果发现P.pastoris表达的人ZP3蛋白可以分泌到培养液中,并且可溶性好。纯化前后的重组人ZP3蛋白均能与兔抗猪ZP3蛋白抗体发生交叉反应,证实表达的目的蛋白具有反应原性。     

人卵透明带蛋白ZP3a和ZP3b肽段的免疫原性及其抗血清体外抑制人精子-半透明带结合

分析大肠杆菌表达的重组人卵透明带-3（r-huZP3）蛋白两个肽段（r-huZP3a^22-176及r-huZP3b^177-348）的免疫原性,比较两者抗血清体外抑制人精子-半透明带结合的能力。以制备性聚丙烯酰胺凝胶电泳（PAGE）纯化的大肠杆菌表达蛋白为抗原,主动免疫新西兰兔产生抗huZP3a^22-176及huZP3b^177-348抗血清：通过ELISA测定比较两者的抗体应答水平;以蛋白印迹和免疫组化方法测定两者抗血清同重组表达蛋白、大然人卵ZP以及卵巢组织的反应性;通过竞争性半透明带结合试验（hemizona assay,HZA）观察两者抗血清体外抑制人精子-ZP结合能力。结果显示：未与人分子蛋白载体耦联的r-huZP3a^22-176和r-huZP3b^177-348抗原都在免疫兔中产生了较高的抗体滴度,而且它们的抗血清可识别人肠杆菌表达的各自重组ZP3肽以及人卵细胞表面天然ZP,两者抗血清也都能在体外抑制人精子-ZP结合。由此可见,r-huZP3^22-176及r-huZP3b^177-348蛋白具有良好免疫原性,所产生抗血清也都显示出细胞和组织特异性。因此,单一或合并两个huZP3肽段均可作为抗原研制检测不明原因性不孕妇女中是否存在抗自身ZP抗体的诊断试剂盒,另外它们的抗血清也能用于鉴定已知huZP3表位肽段的最小B-细胞表位基序。     

猪精子中与卵透明带糖蛋白ZP3结合的蛋白质

依次经ＰＳＬ－Ｓｅｐｈａｒｏｓｅ亲和层析柱和纤维素ＣＭ－５２离子交换层析柱,从猪精子的ＣＨＡＰＳ抽提液分离得４个蛋白质组分。用固相透明带精蛋白结合试验（ＩＺＰＧＢＡ）检测;表明精子蛋白ＳＰ１和ＳＰ２具有结合透明带糖蛋白ＺＰ３的活性,ＳＰ２并显示凝集血球的活性。精子蛋白ＳＰ１与卵预温育明显抑制精卵结合,抑制活性与加入的精子蛋白的浓度呈正相关。用生物素标记的ＺＰ３和蛋白质印迹技术,证明ＳＰ１中的６８ｋＤ精子蛋白与ＺＰ３结合,提示６８ｋＤ精子蛋白参与精卵结合。     

卵透明带ZP3的研究及其应用

卵透明带蛋白围绕在卵母细胞外,在受精过程中起着重要作用。ZP3蛋白是卵透明带蛋白家族中的重要成员,在功能上,ZP3作为初级精子受体,起始精卵结合和顶体反应。由于ZP3在受精中的重要作用,它成为免疫避孕的有效靶点。ZP3蛋白疫苗和DNA疫苗可以诱导机体产生较强的免疫反应,导致生育降低,同时带来一定程度的副作用。本文重点介绍了ZP3的免疫特性及其应用。     

重组猪卵透明带-3α抗原及其抗体的制备

目的:制备重组猪卵透明带-3α(rpZP3α)抗原及其抗体供发展避孕疫苗研究。方法:在2L发酵罐中接种毕赤酵母GS115-pZP3α工程菌,高密度发酵表达rpZP3α蛋白,表达的蛋白经螯合镍离子的亲和柱纯化后,用SDS-PAGE和Western blot进行鉴定,以Quantity One软件对rpZP3α进行定量分析。rpZP3α免疫家兔,间接免疫荧光法检测抗血清对rpZP3α和猪卵透明带的抗体反应。IVF试验体外检测抗rpZP3α抗体对猪、小鼠精卵结合的影响作用。结果:工程菌高密度发酵产物经分离纯化后获得能与抗pZP3抗体反应的46kD成分,平均产量为8mg/L,用其免疫家兔获得抗rpZP3α抗血清,间接免疫荧光法分析显示此抗血清能与猪卵透明带反应,产生亮绿荧光。体外精卵结合试验中,随着兔抗rpZP3α抗血清浓度的加大,其对猪、鼠精卵结合的抑制作用也增强。ELISA结果显示抗rpZP3α抗体与rpZP3α蛋白以及天然pZP3蛋白两种抗原反应的强度和趋势基本一致。结论:通过酵母体系制备的rpZP3α及其抗体具有免疫学活性。     

哺乳动物透明带糖蛋白及其生物活性

透明带在哺乳动物的受精过程中发挥着重要作用,透明带化学成分及其生物活性的研究对于了解精卵识别、顶体反应的诱导等过程的分子机理具有重要意义．近10年来的研究表明透明带主要由糖蛋白（ZPGPs）组成,是在细胞质内合成后转移至透明带的.大多数动物的ZPGPs为4～5种,不同种类动物的ZPGPs的氨基酸序列存在高度同源性.在受精过程中,ZPGPs具有精子受体和诱导顶体反应的双重作用,ZPGPs含有N－连接和O－连接寡聚糖,这些寡糖链是ZPGPs行使生物活性不可缺少的部分．     

犬卵透明带3蛋白原核表达和抗血清的制备

在研发控制流浪犬卵透明带3（CZP3）免疫不育疫苗的过程中,需要制备特异性的CZP3抗血清进行免疫检测。本研究克隆获得CZP3的cDNA（全长1 281 bp）和除去跨膜区和信号肽的核心片段（CZP3C,984 bp）,通过构建原核表达载体pET28a-CZP3C,转化大肠杆菌BL21（DE3）。在28℃下以0.6 mmol·L-1异丙基硫代β-D-半乳糖苷诱导表达4 h,获得CZP3C融合蛋白。利用CZP3C融合蛋白免疫新西兰大白兔,制备特异性的抗血清,并对抗血清的特异性和效价进行评价。SDS-PAGE结果表明,融合蛋白以包涵体形式存在。经过镍柱亲和纯化及8 mmol·L-1尿素复性后,获得了高纯度的CZP3C融合蛋白。用0.8 μg/μL-1的CZP3C融合蛋白免疫新西兰大白兔,免疫后的兔抗血清用Western blot及间接免疫荧光实验检测,结果证明CZP3C兔抗血清具有较高的特异性,ELISA检测效价为1：409 600。本研究所制备的抗血清能够为流浪犬免疫不育疫苗的研制提供检测材料。     

草原兔尾鼠卵透明带3基因原核表达条件的优化

目的：通过大肠杆菌原核表达系统制备草原兔尾鼠卵透明带3（LZP3）融合蛋白,作为草原兔尾鼠疫苗免疫抗生育研究的抗原物质。方法：将LZP3基因的核心片段构建到融合表达载体pGEX-4T-1K中GST的3’端,形成GST-LZP3融合基因,经本科切鉴定和序列分析证实基因序列的正确性。在不同温度,不同时间和不同IPTG浓度进行诱导后,用SDS-PAGE检测融合蛋白的表达,发现有一条分子量约63kD的新增蛋白条带。结果：研究表明在37℃,25℃的不同温度条件下均能诱导出LZP3蛋白,经SDS-PAGE和间接ELISA法检测,初步证明了LZP3基因能够在大肠杆菌中有效表达可溶性的融合蛋白。结论：获得了LZP3融合蛋白表达的最佳诱导条件,为大量诱导产生LZP3融合蛋白奠定了理论基础。     

鼠卵透明带3在Pichia pastoris酵母中的分泌表达

本研究通过酵母Pichiapastoris表达鼠卵透明带 3(mZP3)蛋白 ,获得控制害鼠生育研究的抗原物质。根据国际基因库已发表的mZP3基因序列设计引物 ,并分别在 5’引物和 3’引物中引入EcoRI和XbaI酶切位点。经PCR扩增 ,将mZP3克隆至穿梭质粒pGAPZαA上 ,获得的重组穿梭质粒pGAPZαA mZP3经线性化后 ,采用LiCl法转入毕赤酵母SMD1 1 68菌株中 ,Zeocin+ 筛选阳性克隆 ,酵母发酵上清液进行SDS PAGE和Western blot检测 ,结果表明mZP3在酵母中得到了特异性表达 ,表达产物的分子量约为 60kDa ,说明mZP3能够在酵母真核系统中成功表达 ,并可以用作mZP3免疫不育控制鼠害的检测抗原。     

哺乳动物卵子的透明带

哺乳动物的卵子表面由透明带所包围,已知透明带可被蛋白质分解酶、低PH、氧化剂、还原剂以及热处理等溶解破坏.有关透明带的来源,过去曾认为是由卵子颗粒层细胞,或是卵细胞,以及由两者共同产生.最近的研究表明,透明带是由卵细胞所产生.目前一般认为卵子的透明带在受精时,可起到种间识别、阻止多精子受精以及在着床前保护受精卵的作用.此外,透明带还具     

A guanine nucleotide-binding regulatory protein in human sperm mediates acrosomal exocytosis induced by the human zona pellucida.

Guanine nucleotide-binding regulatory proteins play key intermediary roles in regulating zona pellucida-mediated acrosomal exocytosis in mouse and bull sperm. Since human sperm possess a Gi-like protein and undergo the acrosome reaction in response to the human zona pellucida, we investigated whether this G protein plays a regulatory role in this exocytotic process. Zonae pellucidae isolated from eggs that had been inseminated but had shown no signs of fertilization after retrieval for in vitro fertilization and embryo transfer were pooled into groups of greater than or equal to 50 in order to reduce variability in biological responses due to the possible presence of ZP that had undergone modifications associated with the polyspermy block. Acid-solubilized zonae pellucidae were incubated with capacitated sperm, and the sperm then assessed for the acrosome reaction using both the P. sativum agglutinin and chlortetracycline fluorescence assays; both assays gave similar results. Sperm incubated with solubilized zonae pellucidae at a final concentration of 2, 4, or 6 ZP/microliter underwent acrosomal exocytosis to a similar extent as compared with A-23187. Sperm were incubated with 1 microgram/ml pertussis toxin during capacitation to functionally inactivate the Gi-like protein. Pertussis toxin treatment of sperm did not affect sperm motility and the ability of the cells to bind to structurally intact zonae pellucidae. Pertussis toxin, however, completely inhibited the percentage acrosome reactions induced by solubilized zonae pellucidae. By contrast, the A-23187-induced acrosome reaction was insensitive to PT treatment. Pertussis toxin inhibition of the zona pellucida-induced acrosome reaction occurred in a concentration-dependent manner with maximal effects observed at 100 ng/ml PT.(ABSTRACT TRUNCATED AT 250 WORDS)     

Evidence for a binding site on the sperm plasma membrane which recognizes the murine zona pellucida: A binding site on the sperm plasma membrane

Murine cauda epididymal sperm preincubated in either a modified Krebs-Ringer or M 199 solution bind to cumulus-free, zona pellucida-intact eggs. Pretreatment of such eggs with an affinity purified preparation of the seminal inhibitor binding component (acceptor), isolated from epididymal sperm, reduces in a concentration dependent manner, the number of sperm that bind. Treatment of cauda sperm, preincubated in either of the above two media, with the seminal inhibitor, also reduces the number of sperm able to bind. Incubation of cauda sperm in the Krebs-Ringer solution for up to 4 h does not affect their ability to bind the seminal inhibitor. Omission of bovine serum albumin from the preincubation medium results in a significant reduction in sperm binding. These data are interpreted to mean that the seminal inhibitor acceptor sites on the sperm surface of incubated sperm function in the in vitro binding to the zona pellucida.     

Ultrastructural localization of labeled acrosomal glycoproteins during in vivo fertilization in the rabbit

Rabbit spermatozoa were labeled predominantely in their acrosomal glycoproteins by 1-³H-glucosamine during spermiogenesis. Ova fertilized in vivo by spermatozoa labeled 22 days earlier were analyzed by fine-structure autoradiography for the localization of the label. The latter was found associated with 1) the fused membranes of the acrosomal cap remaining on the zona pellucida surface, 2) the material released on the zona surface after the acrosome reaction and possibly detectable after tannic acid fixation, 3) the equatorial segment of the sperm head and the preequatorial swellings, and 4) other sperm components, eg, the sperm tail. No labeling, on the other hand, was detected on the denuded leading edge of spermatozoa found either in the penetration slit or in the perivitelline space. Our observations suggest the involvement of acrosomal glycoproteins in different mechanisms of sperm/zona pellucida interaction but are not in favor of a major role of (enzymatic) glycoproteins bound to the inner acrosomal membrane during the penetration of the zona pellucida.     

Sperm membrane physiology and relevance for fertilization

This paper aims to overview recent insights in sperm surface remodelling pertinent to fertilization. A basic understanding of this remodelling is required to interpret the high amount of data appearing from high-throughput identification techniques for proteins presently applied in reproductive biology. From the extensive lists of protein candidates identified by proteomics, only a few are recognized to be directly involved in fertilization. Others are indirectly involved, but many are not yet considered to be involved in fertilization. Some of these newly identified and unexpected proteins may shed new light in the current molecular models for fertilization. However, the gathered lists of sperm proteins possibly involved in fertilization do only tell a part of the story regarding how fertilization is accomplished. When considering the identification of proteins involved in fertilization, one also needs to take into account the fundamental mechanisms involved in the redistribution of sperm surface proteins in membrane protein complexes and the involvement of cell signalling events that regulate their post-translational modification status. Both processes are likely requisite for protein configuration and grouping into functional membrane protein complexes necessary to elicit their delicate roles in fertilization. This paper emphasizes biochemical models for membrane surface modelling and their potential involvement for remodelling the sperm surface in the above described processes.     

猪精子凝集素在精卵结合中的作用机制

猪精子凝集素（ＢＳＬ）位于精子头部,既与精子蛋白结合,亦与透明带糖蛋白ＺＰ３结合。并且ＢＳＬ自身分子间亦会聚合。与ＺＰ３结合的片段亦结合于岩藻素－Ｓｅｐｈａｒｏｓｅ亲和柱,但不与胎球蛋白－Ｓｅｐｈａｒｏｓｅ柱结合。该片段具有较强的抑制精卵结合的活性。ＢＳＬ经ＮＢＳ修饰后,血凝活力大大下降,同时推动与精子结合的活性,但不影响它与ＺＰ３的结合。修饰后的ＢＳＬ亦显著抑制精卵结合。表明ＢＳＬ以两个不同的部     

哺乳动物精子中的ZP3结合蛋白研究进展

哺乳动物卵透明带糖蛋白ZP3(zona pellucida3)是介导精卵初级结合、诱发精子发生顶体反应的关键分子。目前已在精子中发现多种ZP3结合蛋白。95kD酪氨酸激酶受体可能通过其酪氨酸激酶活性介导ZP3诱发的顶体反应。β—1,4—半乳糖基转移酶与ZP3的糖基结合后,通过激活下游信号分子诱发顶体反应。精子蛋白sp56可能介导了顶体反应期间顶体基质与ZP之间的相互作用。透明带粘附素(zonadhesin)也是在顶体反应发生之后才与ZP发生相互作用。这些精子蛋白介导的下游信号事件将是下一步研究的热点。