【作者】 单文娟；

【导师】 张富春；

【作者基本信息】 新疆大学 ， 动物学， 2004， 硕士

【摘要】 所有哺乳动物的卵膜外都由一层相对较厚的胞外物质即透明带（zona pellucida ZP）所包围。透明带是初级卵母细胞成熟过程中，在早期由卵母细胞和初级卵母细胞分泌而形成的一层含糖蛋白的嗜酸性膜。鼠透明带由ZP1,ZP2, ZP3三种硫酸化的糖蛋白交联在一起，构成一个网状结构，包围着完全成熟的卵子，保护着卵子和着床前的胚胎。更重要的是鼠ZP3在受精过程中还起着精子受体及精子结合以后诱导精子产生顶体反应的作用。实验证明，把精子和透明带糖蛋白预先培养可以抑制小鼠精子和透明带的结合，并且在透明带的三种糖蛋白成分中，ZP3是这个抑制实验的竞争物；而且抗ZP3的单克隆抗体在体内也可以有效的抑制受精过程的进行，所以，在以ZP3为特异性抗原，运用免疫手段来阻断透明带的功能，降低免疫动物受精率的过程中，表达并制备ZP3抗原对制造抗生育疫苗从而控制新疆特有害鼠种--草原兔尾鼠（Lagurus lagurus）鼠害的研究有着十分重要的意义。毕赤巴斯德酵母(Pichia pastoris)表达系统是最近迅速发展起来的一种高效的真核表达体系，用此系统成功地表达了多种蛋白和多肽，它具有真核细胞翻译后的加工和修饰功能，并且能将所表达的蛋白产物分泌到细胞外的培养基中，因此大大简化了表达产物的回收和纯化过程。本研究的目的就是在于利用Pichia pastoris真核酵母表达系统进行草原兔尾鼠卵明带3（ lZP3）的分泌型表达，并对所表达的蛋白进行免疫原性的检测和部分理化性质的分析。首先构建用于真核酵母表达草原兔尾鼠卵明带3蛋白的重组穿梭质粒pSuperY-lZP3。根据本实验室克隆并在GenBank上注册（注册号为AF515621）的草原兔尾鼠ZP3基因序列设计引物，通过PCR获得lZP3基因的核心片段，用DNA重组定向克隆技术将草原兔尾鼠透明带3（lZP3）目的基因片断插入到真核表达载体pSuperY上，根据选择标记的抗性基因（Kana）筛选到阳性克隆，培养、小提、纯化后，通过限制性内切酶酶切分析初步证明阳性克隆为连接了lZP3目的基因的重组质粒。将质粒pSuperY-lZP3进行序列测<WP=5>定，用DNAMAN软件与在GenBank上已发表的lZP3序列进行比较，进一步证明pSuperY-lZP3构建的正确性，没有发生碱基突变，并且读码框正确。这说明构建的pSuperY-lZP3质粒可以作为真核表达载体，进行下一步草原兔尾鼠卵透明带3蛋白在真核酵母中的表达研究。将构建在真核表达质粒pSuperY-lZP3上的lZP3核心片段在酵母受体菌SMD1168中进行表达。表达质粒用AvrII线性化并经琼脂糖电泳检测线性化完全后，以电击的方法导入受体菌中。整合到酵母基因组中的阳性克隆经Zeocin平板筛选，挑取单菌落在30oC小瓶发酵培养2-3d，取样品上清做SDS-PAGE分析，胶上显示我们得到了约55kD的有适量糖基化的目的蛋白。大量培养表达后，以本实验室自制的以原核表达蛋白为抗原制备的兔多克隆抗血清为第一抗体，进行Western blot检测，得到与SDS-PAGE检测完全一致的条带，证明草原兔尾鼠卵明带3蛋白在真核酵母系统中得到了特异性表达。所获得的目的蛋白直接从SDS-PAGE胶上切下，研磨定量后溶于PBS中，与等体积弗氏佐剂充分混匀免疫新西兰大白兔，得到大量兔多克隆抗血清，利用ELISA和Western blot对抗血清进行检测，得到的特异性的抗血清效价在1：50000以上。为了检测真核酵母表达的草原兔尾鼠卵透明带3蛋白是否具有免疫原性，以未免疫组、pCMV4-lZP3 DNA疫苗免疫组为对照组，以真核酵母表达的lZP3蛋白免疫组和DNA免疫与蛋白免疫相结合的联合免疫组为实验组，对NIH雌性小鼠进行免疫来验证所得酵母表达蛋白是否能使小鼠产生抗生育功能。方法是：DNA免疫疫苗pCMV4-lZP3为本实验室构建并验证其功能正确，于小鼠腿部肌肉三点注射；蛋白免疫是将真核表达的蛋白与等体积弗氏（不）完全佐剂混匀皮下多点注射；联合免疫第一次免疫用DNA疫苗肌肉注射，后两次加强免疫为蛋白皮下多点注射，两周后与成熟雄鼠合笼，未免疫组雌鼠直接与成熟雄鼠合笼，每次免疫前采血，并通过ELISA方法检测小鼠体内的抗体水平。结果显示，lZP3疫苗免疫在小鼠体内产生了相应的抗体，且随着免疫次数的增加抗体水平也有所增高。合笼实验结果表明阳性对照DNA免疫组获得了83.3%的不孕率，实验组中，蛋白免疫组不孕率为87.5%，联合免疫获得了100%的不孕率，而未免疫的阴性对照组的怀孕率为100%。说明我们所得到的真核表达蛋白有很强的免疫原性，可以大规模的制备用作今后的免疫不育控制<WP=6>鼠害研究的免疫抗原物质。将真核酵母表达的草原兔尾鼠卵透明带3（lZP3）蛋白进行硫酸铵沉淀、透析等初步纯化并冷冻干燥浓缩后，利用凝胶等电点聚焦（Mini-IEF）技术对lZP3蛋白的等电点做了初步测定。首先向存在于酵母培养基上清内的lZP3蛋白中依次加入20%、40%、60%、80%饱和度硫酸铵溶液，摸出分离lZP3蛋白的最佳硫酸铵饱和度，将经硫酸铵沉淀的lZP3蛋白样品在20mM的Tris-HCL中透析过夜除去盐离子后，冷冻干燥浓缩样品。利用DNAMAN软件预测lZP3核心片段的等电点及疏水性并用等电点聚焦电泳测定其等电点。实验结果如下：以SDS-PAGE确定80%硫酸铵饱和度时分离真核酵母表达的lZP3蛋白效果最佳，样品经透析除去盐离子后，冷?更多还原

【Abstract】 Zona pellucida (ZP), a complex matrix surrounding oocytes, is comprised of three immunologically distinct glycoproteins (ZP1, ZP2, ZP3). Zona pellucida 3 (ZP3) is a major glycoprotein of the zona pellucida that possesses the sperm receptor function and the acrosome-inducing activity. It induces antibody that can block sperm/oocyte interaction. So ZP3 has long been used as a candidate antigen to develop an immunocontraceptive vaccine. One of the yeast -- Pichia pastoris has been developed to be an outstanding host for the production of foreign proteins. It offers many advantages, for example, it does not have the endotoxin problem associated with bacteria nor the viral contamination problem of proteins produced in animal cell culture. Furthermore, P. pastoris expression system has strong promoter that provides high expresstion. The proteins produced in P. pastoris are typically folded correctly and secreted into the medium. A number of proteins have been produced using this system, such as tetanus toxin fragment, human serum albumin and lysozyme. This study mainly aimed at the expression of Lagurus lagurus zona pellucida3 (lZP3) in Pichia pastoris system, and its purification and characterization. Firstly, a pair of primers were designed according to the lZP3 gene constructed in our lab (GenBank NumberAF515621). The core fragment of lZP3 was amplified by PCR. Then the target gene and the vector pSuperY were linked together at two restrictive sites: EcoRI and XbalI. After kanamycin screening and mini-preparation, the positive clones were testified with the same restrictive enzymes. In addition, the plasmid was sequenced and blasted in GenBank comfirming the correctness of the plasmid used as an expression vector. Then the expression plasmid was transformed into SMD1168 by electroporation after linearized with AvrII. Zeocin was used to screen positive clones that inserted into yeast genome. A band about 55kD was observed on SDS-PAGE, and then Western blot analysis was conducted furtherly to confirm that the band was specific lZP3 glycoprotein. This expressed protein was then used to immunize animal to produce antiserum. Antiserum against the ZP3 was determined with ELISA and Western blot analysis, and the titer was above 1:50000. In order to test immunological activity of the Lagurus lagurus zona pellucida3 (lZP3) expressed in Pichia pastoris, female NIH mice were immunized with lZP3. The NIH <WP=8>mice were chosen and enlisted to 4 groups randomly, and were received immunization with three kinds of vaccine: pCMV4-lZP3 DNA vaccine, lZP3 protein vaccine, pCMV4-lZP3 DNA and lZP3 protein co-immunization. Each injection, per mouse received 100ug/100ul of pCMV4-lZP3 at hind leg or 100ug/100ul lZP3 protein with Freund’s adjuvant under skin. Sera were collected from the postorbital vein after each immunization. lZP3 antiserum levels were detected by ELISA. 5 weeks later, nonimmunized male mice were introduced to immunized females and allowed to mate with them. The result was that the levels of lZP3 anti-serum in mice were rising with the increasing of the immunization times. 83.3%, 87.5%, 100% infertility rate was showed in DNA, protein and co-immunization, respectively, while it was 0% in non-immunized group. There was a significant difference between the number of pups per litter in the immunization group and in the control group (p(0.01). These results encourage us to further ZP3 immunocontraceptive function study and ZP3 vaccine development.In this study, isoelectric point of Lagurus lagurus zona pellucida3 (lZP3) protein expressed in Pichia pastoris was also assayed by Mini-IEF with Model 111 Mini-IEF Cell. Preliminarily purifying of lZP3 was performed with sulfate and dialysis, and 80% sulfate was suitable for separating lZP3 protein. After dialysis in 20mM Tris-HCL overnight to eliminate sulfate, the purified protein was added to the IEF gel to analysis its isoelectric point. A band between pI 6.0 and 7.0 was seen in the gel. This result was consistent with predicted pI (6.34) of lZP3. Put these更多还原