1、1附件 2论文中英文摘要格式作者姓名：吕利霞论文题目：绵羊和绒山羊 PDIA3 cDNA 的克隆、睾丸组织中的表达及其与IZUMO1 之间相互结合的研究作者简介：吕利霞，女，1983 年 7 月出生，2007 年 9 月师从于内蒙古大学邢万金教授，于 2010 年 7 月获硕士学位。中 文 摘 要哺乳动物精卵融合是一个精卵表面众多分子参与的复杂过程。基因敲除实验证明位于精子表面的具有二硫键的免疫球蛋白超家族成员 IZUMO1 是精卵融合的必须因子。体外抑制剂和抗体抑制实验表明蛋白二硫键异构酶（Protein disulfide-isomerase，PDI）家族的部分成员也能影响精卵融合，其中
2、PDIA3 最为重要。但目前尚不知道 IZUMO1 和 PDIA3 影响精卵融合的分子机制。本实验拟克隆绒山羊和绵羊 Pdia3 cDNA，结合本实验室已经克隆的绒山羊和绵羊 Izumo1 基因，探索 PDIA3 能否与 IZUMO1 结合，共同参与精卵融合。实验首先从绵羊和绒山羊睾丸组织中提取总 RNA，根据牛 Pdia3 cDNA 序列设计引物，用 RT-PCR 方法分别成功克隆了绵羊和绒山羊的 Pdia3 cDNA。克隆得到的绵羊 Pdia3 cDNA序列为 1660 bp，绒山羊的 Pdia3 序列为 1591bp，两者的编码区（ CDS）均为 1518 bp，编码505 个氨基酸，两
3、者的核酸和氨基酸序列完全相同，与牛、人、大鼠和小鼠的相似性分别是99.21%、95.05%、89.50% 和 90.89%。将 GenBank 和 Ensembl 数据库中公布的 31 种动物的PDIA3 的完整氨基酸序列与我们克隆的绵羊和绒山羊 PDIA3 氨基酸序列进行了比对分析，结果发现 PDIA3 在不同种动物中的同源性很高，部分基序（包括 CGHC）在所有物种中绝对保守。将克隆得到的绵羊 Pdia3 CDS 全长序列与表达载体 pGEX-4T-1 连接构建表达载体pGEX-Pdia3，转入大肠杆菌 BL21 (DE3) 中，用 IPTG 成功诱导表达出 GST-PDIA3 融合蛋白，
4、经亲和层析和切胶纯化后得到较高纯度的 GST-PDIA3 融合蛋白，以此作为免疫原免疫昆明（KM ）小鼠。四次加强免疫后向小鼠腹腔注射 S180 细胞，成功地制备出小鼠抗绵羊GST-PDIA3 腹水多克隆抗体，经 Western blot 检测，所制备的腹水多克隆抗体能够特异性地识别 PDIA3 蛋白和 GST 蛋白。我们用戊二醛交联法对抗体进行了纯化，去除了其中识别2GST 的抗体，纯化后的抗体仍然能够识别 PDIA3 蛋白。用纯化后的腹水多克隆抗体和绵羊睾丸组织石蜡切片进行免疫组织化学检测，结果显示在绵羊睾丸组织中 PDIA3 普遍存在于从精原细胞到次级精母细胞的细胞质中，并且在 leyd
5、ig 细胞中也表达。精子免疫组织化学检测发现 PDIA3 位于绒山羊精子的赤道区。RNA 原位杂交结果显示 Pdia3 基因在精原细胞、初级精母细胞以及次级精母细胞中转录。本实验还将真核表达载体 BiFC-VC155-PDIA3 和 BiFC-VN173-IZUMO1 共转染人 Hela细胞，收集细胞总蛋白，用免疫共沉淀和 Western blot 首次证明 PDIA3 与 IZUMO1 在细胞中能相互结合。关键词：绵羊；绒山羊；PDIA3；基因克隆；IZUMO1 Molecular cloning and characterization of PDIA3 from sheep and ca
6、shmere goat, identification of its expression in testis and binding to IZUMO1Lv Li-Xia ABSTRACTGamete fusion is a complex process which mediated by many molecules in the sperm and egg membrane. It has been established in knockout mice that IZUMO1, a novel member of immunoglobulin superfamily (IgSF)
7、located on the sperm, is crucial for sperm-egg fusion. Recent studies found that inhibitors of protein disulfide isomerases (PDIs) and specific antibodies against PDIs were able to inhibit sperm-egg fusion in vitro, suggesting that PDIA3 plays an important role in gamete fusion, but the mechanism of
8、 IZUMO1 and PDIA3 in the gamete fusion is still unkown. Our laboratory had cloned sheep and cashmere goat Izumo1 gene, now we cloned the Pdia3 cDNA from sheep and cashmere goat for identification of the potential interaction between PDIA3 and IZUMO1.We first extracted the total RNA from sheep and ca
9、shmere goat testis tissue and designed primers according to the cDNA sequence of bovine Pdia3 gene. The Pdia3 cDNA of sheep and goat were amplified successfully by RT-PCR. The cloned sheep and cashmere goat Pdia3 cDNA are 31660 bp and 1591 bp, respectively. Bioinformatics analysis showed that sheep
10、and goat Pdia3 cDNA have the same nucleotide sequence encoding the same 505 amino acides, with high homologies to those of bovine (99.21%), human (95.05%), rat (89.50%) and mouse (90.89%). We aligned PDIA3 amino acids sequences from 31 species animals released in GeneBank and Ensembl databases with
11、our cloned sheep and cashmere goat PDIA3, the result showed that the PDIA3 of different species share high homology, some mitifs, including the CGHC，are absolutely conserved. Then we inserted the Pdia3 CDS into the prokaryotic expression vector pGEX-4T-1, and expressed GST-PDIA3 fusion protein in E.
12、 coli BL21(DE3). The GST-PDIA3 fusion protein was purified by Glutathione-Sepharose 4B column, and was used as an antigen to immunize the Kunming (KM) mouse. The ascites polyclonal antibody against sheep GST-PDIA3 was harvested after four times immunization followed by celiacly injection of S180 cel
13、ls. Western blot analysis showed that the polyclonal antibody can specifically recognize both PDIA3 and the GST tag. Then the antibody was purified by glutaraldehyde cross linking method to remove the anti-GST component，leaving the polyclonal antibody with much higher specificity to PDIA3.The PDIA3
14、was detected to be expressed in cytoplasm of spermatogenic cells from spermatocytes to the prespermatid phases as well as in leydig cells of sheep testis by immunohistochemistry. It is also found in the equatorial segment of sperm. The results of RNA in situ hybrization showed that the Pdia3 gene tr
15、anscribes in the spermatogonium、spermiocytes and prespermatid.Finally, we transiently cotransfected eukaryotic expression vectors pBiFC-VC155-PDIA3 and pBiFC-VN173-IZUMO1 into the human HeLa cells, and the binding of PDIA3 to IZUMO1 was proved by Co-immunoprecipitation and Western blot analysis.Key words: sheep; cashmere goat; PDIA3; gene cloning; IZUMO1
