人精子蛋白SP22单克隆抗体杂交瘤细胞株的建立及临床应用

发布时间：2018-11-24 12:47

【摘要】：精子蛋白22(sperm protein 22，SP22)是一种由常染色体的持家基因编码的蛋白。大鼠SP22主要分布于心、肺、脑、肝、肾、前列腺、睾丸、输精管、附睾等组织，，以睾丸中含量最多。同样，人各组织均表达SP22，表达量较多的组织是心脏、肌肉组织、胰腺、垂体、肾上腺、甲状腺和唾液腺等。在精子发生过程中，SP22 mRNA在粗线期精母细胞、次级精母细胞和圆形精子期均有转录，但其蛋白却在精子发生后期出现。SP22作为一种新的生殖相关蛋白，既具有酶活性，也具有调节因子和致癌因子的特性。精子表面SP22能与透明带结合，辅助精子穿透卵细胞直接参与顶体反应。若建立SP22的免疫学检测方法并探讨其在男性生殖系统中的具体功能、作用机制以及与男性不育的关系等，就必须得到大量的纯化SP22抗原和相应的抗体。本研究第一部分，应用已有的重组菌株诱导表达。并用镍离子亲和层析柱对重组SP22蛋白进行纯化，行SDS-PAGE鉴定，结果获得了高纯度较的SP22重组蛋白。第二部分，采用杂交瘤技术将无分泌功能的骨髓瘤细胞SP2／0与分泌SP22抗体的小鼠脾细胞融合成杂交瘤细胞，利用瘤细胞无限增殖及免疫细胞分泌抗体的能力，大量制备针对SP22抗原的单克隆抗体(McAb)。首先以重组SP22作为抗原免疫BALB／c小鼠，免疫成功后取小鼠脾细胞与小鼠骨髓瘤细胞SP2／0融合，通过阳性克隆有限稀释法，得到3株IgG1类SP22 McAb的杂交瘤细胞株；再将生长良好的杂交瘤细胞接种到石蜡油预处理的BALB／c小鼠腹腔中，制备腹水型抗人SP22 McAb。酶联免疫吸附试验(ELISA)证明混合腹水的滴度可达1∶1000～1∶3200；Western印迹鉴定表明该McAb能特异性地结合精子膜蛋白中分子量(Mr)为22000的SP22。
[Abstract]:Sperm protein 22 (SP22) is a protein encoded by autosomal housekeeping genes. SP22 was mainly distributed in heart, lung, brain, liver, kidney, prostate, testis, vas deferens and epididymis. Similarly, the tissues in which the expression of SP22, was higher in human tissues were heart, muscle, pancreas, pituitary gland, adrenal gland, thyroid gland and salivary gland. During spermatogenesis, SP22 mRNA was transcribed in spermatocytes, secondary spermatocytes and round spermatozoa, but its protein appeared in the late stage of spermatogenesis. SP22, as a new reproductive related protein, has enzyme activity. It also has the characteristics of regulatory factors and carcinogenic factors. Sperm surface SP22 can bind to the pellucida and assist sperm to penetrate the egg cells directly to participate in acrosome reaction. If the immunological detection method of SP22 is established and its specific function, mechanism and relationship with male infertility are discussed, a large number of purified SP22 antigens and corresponding antibodies must be obtained. In the first part of this study, the recombinant strain was used to induce expression. The recombinant SP22 protein was purified by nickel ion affinity chromatography and identified by SDS-PAGE. A high purity recombinant SP22 protein was obtained. In the second part, hybridoma technique was used to fuse non-secreting myeloma cells (SP2/0) with murine spleen cells secreting SP22 antibodies to form hybridoma cells, and to utilize the ability of tumor cells to proliferate and immune cells to secrete antibodies. Preparation of Monoclonal Antibody (McAb). Against SP22 Antigen in large quantities At first, BALB/c mice were immunized with recombinant SP22 as antigen. After successful immunization, the spleen cells of mice were fused with mouse myeloma cells SP2/0, and three IgG1 SP22 McAb hybridoma cell lines were obtained by positive clone limited dilution method. Then the well-growing hybridoma cells were inoculated into the peritoneal cavity of BALB/c mice pretreated with paraffin oil to prepare ascites anti-human SP22 McAb.. Enzyme-linked immunosorbent assay (ELISA) proved that the titer of mixed ascites was 1: 1000 / 320. Western blot indicated that the McAb could specifically bind to SP22. with a molecular weight of 22000 in sperm membrane protein.
【学位授予单位】：南京医科大学
【学位级别】：硕士
【学位授予年份】：2006
【分类号】：R392

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