人精子蛋白SP22單克隆抗體雜交瘤細(xì)胞株的建立及臨床應(yīng)用
發(fā)布時(shí)間：2018-11-24 12:47
【摘要】：精子蛋白22(sperm protein 22，SP22)是一種由常染色體的持家基因編碼的蛋白。大鼠SP22主要分布于心、肺、腦、肝、腎、前列腺、睪丸、輸精管、附睪等組織，，以睪丸中含量最多。同樣，人各組織均表達(dá)SP22，表達(dá)量較多的組織是心臟、肌肉組織、胰腺、垂體、腎上腺、甲狀腺和唾液腺等。在精子發(fā)生過程中，SP22 mRNA在粗線期精母細(xì)胞、次級(jí)精母細(xì)胞和圓形精子期均有轉(zhuǎn)錄，但其蛋白卻在精子發(fā)生后期出現(xiàn)。SP22作為一種新的生殖相關(guān)蛋白，既具有酶活性，也具有調(diào)節(jié)因子和致癌因子的特性。精子表面SP22能與透明帶結(jié)合，輔助精子穿透卵細(xì)胞直接參與頂體反應(yīng)。若建立SP22的免疫學(xué)檢測(cè)方法并探討其在男性生殖系統(tǒng)中的具體功能、作用機(jī)制以及與男性不育的關(guān)系等，就必須得到大量的純化SP22抗原和相應(yīng)的抗體。 本研究第一部分，應(yīng)用已有的重組菌株誘導(dǎo)表達(dá)。并用鎳離子親和層析柱對(duì)重組SP22蛋白進(jìn)行純化，行SDS-PAGE鑒定，結(jié)果獲得了高純度較的SP22重組蛋白。 第二部分，采用雜交瘤技術(shù)將無(wú)分泌功能的骨髓瘤細(xì)胞SP2／0與分泌SP22抗體的小鼠脾細(xì)胞融合成雜交瘤細(xì)胞，利用瘤細(xì)胞無(wú)限增殖及免疫細(xì)胞分泌抗體的能力，大量制備針對(duì)SP22抗原的單克隆抗體(McAb)。首先以重組SP22作為抗原免疫BALB／c小鼠，免疫成功后取小鼠脾細(xì)胞與小鼠骨髓瘤細(xì)胞SP2／0融合，通過陽(yáng)性克隆有限稀釋法，得到3株IgG1類SP22 McAb的雜交瘤細(xì)胞株；再將生長(zhǎng)良好的雜交瘤細(xì)胞接種到石蠟油預(yù)處理的BALB／c小鼠腹腔中，制備腹水型抗人SP22 McAb。酶聯(lián)免疫吸附試驗(yàn)(ELISA)證明混合腹水的滴度可達(dá)1∶1000～1∶3200；Western印跡鑒定表明該McAb能特異性地結(jié)合精子膜蛋白中分子量(Mr)為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.
【學(xué)位授予單位】：南京醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2006
【分類號(hào)】：R392
本文編號(hào)：2353769
[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.
【學(xué)位授予單位】：南京醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2006
【分類號(hào)】：R392
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