人睾丸特异性新基因hTSC29合成肽单克隆抗体制备与鉴定
发布时间：2019-06-10 05:58
【摘要】：目的:制备和初步鉴定人睾丸特异性新基因h TSC29(Gen Bank登录号:BC032957)合成肽的单克隆抗体(m Ab)。方法:利用生物信息学方法对h TSC29的蛋白质序列进行分析和预测,根据免疫原性、表露性、亲疏水性及柔韧性4个参数,合成一段由18个氨基酸组成的多肽,并以此为抗原免疫BALB/c小鼠,取其脾细胞和小鼠骨髓瘤细胞融合后,通过有限稀释法筛选阳性克隆,采用ELISA法、Western blot、免疫组化和免疫荧光法鉴定h TSC29 m Ab的特异性。结果:获得了1株稳定分泌h TSC29 m Ab的杂交瘤细胞株,分泌的抗体属Ig G2b(κ)亚型,杂交瘤细胞培养上清效价达到1∶104;Western blot鉴定表明,制备h TSC29 m Ab可特异性地识别人睾丸总蛋白中Mr约为60 000处的蛋白;免疫组织化学和免疫荧光结果显示h TSC29蛋白主要定位在正常人睾丸组织中的精母细胞和精子细胞。结论:成功建立了1株稳定分泌抗h TSC29 m Ab的杂交瘤细胞株,其分泌的抗体效价高、特异性强,为进一步研究h TSC29的功能提供了特异的检测工具。
[Abstract]:Objective: to prepare and identify the monoclonal antibody (m Ab). Against the synthetic peptide of human testicular specific gene h TSC29 (Gen Bank accession number: BC032957. Methods: the protein sequence of h TSC29 was analyzed and predicted by bioinformatics method. According to the four parameters of immunogenicity, disclosure, hydrophilicity and flexibility, a polypeptide composed of 18 amino acids was synthesized. BALB/c mice were immunized with this antigen. After fusion of spleen cells and mouse myeloma cells, the positive clones were screened by finite dilution method. The specificity of h TSC29 m Ab was identified by ELISA, Western blot, immunohistochemistry and immunofluorescence. Results: a hybridoma cell line stably secreting h TSC29 m Ab was obtained. The antibody secreted belonged to Ig G2b (k) subgroup, and the titer of hybridoma cell culture was 1 鈮，
本文编号：2496226
[Abstract]:Objective: to prepare and identify the monoclonal antibody (m Ab). Against the synthetic peptide of human testicular specific gene h TSC29 (Gen Bank accession number: BC032957. Methods: the protein sequence of h TSC29 was analyzed and predicted by bioinformatics method. According to the four parameters of immunogenicity, disclosure, hydrophilicity and flexibility, a polypeptide composed of 18 amino acids was synthesized. BALB/c mice were immunized with this antigen. After fusion of spleen cells and mouse myeloma cells, the positive clones were screened by finite dilution method. The specificity of h TSC29 m Ab was identified by ELISA, Western blot, immunohistochemistry and immunofluorescence. Results: a hybridoma cell line stably secreting h TSC29 m Ab was obtained. The antibody secreted belonged to Ig G2b (k) subgroup, and the titer of hybridoma cell culture was 1 鈮，
本文编号：2496226
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