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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。
OBJECTIVE: To prepare and preliminarily identify the monoclonal antibody (m Ab) of synthetic peptide of human testis-specific new gene h TSC29 (Gen Bank accession number: BC032957). Methods: The protein sequence of h TSC29 was analyzed and predicted by bioinformatics method. A polypeptide consisting of 18 amino acids was synthesized according to the four parameters of immunogenicity, apparent property, hydrophilicity and hydrophobicity and flexibility. BALB / c mice were immunized and the spleen cells were fused with mouse myeloma cells. The positive clones were screened by limiting dilution. The specificity of h TSC29 m Ab was identified by ELISA, Western blot, immunohistochemistry and immunofluorescence . Results: One hybridoma cell line stably secreting h TSC29 m Ab was obtained. The secreted antibody was Ig G2b (κ) subtype. The titer of hybridoma cell culture supernatant reached 1:104. Western blot showed that h TSC29 m Ab specifically recognized about 60 000 Mr in total human testis protein; immunohistochemistry and immunofluorescence results showed that h TSC29 protein mainly localized in spermatocytes and sperm cells in normal human testicular tissue. CONCLUSION: A hybridoma cell line stably secreting anti-h TSC29 m Ab has been successfully established. The secreted antibody has high titer and high specificity, which provides a specific tool for further study on the function of h TSC29.