【摘要】：目的:制備和初步鑒定人睪丸特異性新基因h TSC29(Gen Bank登錄號(hào):BC032957)合成肽的單克隆抗體(m Ab)。方法:利用生物信息學(xué)方法對(duì)h TSC29的蛋白質(zhì)序列進(jìn)行分析和預(yù)測(cè),根據(jù)免疫原性、表露性、親疏水性及柔韌性4個(gè)參數(shù),合成一段由18個(gè)氨基酸組成的多肽,并以此為抗原免疫BALB/c小鼠,取其脾細(xì)胞和小鼠骨髓瘤細(xì)胞融合后,通過有限稀釋法篩選陽性克隆,采用ELISA法、Western blot、免疫組化和免疫熒光法鑒定h TSC29 m Ab的特異性。結(jié)果:獲得了1株穩(wěn)定分泌h TSC29 m Ab的雜交瘤細(xì)胞株,分泌的抗體屬Ig G2b(κ)亞型,雜交瘤細(xì)胞培養(yǎng)上清效價(jià)達(dá)到1∶104;Western blot鑒定表明,制備h TSC29 m Ab可特異性地識(shí)別人睪丸總蛋白中Mr約為60 000處的蛋白;免疫組織化學(xué)和免疫熒光結(jié)果顯示h TSC29蛋白主要定位在正常人睪丸組織中的精母細(xì)胞和精子細(xì)胞。結(jié)論:成功建立了1株穩(wěn)定分泌抗h TSC29 m Ab的雜交瘤細(xì)胞株,其分泌的抗體效價(jià)高、特異性強(qiáng),為進(jìn)一步研究h TSC29的功能提供了特異的檢測(cè)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，

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