【摘要】 目的:通过制备抗天冬酰胺合成酶(ASNS)单克隆抗体(mAb),检测ASNS在不同肿瘤中的表达,探索其表达的意义。方法:诱导表达融合蛋白MS2-ASNS,以纯化的MS2-ASNS蛋白免疫BALB/c小鼠,取脾细胞与Sp2/0融合,以融合蛋白MS2-ASNS和MS2-PAI通过间接ELISA双筛和有限稀释克隆化,获得稳定分泌抗ASNS mAb的杂交瘤细胞株。间接ELISA和Western blot方法对抗ASNS mAb效价、特异性及亲和力进行鉴定。IHC技术检测ASNS在肿瘤细胞株及肿瘤组织中的表达。结果:获得2株能高效分泌特异性抗ASNS mAb的杂交瘤细胞株F4-15、F4-16,亚类均为IgG2a。间接ELISA结果表明制备的抗体有很好的特异性,抗体效价均达到1∶5×105。Western blot鉴定抗ASNS mAb能特异性识别肿瘤细胞株中的ASNS。IHC技术检测天然ASNS在胃癌细胞株SGC-7901和肝癌细胞株SMMC-7721、BEL-7402、HepG2及肺癌、食管癌组织中均为胞质表达。结论:制备的抗ASNSmAb特异性强,可检测ASNS在多种肿瘤中的表达,为进一步研究ASNS在特定肿瘤如中线鼻/鼻型NK/T细胞淋巴瘤中的表达奠定了基础。更多还原

【Abstract】 AIM:To prepare and characterize monoclonal antibodies against ASNS for the research of ASNS function.METHODS: To induce the expression of the fusion protein MS2-ASNS,Hybridomas were generated by the fusion with Sp2/0 myelomas and spleen cells,which were obtained from mice immunized with MS2-ASNS recombinant proteins.The specificity and titer of mAb were identified by ELISA methods,and then used to detect the affinity of ASNS in cancer cells by Western blot.The expression of ASNS was detected in some cancer lines and tissues by IHC.RESULTS: Two hybridoma cell lines F4-15 and F4-16,which stably secret anti-ASNS mAbs were produced.Both cell lines produce IgG2a monoclonal antibody.ELISA demonstrated that anti-ASNS mAbs had high specificity and titer.The titers of anti-ASNS mAbs produced by hybridoma cell lines were up to 1∶5×105.Western blot demonstrated that ASNS was expressed in some cancer lines,including human lymphoma cell line K562 and cervical cancer cell line HeLa.The expression of ASNS was also detected by IHC in several tumor cell lines,such as stomach cancer cell line SGC-7901 and liver cancer cell lines SMMC-7721,BEL-7402,HepG2 as well as lung and esophageal carcinoma tissue.CONCLUSION: The monoclonal antibodies against ASNS have been successfully prepared,which provides a tool for the following research of nasal type NK/T cell lymphoma.更多还原