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目的:获得原核表达的新的肿瘤抗原CML66蛋白,并制备兔多克隆抗体。方法:采用RT-PCR技术从睾丸中获得cML66的cDNA,亚克隆至pGEMT载体中,经测序确证后,将该基因插入原核表达载体pET32b(+)中,通过电穿孔技术转化E.coli表达菌BL21(DE3),以IPTG诱导6×His融合蛋白的表达,并经Ni2+亲和柱层析纯化。通过SDS-PAGE、Westernblot鉴定后,应用纯化蛋白免疫家兔,制备多克隆抗体。结果:获得的CML cDNA序列与GenBank登录的cDNA序列 一致。用纯化的目的蛋白免疫家兔后,获得高滴度的特异性兔抗血清。结论:成功地克隆CML66基因,建立了原核表达、纯化体系。制备纯化的CML66蛋白和高滴度、特异的兔抗血清。
Objective: To obtain the prokaryotic expression of a new tumor antigen CML66 protein, and prepare rabbit polyclonal antibody. Methods: cDNA of cML66 was obtained from testis by RT-PCR and subcloned into pGEMT vector. After sequencing, the gene was inserted into prokaryotic expression vector pET32b (+) and transformed into E.coli by electroporation BL21 (DE3). The expression of 6 × His fusion protein was induced by IPTG and purified by Ni2 + affinity column chromatography. After identification by SDS-PAGE and Western blot, the purified protein was used to immunize rabbits to prepare polyclonal antibodies. Results: The cDNA sequence of CML obtained was consistent with that of GenBank. After the rabbit was immunized with the purified protein of interest, a high titer of specific rabbit antiserum was obtained. Conclusion: The CML66 gene was successfully cloned and the prokaryotic expression and purification system was established. Purified CML66 protein and high titer, specific rabbit antiserum were prepared.