CML66基因的克隆、原核表达及多克隆抗体的制备

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。

Cloning, prokaryotic expression of CML66 and preparation of its polyclonal antibody

AIM: To clone and express CML66 cDNA and to prepare rabbit anti-CML66 antibody. METHODS: cDNA isolated from the testis using RT-PCR was cloned into pGEMT. After sequencing, the cDNA was inserted into prokaryotic expression vector pET32b ( ). The recombinant vector was transformed into BL-21(DE3) through eiectroporation. 6 x His-tagged CML66 expression was then induced by IPTG. The protein was purified through Ni2 affinity chromatogra-phy column and characterized by SDS-PAGE and Western blot. The purified protein was injected into rabbits to prepare polyclonal antibody. RESULTS: The cloned cDNA sequence was identical with that previously reported. The target protein was successfully purified. And rabbit's anti serum with high titer was obtained. CONCLUSION: We have cloned CML66 successfully, expressed and purified the protein in E. coll Furthermore, rabbit polyclonal antibody has been obtained.