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目的:构建人谷丙氨酸氨基转移酶(ALT1)的原核表达载体,并用纯化的ALT1重组蛋白免疫家兔,制备ALT1抗血清.方法:从HepG2细胞中提取总RNA,RT-PCR扩增alt1基因,并将其克隆入pMD19-T载体中测序,将测序正确的目的基因克隆至PET-32a(+)表达载体中,并诱导其在大肠杆菌BL21(DE3)中表达;所获得的包涵体蛋白经亲和层析纯化、透析复性、Western Blot鉴定后,免疫家兔制备抗血清,抗体效价和特异性分别采用ELISA,Western Blot进行检测.结果:测序证实克隆的基因序列与GenBank中的ALT1序列相符;SDS-PAGE,Western blot结果证实获得Mr为75×103的ALT1融合蛋白,其表达形式为不溶性包涵体;此包涵体蛋白经Ni2+亲和层析能有效纯化,以该蛋白免疫家兔制备抗血清,抗体效价为1∶100000,Western Blot检测证实该抗体能与目的蛋白发生特异性结合.结论:获得了ALT1重组蛋白及特异性多克隆抗体.
Objective: To construct prokaryotic expression vector of human alanine aminotransferase (ALT1) and immunize rabbit with purified recombinant ALT1 protein to prepare ALT1 antiserum.Methods: Total RNA was extracted from HepG2 cells and amplified by RT-PCR The gene was cloned into pMD19-T vector and sequenced. The correct target gene was cloned into PET-32a (+) expression vector and induced to express in E. coli BL21 (DE3). The obtained inclusion body The protein was purified by affinity chromatography, dialyzed and refolded, and identified by Western Blot, the rabbits were immunized to prepare antiserum.The antibody titers and specificities were detected by ELISA and Western Blot respectively.Results: Sequencing confirmed that the cloned gene sequence was identical with GenBank The ALT1 fusion protein was confirmed to be 75 × 103 by SDS-PAGE and Western blot. The fusion protein was expressed as insoluble inclusion body. The inclusion protein was effectively purified by Ni2 + affinity chromatography. Rabbit prepared antiserum with antibody titer of 1: 100,000, Western Blot analysis confirmed that the antibody could specifically bind with the target protein.Conclusion: ALT1 recombinant protein and specific polyclonal antibody were obtained.