一个新的人信号识别微粒受体基因APMCF1的克隆、表达和抗体制备

Cloning, expression and preparation of polyclonal antibody of APMCF1, a novel human gene of signal recognition particle receptor

目的 :克隆人类新基因APMCF1 ,原核表达并制备其特异性多克隆抗体 .方法 :运用 5′末端快速扩增方法 ,结合Genbank数据库进行电子拼接 ,完善APMCF1的 5′端序列 ;利用RT PCR方法 ,扩增出APMCF1的蛋白编码cDNA序列 ,克隆入pGEX KG中 ,构建APMCF1的原核表达载体 ,在大肠杆菌中诱导表达后获得了GST APMCF1融合蛋白 ,以此融合蛋白为免疫原制备兔抗血清 ,用Westernblot鉴定抗体特异性 .结果 :通过RACE及电子拼接 ,完善了APMCF1的 5′端序列 ,其cDNA长度为 1 74 5bp,与鼠信号识别微粒受体 β亚基序列同源性较高 ,染色体定位于 3q2 2 .2 ;克隆了APMCF1 81 6bp的开放读框序列并表达了GST APMCF1融合蛋白 .制备了特异性兔抗GST APMCF1多克隆抗体 .结论 :APMCF1可能是编码人类信号识别微粒受体β亚基的基因 .该分子多克隆抗体的初步制备 。

AIM: To clone the human novel gene APMCF1 and to express and prepare its specific polyclonal antibody. METHODS: With the method of 5′rapid amplification of cDNA end (RACE) and electronic splicing in Genbank, we extended the length of APMCF1 at 5′end. Then the sequence encoding the APMCF1 protein was amplified by RT PCR from the total RNA of apoptotic MCF 7 cells and was cloned into the prokaryotic expression vector pGEX KG so as to construct the recombinant expression vector pGEX APMCF1. The protein was expressed in E. coli and the GST APMCF1 fusion protein was used to immunize rabbits to get the rabbit anti APMCF1 serum. The specificity of the ployclonal anti APMCF1 antibody was determined by Western blot. RESULTS: We got a cDNA fragment with the length of 1745 bp. It was homologous to mouse signal recognition particle receptor β,(SRβ). Chromosome location was at 3q22.2 and the coding region covered 816 bp. GST APMCF1 fusion protein was expressed successfully. Preliminary western blot results indicated that the ployclonal anti APMCF1 antibody showed high specificity against APMCF1. CONCLUSION: APMCF1 may be the gene coding for human signal recognition particle receptor β. Preliminary preparation of polyclonal antibody of human novel gene APMCF1 is of help in the further study of this gene.