斑马鱼心脏标记基因VMHC多抗血清的制备

Preparation of Polyclonal Antiserum of Heart Marker Gene VMHC in Zebrafish

斑马鱼基因VMHC(Ventricular myosin heavy chain)是心脏发育早期的标志基因,制备该基因多抗血清,首先通过生物信息学方法,选择VMHC基因中特异性强、具亲水性的一段核苷酸序列(3 772～4 221 bp),通过PCR扩增,将片段重组到原核表达载体pGEX-4T-1,通过原核诱导表达获得含有表达目的片段的融合蛋白,以该融合蛋白免疫小鼠,获得VMHC多克隆抗鼠血清.对该多抗血清抗体进行验证,结果具有很好特异性和较高效价,可以用作western-blot、免疫印迹等试验分析.

Zebrafish gene VMHC （Ventricular myosin heavy chain） is a marker gen in the early development heart. In order to get the polyclonal antiserum of VMHC, a VMHC-specifie, hydrophilie nucleotide sequence （ CDS from 3 772 to 4 221 bp） was selected by using bioinformatics methods, and the sequence was amplified by PCR. The PCR product was inserted into the prokaryotic expression vector pGEX-4T-1 and the fusion protein was induced to express in BL21 cells. The mouse was immunized by injecting appropriate amount of fusion protein and the VMHC polyclonal antiserum was obtained. The antiserum＇s titer and specification was identified. The results showed that the VMHC polyclonal antiserum was practical and available for western-blotting analysis and immueprecipitation.