A study on cell proliferation and differentiation using antibody against human homeodomain protein.

将同源框4 B基因的全长cDNA克隆到原核表达载体pIH 888中，转化大肠杆菌JM 109。在该实验中，未获得转化体。同源框序列的表达被怀疑是有毒的大肠杆菌。因此，去除了含有同源框序列的Hox 4 B基因的第二个外显子，并将第一个外显子插入pIH 888中（命名为pIH 4 Bb）。通过将pIH 4 Bb转染至JM 109获得转化体。然后，在Hox 4 B的第一个外显子末端插入翻译终止密码子TAG（pIH 4 Bc）。核苷酸序列分析表明，一个接头，Hox 4 B的第一个外显子，终止密码子和一个接头连接在框内的翻译到融合蛋白。通过将pIH 4 Bc质粒转染到JM 109中获得转化体。通过SDS-PAGE分析来自细菌周膜的蛋白。在染色的凝胶中观察到57 kDa的致密条带。通过制备性丙烯酰胺凝胶电泳纯化57 kDa的蛋白。用该融合蛋白免疫家兔，获得了抗Hox 4 B的抗体。用亲和柱纯化后使用抗体。用小鼠胚胎匀浆进行免疫印迹分析，证明了抗体的特异性。免疫印迹显示64 kDa的单一条带。该抗体用于小鼠胚胎石蜡切片的免疫染色。未成熟的成软骨细胞，脊髓神经元，和肾上腺髓质使用这种抗体染色。

A whole cDNA fragment of homeobox 4B was inserted into the expression vector pIH888 and the recombinant plasmid was transfected to E.coli JM109. In this experiment, transformants were not obtained. The expression of homeobox sequence was suspected to be toxic to E.coli. Therefore, the second exon of Hox4B gene, which contains the homeobox sequence, was removed and the first exon was inserted into pIH888 (designated pIH 4Bb). Transformants were obtained by transfection of the pIH4Bb to JM109. Thereafter, translation termination codonTAG was inserted at the end of the first exon of Hox4B(pIH 4Bc). The nucleotide sequence analysis revealed that a linker, the first exon of Hox4B, termination codon and a linker were ligated in-frame for the translation to the fusion protein. Transformants were obtained by transfection of the pIH 4Bc plasmid to JM109. Proteins from bacterial periplasms were analyzed by SDS-PAGE. A dense band of 57 kDa was observed in the stained gel. The protein of 57 kDa was purified by preparative acrylamide gel electrophoresis. The antibody against Hox4B was obtained by immunization of the fusion protein to rabbits. The antibody was used after purification with an affinity column. The specificity of the antibody was demonstrated by immunoblot analysis using mouse embryo homogenate. The immunoblot revealed a single band of 64 kDa. This antibody was used for the immunostaining of paraffin sections of mouse embryos. Immature chondroblasts, spinal neurons, and medulla of adrenal glands were stained using this antibody.