Research for noles of oligosaccharide on human prolactin

1.制备人催乳素（PRL）cDNA，并将其插入真核细胞表达载体。将含有PRL cDNA的载体转染COS-1细胞，用~（13 S）-蛋氨酸标记筛选PRL产生克隆<35>。用β-<35>S-甲硫氨酸标记克隆12小时，用抗PRL抗体免疫沉淀细胞裂解物或培养基中合成和分泌的PRL。沉淀的PRL在SDS-聚丙烯酰胺凝胶电泳上进行分析。在细胞裂解液和培养基中均观察到含或不含寡糖的PRL。细胞裂解液和培养液中不加寡糖的PRL含量均高于加寡糖的PRL含量。通过这些结果，我们不能阐明人PRL中寡糖对细胞分泌PRL的生理意义等.将人PRL cDNA插入到MV 1190噬菌体中，将含有PRL cDNA的单链DNA插入到MV 1190噬菌体中，制备含有PRL cDNA的单链DNA。进行定点突变，将天冬酰胺密码子（AAC）改变为天冬氨酸密码子（GAC），并通过DNA测序确认核苷酸突变。为了进一步的实验，将突变体DNA插入表达载体中，并制备纯化的不含寡糖的PRL。该突变体可用于分析寡糖对PRL生理功能的影响。

1. Human prolactin (PRL) cDNA was prepared, and inserted into the expression vector for eukariotic cell. The vector containing PRL cDNA was tranfected into COS-1 cells, and the PRL producing clones were screened by the labeling with ^<35>S-methionine. The clones were labeled with ^<35>S-methionine for 12 hours, and synthesized and secreted PRL in cell lysate or culture medium were immunoprecipitated with anti-PRL antibody. The precipitated PRL was analyzed on SDS-polyacrylamide gel electrophoresis. The PRLs either with or without oligosaccharide were observed in both cell lysate and medium. The amount of PRL without oligosaccharide was larger than that with oligosaccharide both in cell lysate and medium. By these results, we can not clarify the physiological significanses or the oligosaccharide in human PRL on secretion of PRL from cell, and so on.2. Human PRL cDNA was inserted into MV1190 phage, and single-stranded DNA containing PRL cDNA was inserted into MV1190 phage, and singe-straned DNA containing PRL cDNA was prepared. Site-directed mutagenesis that altered asparagine codons (AAC) to aspartic acid codons (GAC) was performed, and the mutation in nucleotides was confirmed by DNA sequencing. For further experiments, the mutant DNA will be inserted into the expression vector, and purified PRL without oligosaccharide will be prepared. This mutant PRL can be useful for the analysis of the roles of the oligosaccharide on the physiology of PRL.