Research for noles of oligosaccharide on human prolactin

1.制备人催乳素(PRL)基因，并将其插入真核细胞表达载体中。将含催乳素基因的载体导入COS-1细胞，用~(35)&t；35&gt；S-蛋氨酸标记筛选产生催乳素的克隆。用~(35)&gt；S-蛋氨酸标记克隆12h，用抗催乳素抗体免疫沉淀细胞裂解液或培养上清液中合成和分泌的催乳素。用十二烷基硫酸钠-聚丙烯酰胺凝胶电泳法对沉淀的PRL进行分析。无论有无低聚糖，在细胞裂解液和培养液中都能观察到PRL。无论是在细胞裂解液中还是在培养上清液中，无寡糖的PRL含量都大于有寡糖的PRL。这些结果并不能阐明人PRL中寡糖对细胞分泌PRL的生理意义等。将人催乳素基因插入到MV1190噬菌体中，再将含有催乳素基因的单链DNA插入到MV1190噬菌体中，制备出含有催乳素基因的单链DNA。通过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测序证实了突变的存在。进一步的实验将突变的DNA插入到表达载体中，制备纯化的不含寡糖的催乳素。该突变体可用于分析寡糖在PRL生理中的作用。

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.