小儿扁桃体淋巴细胞Poly(A)-RNA的制备及其cDNA的合成

Preparation of Poly(A)-BNA of Children Tonsil Lymphocytes and Synthesis of Its Total ds-cDNA

采用盐酸胍法从经PHA诱导的小儿扁桃体淋巴细胞中提取总RNA,经Oligo(dT)-纤维素柱层析制备了Poly(A)-RNA。经蔗糖线性密度梯度超速离心步骤,富集了含IL-2mRNA的Poly(A)-RNA。经麦胚无细胞体系翻译实验表明,富集后的Poly(A)-RNA具有良好的模板翻译活性,其~3H-亮氨酸参入活性是对照的5.1倍。又以此富集了的Poly(A)-RNA为模板,按照Watson和Jackson的最新方法合成了小儿扁桃体淋巴细胞的双链cDNA。经碱性凝胶电泳检查,用此方法合成的cDNA长度最长的超过了IL-2的cDNA长度。

The total cellular RNA was isolated from the PHA-induced children tonsil lymphocytes by Guaniding·HCl method. The poly(A)-RNA was prepared by the oligo (dt)-cellulose affinity chromatography. The poly(A)-RNA containing the Interleukin 2 (IL-2) mRNA was enriched with sucrose linear density gredient ultracentrifugation. Aliqu-ots of the enriched mRNA were assayed by in vitro translation in a wheat germ system. The incorporation activity of 2H-leucine was 5.1 fold over the control. The total cDNAs of the children tonsil lymphocytes were synthesized according to the Watson and Jackson method. The total synthesized cDNAs were identified on alkaline agarose gel eleclrophoresis. The largest sizes of the cDNAs synthesized were longer then 800 base pairs.