Sogawa K, Ichihara Y, Takahashi K, Fujii-Kuriyama Y, Muramatsu M
J Biol Chem. 1981 Dec 10;256(23):12561-5.
Poly(A)-containing RNA was isolated from total RNA of swine gastric mucosa by oligo(dT)-cellulose chromatography, and was translated in a wheat germ cell-free system. Analysis of the translation products by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that pepsinogen was a major translation product and was synthesized as two different molecular forms with apparent molecular weights of 45,000 and 43,000. The pepsinogen translated in the in vitro translation system had no autocatalytic activity. The pepsinogen mRNA was further purified by sucrose density gradient centrifugation, where the mRNA activity for pepsinogen was located around 15 S. At this stage, the translation product of the pooled fractions appeared to be almost exclusively pepsinogen. The peptide maps of the pepsinogen which was translated in vitro and digested by alpha-chymotrypsin and by Staphylococcus aureus V8 protease were nearly identical with the corresponding peptide maps of standard pepsinogen. The double-stranded complementary DNA which had been synthesized from the partially purified mRNA by avian myeloblastosis virus reverse transcriptase was cloned in Escherichia coli chi 1776, using plasmid pBR322 as a cloning vector. A colony carrying pepsinogen cDNA sequence was identified by in situ colony hybridization using the cDNA synthesized from the partially purified mRNA as a probe and further by a positive hybridization-translation assay. One of the recombinant clones (pSPcA1) had an insert of about 850 base pairs, and the nucleotide sequence analysis revealed that pSPcA1 codes for swine pepsinogen.
通过寡聚（dT）-纤维素柱层析从猪胃黏膜总RNA中分离出含Poly（A）的RNA，并在麦胚无细胞体系中进行翻译。用十二烷基硫酸钠-聚丙烯酰胺凝胶电泳分析翻译产物，结果显示胃蛋白酶原是主要的翻译产物，且以两种不同的分子形式合成，表观分子量分别为45,000和43,000。在体外翻译体系中翻译的胃蛋白酶原没有自催化活性。胃蛋白酶原mRNA通过蔗糖密度梯度离心进一步纯化，其中胃蛋白酶原的mRNA活性位于约15S处。在此阶段，合并组分的翻译产物似乎几乎全是胃蛋白酶原。体外翻译并用α-胰凝乳蛋白酶和金黄色葡萄球菌V8蛋白酶消化的胃蛋白酶原的肽图与标准胃蛋白酶原的相应肽图几乎相同。用禽成髓细胞瘤病毒逆转录酶从部分纯化的mRNA合成的双链互补DNA，以质粒pBR322作为克隆载体，克隆到大肠杆菌chi 1776中。用从部分纯化的mRNA合成的cDNA作为探针，通过原位菌落杂交以及进一步通过阳性杂交-翻译试验，鉴定出携带胃蛋白酶原cDNA序列的菌落。其中一个重组克隆（pSPcA1）有一个约850个碱基对的插入片段，核苷酸序列分析表明pSPcA1编码猪胃蛋白酶原。
