Smith E M, Galin F S, LeBoeuf R D, Coppenhaver D H, Harbour D V, Blalock J E
Department of Psychiatry and Behavioral Sciences, University of Texas Medical Branch, Galveston 77550.
Proc Natl Acad Sci U S A. 1990 Feb;87(3):1057-60. doi: 10.1073/pnas.87.3.1057.
To determine the degree of similarity between pituitary and lymphocyte proopiomelanocortin, the lymphocyte mRNA was reverse transcribed, cloned, and sequenced. Murine lymphocyte mRNA was first purified by oligo(dT)-cellulose affinity chromatography and was reverse transcribed by using a selective 3' antisense oligonucleotide primer directed at the boundary between the translated/nontranslated region on the 3' end of exon 3. This cDNA was then amplified in a polymerase chain reaction with selective primers containing Sal I and Kpn I restriction endonuclease sites. Amplified cDNA was then directionally ligated into M13mp18 and M13mp19 bacteriophage and was sequenced. The nucleotide sequence encoding this peptide was identical to that of mouse pituitary corticotropin (ACTH). Elevated levels of lymphocyte immunoreactive ACTH were then induced with bacterial lipopolysaccharide and the peptide(s) was purified by antibody affinity chromatography and reverse-phase high-performance liquid chromatography. The predominant immunoreactive ACTH species was approximately 3 kDa and its sequence was identical to pituitary ACTH(1-25). These results conclusively demonstrate that lymphocytes produce authentic ACTH and harbor its mRNA.
为了确定垂体促阿黑皮素原和淋巴细胞促阿黑皮素原之间的相似程度，对淋巴细胞信使核糖核酸进行了逆转录、克隆和测序。小鼠淋巴细胞信使核糖核酸首先通过寡聚（dT）-纤维素亲和层析进行纯化，然后使用针对外显子3 3'端翻译/非翻译区域边界的选择性3'反义寡核苷酸引物进行逆转录。然后，该互补脱氧核糖核酸在聚合酶链反应中用含有Sal I和Kpn I限制性内切酶位点的选择性引物进行扩增。扩增后的互补脱氧核糖核酸然后定向连接到M13mp18和M13mp19噬菌体中并进行测序。编码该肽的核苷酸序列与小鼠垂体促肾上腺皮质激素（ACTH）的序列相同。然后用细菌脂多糖诱导淋巴细胞免疫反应性促肾上腺皮质激素水平升高，并通过抗体亲和层析和反相高效液相色谱法纯化该肽。主要的免疫反应性促肾上腺皮质激素种类约为3 kDa，其序列与垂体促肾上腺皮质激素（1-25）相同。这些结果确凿地证明淋巴细胞产生真正的促肾上腺皮质激素并含有其信使核糖核酸。
