Chen L M, Richards G P, Chao L, Chao J
Department of Biochemistry and Molecular Biology, Medical University of South Carolina, Charleston 29425, USA.
Biochem J. 1995 Apr 15;307 ( Pt 2)(Pt 2):481-6. doi: 10.1042/bj3070481.
We have cloned and characterized a full-length cDNA encoding tissue kallikrein from a human colon carcinoma cell line (T84). The nucleic acid sequence of the colon kallikrein cDNA is identical to that of renal/pancreatic or tissue kallikrein cDNA. Reverse-transcription PCR followed by Southern-blot analysis using specific oligonucleotide probes showed expression of tissue kallikrein in human colon, pancreas and kidney. Tissue kallikrein mRNA was localized in glandular epithelial cells (goblet cells) in colon by in situ hybridization histochemistry. Human colon kallikrein was purified to apparent homogeneity by DEAE-Sepharose Cl-6B, aprotinin-affinity, and HQ/M perfusion chromatography. The purified colon kallikrein migrated as a broad, 40-45 kDa band in SDS/PAGE and was recognized by antibodies to human tissue kallikrein. The linear displacement curves for the colon kallikrein in an RIA were parallel with the human tissue kallikrein standard curve, indicating their immunological identity. The N-terminal sequence of the purified colon kallikrein matches completely with that of purified urinary or tissue kallikrein. These results indicate that human colon kallikrein is transcribed from the tissue kallikrein gene.
我们从人结肠癌细胞系（T84）中克隆并鉴定了编码组织激肽释放酶的全长cDNA。结肠激肽释放酶cDNA的核酸序列与肾/胰腺或组织激肽释放酶cDNA的序列相同。使用特异性寡核苷酸探针进行逆转录PCR，随后进行Southern印迹分析，结果显示组织激肽释放酶在人结肠、胰腺和肾脏中表达。通过原位杂交组织化学方法，发现组织激肽释放酶mRNA定位于结肠的腺上皮细胞（杯状细胞）中。利用DEAE-琼脂糖凝胶CL-6B、抑肽酶亲和柱和HQ/M灌注色谱法，将人结肠激肽释放酶纯化至表观均一性。纯化后的结肠激肽释放酶在SDS/PAGE中迁移为一条宽的40 - 45 kDa条带，并能被抗人组织激肽释放酶的抗体识别。在放射免疫分析中，结肠激肽释放酶的线性位移曲线与人类组织激肽释放酶标准曲线平行，表明它们具有免疫同一性。纯化后的结肠激肽释放酶的N端序列与纯化后的尿激肽释放酶或组织激肽释放酶的N端序列完全匹配。这些结果表明，人结肠激肽释放酶是从组织激肽释放酶基因转录而来的。
