Jing H L, Shang H S, Zhang Q Q, Xu Z K
National Laboratory of Plant Molecular Genetics, Shanghai Institute of Plant Physiology, Chinese Academy of Sciences.
Shi Yan Sheng Wu Xue Bao. 1995 Mar;28(1):77-83.
Antifreeze peptide (AFP) produced in some animals is able to lower freezing temperature to prevent serum from freezing. There is a great potential to apply AFP in various circumstances wherever tolerance to cold environment is required. cDNA copies of AFP and proAFP coding sequence were synthesized according to the published sequence from a winter flounder (Pseudopleuronectes americanus). Expression of AFP or proAFP cDNA was initially tested in E. coli using a procaryotic expression vector. Level of the expression was essentially low indicated by SDS-PAGE and protein staining procedures. In order to detect the low level expression, the AFP (or proAFP) cDNA was fused, in frame, to the 5'-end of the CAT reporter gene resulting in a chimeric AFP/CAT (or proAFP/CAT) cDNA. The product translated from such a chimeric mRNA must contain a N'-terminal AFP (or proAFP) portion and C'-terminal CAT portion. Thus, low level of expression of AFP (or proAFP) in the fused form may be detected indirectly by sensitive CAT assay as long as the chimeric CAT still remains the activity. As expected, CAT activity has been clearly detected in protein extract from induced E. coli indicating that AFP (or proAFP) cDNA clones be expressed in E. coli.
一些动物体内产生的抗冻肽（AFP）能够降低冰点温度，防止血清冻结。在任何需要耐受寒冷环境的情况下，应用AFP都具有巨大的潜力。根据美洲拟鲽（Pseudopleuronectes americanus）已发表的序列合成了AFP和proAFP编码序列的cDNA拷贝。最初使用原核表达载体在大肠杆菌中测试AFP或proAFP cDNA的表达。SDS-PAGE和蛋白质染色程序表明表达水平基本较低。为了检测低水平表达，将AFP（或proAFP）cDNA与CAT报告基因的5'端进行读框融合，产生嵌合的AFP/CAT（或proAFP/CAT）cDNA。从这种嵌合mRNA翻译的产物必须包含N端的AFP（或proAFP）部分和C端的CAT部分。因此，只要嵌合的CAT仍然保持活性，就可以通过灵敏的CAT测定间接检测融合形式的AFP（或proAFP）的低水平表达。正如预期的那样，在诱导的大肠杆菌的蛋白质提取物中已清楚地检测到CAT活性，表明AFP（或proAFP）cDNA克隆在大肠杆菌中表达。
