人羧肽酶A的cDNA克隆和原核表达

目的:获得人羧肽酶A基因,为进一步构建、表达抗人精浆蛋白单链抗体/羧肽酶A融合蛋白,用于前列腺癌的抗体导向酶-前体药物疗法奠定基础。方法:从人正常肺组织中提取总RNA,经反转录PCR分离人羧肽酶A基因,并克隆入pUC19质粒中,用全自动荧光测序仪测定其序列。将该目的基因插入pBV220载体,并转入大肠杆菌DH5α中,经热诱导表达重组蛋白。结果:获得了人羧肽酶A基因(由1251bp组成,可编码417个氨基酸),与国外发表的人羧肽酶AcDNA序列一致。表达的重组蛋白以SDSPAGE分析,在Mr为49000左右处出现1条新生蛋白带,表达量约占菌体总蛋白的17%。结论:成功地克隆人羧肽酶A基因,为构建表达抗人精浆蛋白/羧肽酶A的双功能抗体奠定了基础。

Mast cell tryptase and carboxypeptidase A expression in body fluid and gastrointestinal tract associated with drug-related fatal anaphylaxis

AIM: To investigate the expression of mast cell tryptase and carboxypeptidase A in drug-related fatal anaphylaxis.METHODS: The expression of mast cell tryptase and carboxypeptidase A in 15 autopsy cases of drugrelated fatal anaphylaxis and 20 normal autopsy cases were detected. First, the expression of mast cell tryptase was determined in stomach, jejunum, lung, heart, and larynx by immunofluorescence. Different tissues were removed and fixed in paraformaldehyde solution, then paraffin sections were prepared for immunofluorescence. Using specific mast cell tryptase and carboxypeptidase A antibodies, the expression of tryptase and carboxypeptidase A in gastroenterology tract and other tissues were observed using fluorescent microscopy. The postmortem serum and pericardial fluid were collected from drug-related fatal anaphylaxis and normal autopsy cases. The level of mast cell tryptase and carboxypeptidase A in postmortem serum and pericardial fluid were measured using fluor enzyme linked immunosorbent assay(FEIA) and enzyme linked immunosorbent assay(ELISA) assay. The expression of mast cell tryptase and carboxypeptidase A was analyzed in drug-related fatal anaphylaxis cases and compared to normal autopsy cases.RESULTS: The expression of carboxypeptidase A was less in the gastroenterology tract and other tissues from anaphylaxis-related death cadavers than normal controls. Immunofluorescence revealed that tryptase expression was significantly increased in multiple organs, especially the gastrointestinal tract, from anaphylaxis-related death cadavers compared to normal autopsy cases(46.67 ± 11.11 vs 4.88 ± 1.56 in stomach, 48.89 ± 11.02 vs 5.21 ± 1.34 in jejunum, 33.72 ± 5.76 vs 1.30 ± 1.02 in lung, 40.08 ± 7.56 vs 1.67 ± 1.03 in larynx, 7.11 ± 5.67 vs 1.10 ± 0.77 in heart, P < 0.05). Tryptase levels, as measured with FEIA, were significantly increased in both sera(43.50 ± 0.48 μg/L vs 5.40 ± 0.36 μg/L, P < 0.05) and pericardial fluid(28.64 ± 0.32 μg/L vs 4.60 ± 0.48 μg/L, P < 0.05) from the anaphylaxis group in comparison with the control group. As measured by ELISA, the concentration of carboxypeptidase A was also increased more than 2-fold in the anaphylaxis group compared to control(8.99 ± 3.91 ng/m L vs 3.25 ± 2.30 ng/m L in serum, 4.34 ± 2.41 ng/m L vs 1.43 ± 0.58 ng/m L in pericardial fluid, P < 0.05).CONCLUSION: Detection of both mast cell tryptase and carboxypeptidase A could improve the forensic identification of drug-related fatal anaphylaxis.

羧肽酶A的脱毒功能及其应用前景

赭曲霉毒素A(Ochratoxin A,OTA)是由曲霉菌和青霉菌产生的一种真菌毒素,谷物、豆类、葡萄酒和咖啡等制品常常受到其污染,对食品安全构成了极大的威胁。随着全球气候的变化,OTA的污染情况日益严重,为保障人类及畜禽动物的安全,不少研究人员正致力于脱除或降解食品及其原料中的OTA。目前很多真菌和细菌被报道能降解OTA,然而它们产生的降解酶很少被定性和纯化,降解产物也不明确,无法应用于工业中。羧肽酶A(Carboxypeptidase A,CPA)对OTA的脱毒能力已被广泛证明,同时它的性质、结构及作用机理都极为清晰,降解产物也对人体无害,是脱除OTA的理想产品。因此通过基因工程和蛋白质工程大量表达获得稳定并效率高的羧肽酶A是降解OTA的新途径。将从OTA的污染状况及其生物脱毒机制、CPA的脱毒能力、CPA作为脱毒剂的发展前景等方面进行综述,旨在为真菌毒素降解酶制剂的开发和应用奠定理论基础。