摘要
本文将已构建好的带有乳糖酶基因的杆状病毒(AcNPV p10 Z-9)作为载体,经口服感染人工饲养的银纹夜蛾(Argyrogra(?) agnata)幼虫,在昆虫体内高效表达乳糖酶蛋白,表达水平可占虫体可溶性蛋白的20％。将虫匀浆后,经高速离心、丙酮沉淀及冷冻干燥等方法,即可制备成粗酶。进一步经FPLC β-aminophenyl-β-D-thiogalactopyranoside亲和柱层析方法制备成精制酶,可作为试剂及药物应用.经测定纯化后的酶比活为150000u/mg蛋白。酶活力的最适pH值是6.0～8.0,最适温度为30～40℃。该酶水解对硝基苯-β-D-半乳糖(ONPG)的米氏常数(Km)值为7.14×10^(4)mol/L,乳糖可竞争性地抑制乳糖酶水解ONPG的能力,Ki值为1.30×10^(-3)mol/L。
The recombinant Aulographa californica nuclear polyhedrosis virus ( AcNPV) with β-galac-tosidase gene were used as a vector. The Argyrogrmma agnala larvae were infected with the AcNPV by oral to high level expression of β-galactosidase. The expression level in vivo of the insect larvae may be 20% of lysing proteins. The recombinant β-galactosidase has been purified 14-fold with an activity recovery of 79% from the larvae extract through acetone precipitation and FPLC β-arainophenyl-β-D- thiogalactopyranoside affinity chromatography. Final specific activities of 1 50000u/mg were obtained. The enzyme activities were optimun from pH 6. 0 to 8. 0 and temperature extent from 30℃ to 40℃. The enzyme consists of two binds with apparent subunit weights of 120kd and 70kd determined by SDS-PAGE. The β-galactosidase hydrolyzed O-nitro-phenyl-β-galactonoside (ONPG) with which the Km values was 7. 14× 10-4mol/L. ONPG and lactose both competitively inhibited the enzyme activity. The Ki values calculated by Line weaver-Burk plot was 1. 30× 10-3mol/L.
出处
《中山大学学报论丛》
1995年第2期106-111,159,共7页
Supplement to the Journal of Sun Yatsen University
