用Blue Sepharose CL-6B快速纯化天花粉蛋白

差光谱显示染料cibacron blue F3GA与天花粉蛋白(TCS)有特异性结合,复合物在可见光部分的最大吸收波长在690 nm,摩尔消光系数ε=2.6×10^-3(mol/L)^-1·cm^-1,解离常数K_d=1.8 μmol/L,0.5 mol/L NaCl可使复合物解离.根据这一特点,用Blue-Sepharose CL-6B凝胶从栝篓块茎中亲和纯化了TCS.此法快速、简便、高效,易于大量制备.     

A ketoreductase gene from Streptomyces mycarofaciens 1748 DNA involved in biosynthesis of a spore pigment

An efficient plasmid transformation system forS. mycarofaciens 1748 has been established. In order to determine the function of MKR gene in S.mycarofaciens 1748, the gene disruption experiment was carried out. For this purpose the plasmid pKC1139 was used. A recombinant strain with white spore appeared, in contrast to the grey-colour spore of S.myarofaciens 1748. This suggested that homologous recombination between plasmidborne MKR gene sequence and the chromosome of S.mycarofaciens 1748 had occurred. A Southern hybridization experiment using a-³²P-labelled MKR gene as probe indicated that the desired integration event had occurred in the recombinant. The result of gene disruption showed that the alteration of this gene in the chromosome of S.mycarofaciens 1748 made sporulating colonies remain white instead of taking on the typical grey colour of sporulating wild type colonies, suggesting that MKR gene is involved in the biosynthesis of a spore pigment. The recombinant strain was incubated with fermentation medium optimised for midecamycin production. A TLC assay showed that the recombinant strain produced midecamycin in quantities comparable to that ofS. mycarofaciens 1748. A pCN8B12 was a clone from genomic library of midecamycin producing strain which contained a 28-kb DNA insert. The 28-kb DNA fragment contained act I-homologous and act III-homologous regions. he PKS (act I-homologous) and MKR (act III-homologous) genes that define spore pigment of midecamycin producing strain were Jocalized by restriction endonuclease digestion with pCN8B12, indicating that they are separated by about 10 kb DNA. The polyketide synthase gene cluster of simila; organization has not been reported yet. Project supported by the National Natural Science Foundation of China.     

蓖麻蚕蛹卵巢发育过程中亚细胞组分核酸和蛋白质的代谢与脑激素的影响

在20℃条件下,蓖麻蚕蛹期发育需14天,接近发育完成的卵巢中各亚细胞组分中DNA、RNA和蛋白质含量迅速增长。经手术去脑后的蓖麻蚕无脑蛹卵巢不发育,其卵巢中上述成分的含量皆处于蛹早期水平。经脑激素处理后的无脑蛹卵巢即可迅速发育,在处理后18—20天,卵巢发育可接近正常水平,但较晚。在电镜下观察到的游离多核糖体呈二到七聚体,其紫外光谱特性为A260/A280=1.70;A240/A280=1.27。将脑激素注射到无脑蛹中去,可促使卵巢中游离多核糖体含量的增加。     

拟除虫菊酯类农药对茶树害虫的生物活性与残留降解

本文报道了溴氰菊酯、联苯菊酯、氯氰菊酯、顺式氯氰菊酯、杀灭菊酯、功夫菊酯和二氯苯醚菊酯等七种拟除虫菊酯类农药对茶树主要害虫的杀虫效果和在茶树叶片上的降解动态试验结果.上述七种拟除虫菊酯类农药对鳞翅目茶树害虫的幼虫都具有极高的毒力,但对其他害虫的活性谱则表现有差异.它们在茶树芽梢上的降解速率常数为0.20—0.28天^-1,在茶树上的半衰期为2.5—3.5天,明显较有机磷农药稳定.在影响降解的因素中,挥发、热分解、雨水淋洗的作用不大,由茶梢生长稀释所起的作用较为明显.鲜叶中的农药残留在加工过程中的降解率为15—45%,平均20%,远低于有机磷农药.成茶中的残留农药在泡茶过程中由于他们的低水溶性,因此浸出率很低,一般仅有1—4.5%.     

玉米螟人工饲养中应用昆虫保幼激素类似物的初步试验

Lewis等1973年报道以九种昆虫保幼激素类似物;Shigemi等1976年报道以一种昆虫保幼激素类似物和一种天然保幼激素对玉米螟(Ostrinia nubilalis Hub.)幼虫的活性测定。目的在于明确对幼虫生长发育和变态的抑制效应。1978年在玉米螟人工饲养中,我们用另外五种昆虫保幼激素类似物进行了试验:供试的五种昆虫保幼激素类似物对玉米螟幼虫均有活性。高浓度可以阻止正常变态和导致异常变态。低浓度的734-Ⅱ能使玉米螟的生长发育和繁殖力获得有益的调节。     

去N端信号肽的水痘-带状疱疹病毒糖蛋白E的原核可溶性表达及其免疫原性评价

水痘-带状疱疹病毒(varicella zoster virus,VZV)糖蛋白E(glycoprotein E,gE)是VZV亚单位疫苗的主要候选蛋白,但目前原核表达系统制备的gE蛋白以包涵体形式为主,可溶性差。本研究采用去除第1～30氨基酸序列的VZV gE胞外域基因,将其与原核表达载体pET32a连接,并转化至感受态细胞BL21(DE3)中。使用异丙基-β-D-硫代半乳糖苷(Isopropylβ-D-thiogalactoside,IPTG)诱导表达,His-tag柱纯化重组gE蛋白,蛋白质印迹法(Western blot,WB)检测其特异性。用该重组gE蛋白免疫BALB/c小鼠制备多克隆抗体,酶联免疫吸附试验(enzyme linked immunosorbent assay,ELISA)和间接免疫荧光法检测多克隆抗体效价及特异性。结果显示,BL21/pET32a-VZV gE工程菌可以表达可溶性重组gE蛋白,纯化后纯度约为90%。WB鉴定该重组蛋白具有良好的免疫反应性。ELISA检测显示小鼠抗VZV gE多克隆抗体效价＞1∶10 000,间接免疫荧光实验结果显示该抗体特异性较高。结果表明,本研究在原核表达系统中成功表达可溶性重组VZV gE蛋白,同时该蛋白具有较强的免疫原性,这为VZV gE亚单位疫苗的研制和大规模生产奠定了基础。