雌激素受体ERβ配体结合区的异源表达与纯化

为研究环境化合物与雌激素受体的相互作用,在大肠杆菌中异源表达了人雌激素受体ERβ的配体结合区(hERβ-LBD)蛋白。在诱导剂(IPTG)浓度为0.2mmol.L-1,培养温度25℃,诱导时间为8h的优化表达条件下,经亲和色谱法纯化后,hERβ(LBD蛋白的产量可达236mg.L-1.     

Biosynthesis of selenosubtilisin: A novel way to target selenium into the active site of subtilisin

Glutathione peroxidase （GPx, EC1.11.1.9）, an important anti-oxidative selenoenzyme, can catalyze the reduction of harmful hydroperoxides with concomitant glutathione, thereby protecting cells and other biological issues against oxidative damage. It captures considerable interest in redesign of its function for either the mechanism study or the pharmacological development as an antioxidant. In order to develop a general strategy for specifically targeting and operating selenium in active sites of enzymes, the catalytically essential residue selenocysteine （Sec） was first successfully bioincorporated into the catalytic center of subtilisin by using an auxotrophic expression system. The studies of the catalytic activity and the steady-state kinetics demonstrated that selenosubtilisin is an excellent GPx-like biocatalyst. In comparison with the chemically modified method, biosynthesis exhibits obvious advantages： Sec could be site-directly incorporated into active sites of enzymes to overcome the non-specificity generated by chemical modification. This study provides an important strategy for specifically targeting and operating selenium in the active site of an enzyme.     

不同培养条件对基因工程疫苗菌抗原蛋白表达的影响

基因工程菌的表达条件研究在现代生物高技术产业化的发展中具有重要的意义。影响基因工程菌表达的因素主要有pH值、温度、诱导剂IPTG的浓度、诱导时间等。笔者分别就以上因素对基因工程菌抗原蛋白表达的影响进行了初步研究。在摇床培养条件下，外源基因表达蛋白的最适表达条件为终浓度O．8mmol/L的IPTG，32℃，诱导6h。     

线性型研究中的范式问题

文〔１〕在研究线性型的问题时，提出了范式的概念。由于范式在研究线性型时的重要作用，并有其自身独立的意义，我们将在这里给出范式的完全明确的解答。     

Expression,purification and spectra characterization of neuroglobin

The expression, purification and spectra characterization of recombinant human neuroglobin （NGB） are reported. The pET3a plasmid with the gene of NGB was transformed to E. coli BL21 （DE3） plys cells and expressed in TB culture medium. The results indicated that the expression amount of NGB is about 10 percent of the total protein in cells. The NGB protein was purified by ammonium sulfate precipitation, DEAE-Sepharose anion exchange column, Hiload 16/60 superdex 75 size exclusion chromatography and a Hiprep 16/10 Q FF anion exchange column, and a red soluble protein was obtained which showed a single band in electrophoresis. Electrospray ionization mass spectrometry （ESI-MS） showed that its molecular weight is 16930.0 Da. UV-spectra indicated that the reduced NGB has a strong absorption peak at 425 nm, and two weak peaks at 531 and 559 nm, which can be assigned to γ, β and α bands of porphyrin, respectively, and the oxidized NGB has a strong absorption peak at 413 nm which corresponds to the transition of π electrons in the porphyrin ring. The fluorescence maximal excitation wavelength is at 281 nm and its maximal emission wavelength is at 338 nm. CD spectra indicated that its secondary structure is a typical α helix, and has a positive peak at 410 nm induced by heme, The NGB protein is stable when the pH is higher than 4.     

面向网络流的自适应正则表达式分组匹配算法

针对当前的多正则表达式匹配算法占用较大的系统资源,且吞吐量较低的问题,在分析典型的正则表达式匹配算法的基础上,提出了一种自适应的多正则表达式分组匹配算法.该算法通过对正则表达式进行高效分组,将相互之间存在交叠且容易引起状态数指数增长的表达式相互隔离;将每个分组构造为一个确定性有限自动机(DFA),按匹配概率大小建立伸展树进行调度.仿真结果表明,该算法不仅大大节省了存储空间,而且吞吐量提高了大约3倍.     

栖热菌噬菌体TSP4密码子偏嗜性及其对基因异源表达的影响

 为了探索噬菌体TSP4、栖热菌模式菌株Thermus thermophilus HB27中6种蛋白编码序列和Escherichia coli基因组遗传密码子使用偏嗜性,探索TSP4基因密码子偏嗜性对其基因异源表达的影响本研究利用生物学软件Editseq和RSCU算法统计噬菌体TSP4密码子使用频率并分析其偏嗜性,与栖热菌HB27的同源基因以及常温菌E.coli基因组的密码子使用偏嗜性进行对比分析.结果显示,噬菌体TSP4与栖热菌HB27优势密码子相似度高达85%,而与E.coli的相似性仅有65%.为了验证分析结果,选取TSP4基因组中一个潜在分子伴侣基因序列在E.coli BL21和E.coli Rosetta(补充了BL21菌株中缺失的6个稀有密码子)中进行异源表达.噬菌体TSP4与其宿主菌HB27之间密码子高相似性说明在长期的进化过程中TSP4在基因水平上形成对宿主的一种适应性机制.异源表达结果显示,分子伴侣基因在E.coli BL21中未见明显表达,但在Rosetta中高效表达,说明Rosetta中补充的 6个稀有密码子明显有助于分子伴侣基因的表达,该结果也进一步证明密码子偏嗜性是否一致在很大程度上影响基因的表达.     

一种基于GEP演化布尔函数的流密钥算法

密钥产生器是流密码系统的核心部件,根据非线性组合流密钥生成器的构造思想,本文提出了一种新的RSA-LFSR密钥流产生器,该产生器将RSA与LFSR算法相结合所产生的随机序列用一种基于基因表达式程序设计(GEP)的演化布尔函数进行组合运算输出密钥流;并对此产生器生成的不同长度密钥序列进行安全性检验.通过游程检验、频数检验、序列测试和线性复杂度计算的实验证明,使用基于GEP演化布尔函数的RSA-LFSR密钥流产生器要比传统的非线性组合密钥生成器更具优越性和安全性.     

沉默白细胞介素10基因对猪繁殖与呼吸综合征病毒（PRRSV）感染的影响

为了研究藏猪免疫细胞中IL-10基因表达抑制后对RNA病毒感染及其免疫应答的影响,本实验构建和筛选了抑制猪IL-10基因表达的shRNA干扰分子及其表达载体,并以新型的壳聚糖接枝聚乙二醇和聚乙烯亚胺修饰分子(CS-N-PEI-PEG)通过离子交联法进行分子包装,制备DNA-壳聚糖纳米分子,体外转染藏猪免疫细胞,观察猪繁殖和呼吸障碍综合征病毒(PRRSV)诱导免疫细胞IL-10基因的表达及其增殖感染水平.实验结果表明:与未转染纳米分子或者纳米分子不表达shRNA的空白对照组相比较,shRNA实验组的IL-10基因表达在24～72h内明显抑制(P<0.05),免疫细胞中PRRSV的增殖水平显著下降(P<0.05);同时IL-2、IL-4、IL-6和IFN-γ基因的mRNA水平明显升高(P<0.05).这些提示IL-10的shRNA转染能使免疫细胞的抗感染能力显著增强,甚至可完全抑制PRRSV的复制.