嗜水气单胞菌TPS-30株丝氨酸蛋白酶基因与溶血素基因在大肠杆菌中的融合表达

嗜水气单胞菌丝氨酸蛋白酶和溶血素是该菌重要的致病因子与保护性抗原。致病性嗜水气单胞菌TPS-30株为江浙一带鱼类暴发病病原主要血清型O:9的代表株。研究利用PCR方法扩增嗜水气单胞菌TPS-30株的丝氨酸蛋白酶基因(Spe)和溶血素基因(Hly),将基因Spe和Hly通过柔性片段进行融合,并将融合片段插入pET32a的多克隆位点,构建成重组融合表达载体pET32a-Spe-Hly。将重组载体转化大肠杆菌BL21(DE3),经异丙醛-β-D-硫代半乳糖苷(IPTG)诱导表达,获得融合蛋白Spe-Hly。表达产物经SDS-PAGE检测,显示与预期大小约130kD相吻合的融合蛋白带。纯化融合蛋白并对鲫鱼进行免疫攻毒试验。结果表明,丝氨酸蛋白酶基因和溶血素基因融合表达载体构建成功,并成功获得了融合蛋白Spe-Hly,对鲫鱼的免疫保护率达81.4%。这为基因工程亚单位多价疫苗的开发提供基础。       

Discovery and optimization of orally active cyclohexane-based prolylcarboxypeptidase (PrCP) inhibitors

The synthesis, SAR, binding affinities and pharmacokinetic profiles are described for a series of cyclohexane-based prolylcarboxypeptidase (PrCP) inhibitors discovered by high throughput screening. Compounds show high levels of ex vivo target engagement in mouse plasma 20 h post oral dose.     

24(S)-hydroxycholesterol induces neuronal cell death through necroptosis, a form of programmed necrosis

24(S)-Hydroxycholesterol (24S-OHC) produced by cholesterol 24-hydroxylase expressed mainly in neurons plays an important physiological role in the brain. Conversely, it has been reported that 24S-OHC possesses potent cytotoxicity. The molecular mechanisms of 24S-OHC-induced cell death have not yet been fully elucidated. In this study, using human neuroblastoma SH-SY5Y cells and primary cortical neuronal cells derived from rat embryo, we characterized the form of cell death induced by 24S-OHC. SH-SY5Y cells treated with 24S-OHC exhibited neither fragmentation of the nucleus nor caspase activation, which are the typical characteristics of apoptosis. 24S-OHC-treated cells showed necrosis-like morphological changes but did not induce ATP depletion, one of the features of necrosis. When cells were treated with necrostatin-1, an inhibitor of receptor-interacting serine/threonine kinase 1 (RIPK1) required for necroptosis, 24S-OHC-induced cell death was significantly suppressed. The knockdown of RIPK1 by transfection of small interfering RNA of RIPK1 effectively attenuated 24S-OHC-induced cell death. It was found that neither SH-SY5Y cells nor primary cortical neuronal cells expressed caspase-8, which was regulated for RIPK1-dependent apoptosis. Collectively, these results suggest that 24S-OHC induces neuronal cell death by necroptosis, a form of programmed necrosis.     

大肠杆菌丝氨酸羟甲基转移酶基因(glyA)的克隆和表达

利用插入失活及营养缺陷型互补法将大肠杆菌K12 13kb的glyA基因克隆到质粒pBR329中。将重组质粒酶切,亚克隆,确定2.6kb PstI-EcoRI亚克隆片段带有完整的glyA基因。共获得12株glyA基因重组菌,对重组质粒进行了酶切鉴定。不同重组菌丝氨酸羟甲基转移酶(SHMT)活性及其酶表达量均不相同。受体菌未检测到丝氨酸的产生。重组菌株JM109(pSM13)、K12(pSM13)、K12(pSM14)和K12(pSM15)SHMT酶表达量分别占全菌可溶性蛋白的15.7％、15.4％、11.8％和9.48％。     

“两广二号”家蚕抗病毒基因Bmlipase-1和BmSP-2克隆与原核表达

本研究以优良杂交品种"两广二号"家蚕为试材,克隆了该杂交品种家蚕两个抗家蚕核型多角体病毒(BmNPV)基因:脂肪酶基因Bmlipase-1和丝氨酸蛋白酶基因BmSP-2,测序并分别与不同品种蚕的同源基因序列进行比较。结果显示,"两广二号"家蚕Bmlipase-1基因ORF长度为885bp,编码294个氨基酸,BmSP-2扩增长度为855bp,编码284个氨基酸;它们的核苷酸和推导氨基酸序列同源性皆达92%以上,Bmlipase-1更保守,同源性大于99%";两广二号"家蚕的Bmlipase-1基因脂肪酶活化部位和BmSP-2基因酶催化三联体位点的氨基酸残基与不同品种蚕的完全相同。以上结果说明这两个抗病毒基因在蚕的遗传进化过程中高度保守,提示其可能在机体消化或者免疫防御方面起着重要生理作用。将这两个抗病毒基因在大肠杆菌BL21中进行融合表达,获得的融合Bmlipase-1和BmSP-2蛋白分子量分别为47kD和42kD左右。     

In silico and pharmacological screenings identify novel serine racemase inhibitors

d-Serine is a coagonist of the N-methyl-d-aspartate (NMDA)-type glutamate receptor and its biosynthesis is catalyzed by serine racemase (SR). The overactivation of the NMDA receptor has been implicated in the development of neurodegenerative diseases, strokes, and epileptic seizures, thus, the inhibitors of SR have potential against these pathological states. Here, we have developed novel inhibitors of SR by in silico screening and in vitro enzyme assay. The newly developed inhibitors have lower IC₅₀ value comparing with that of malonate, one of the standard SR inhibitor. The structural features of novel inhibitors suggest the importance of central amide structure having a phenoxy substituent in their structure for the SR inhibitory activity. The present findings suggest the importance and rational development of new drugs for diseases of NMDAR overactivation.     

Purification and characterization of a keratinolytic serine protease from Purpureocillium lilacinum LPS # 876

A keratinolytic serine protease secreted by Purpureocillium lilacinum (formerly Paecilomyces lilacinus) upon culture in a basal medium containing 1% (w/v) hair waste as carbon and nitrogen source was purified and characterized. After purification the keratinase was resolved by SDS-PAGE as a homogeneus protein band of molecular mass 37.0 kDa. The extracellular keratinase of P. lilacinum was characterized by its appreciable stability over a broad pH range (from 4.0 to 9.0), and up to 65 °C, along with its strong inhibition by phenylmethylsulphonyl fluoride among the protease inhibitors tested (98.2% of inhibition), thus suggesting its nature as a serine protease. The enzyme was active and stable in the presence of organic solvents such as dimethylsulfoxide, methanol, and isopropanol; certain surfactants such as Triton X-100, sodium dodecylsulfate, and Tween 85; and bleaching agents such as hydrogen peroxide. These biochemical characteristics suggest the potential use of this enzyme in numerous industrial applications.