重组葡激酶对小型猪冠脉血栓的溶栓作用研究*

目的: 评价重组葡激酶的溶栓效力,并与相同作用方式的重组链激酶进行比较。方法: 30只中国实验小型猪分成5组,分别为溶剂对照组、阳性药对照组和三个重组葡激酶组,每组6只,采用麻醉动物、手术开胸、直流电刺激形成冠脉血栓;在冠脉血栓形成30 min后开始静脉给药,采用先推注、再蠕动泵恒速输注的方式给药;溶剂对照组静脉推注对照液,阳性药对照组静脉给予重组链激酶4 mg·kg^-1,三个重组葡激酶组分别静脉给予4 mg·kg^-1、2 mg·kg^-1、1 mg·kg^-1重组葡激酶,静脉推注体积为5 ml,1 min内注毕,输注速度为0.5 ml·min^-1,60 min内输毕,120 min后放血处死动物。于给药前及给药后30、60、120 min取静脉血,实验结束后取血栓形成部位的冠脉血管段,分别检测优球蛋白溶解时间(ELT)、血纤维蛋白原含量(Fbg)、纤维蛋白(原)降解产物(FDP)和伤口出血量,检测冠脉血栓溶解率、心肌缺血程度及缺血范围。结果: 与溶剂对照组相比,试验组ELT明显缩短(P＜0.05或P＜0.01),FDP 明显升高(P＜0.05或P＜0.01),较少量实验动物Fbg降解超过20%,对小型猪血压及心率无明显影响。与对照组相比,试验组高、中2个剂量组,最大血栓面积分别减少34.3%、15.4%(P＜0.05)。与等剂量的重组链激酶相比,重组葡激酶对电刺激引起的冠脉血栓具有更强的溶栓作用(P＜0.05或P＜0.01),引起的出血副反应少。结论: 重组葡激酶对小型猪冠脉血栓有较好的溶栓作用,相比重组链激酶,溶栓速度快、具有更高的纤维蛋白专一性,出血副反应较少。综合比较, 2 mg·kg^-1重组葡激酶具有较好的临床疗效和安全性保障。     

Reduction of medium consumption in perfusion mammalian cell cultures using a perfusion rate equivalent concentrated nutrient feed

Media preparation for perfusion cell culture processes contributes significantly to operational costs and the footprint of continuous operations for therapeutic protein manufacturing. In this study, definitions are given for the use of a perfusion equivalent nutrient feed stream which, when used in combination with basal perfusion medium, supplements the culture with targeted compounds and increases the medium depth. Definitions to compare medium and feed depth are given in this article. Using a concentrated nutrient feed, a 1.8-fold medium consumption (MC) decrease and a 1.67-fold increase in volumetric productivity (PR) were achieved compared to the initial condition. Later, this strategy was used to push cell densities above 100 × 10⁶ cells/ml while using a perfusion rate below 2 RV/day. In this example, MC was also decreased 1.8-fold compared to the initial condition, but due to the higher cell density, PR was increased 3.1-fold and to an average PR value of 1.36 g L⁻¹ day⁻¹ during a short stable phase, and versus 0.46 g L⁻¹ day⁻¹ in the initial condition. Overall, the performance improvements were aligned with the given definitions. This multiple feeding strategy can be applied to gain some flexibility during process development and also in a manufacturing set-up to enable better control on nutrient addition.     

A detailed model and Monte Carlo simulation for predicting DIP genome length distribution in baculovirus infection of insect cells

Baculoviruses have enormous potential for use as biopesticides to control insect pest populations without the adverse environmental effects posed by the widespread use of chemical pesticides. However, continuous baculovirus production is susceptible to DNA mutation and the subsequent production of defective interfering particles (DIPs). The amount of DIPs produced and their genome length distribution are of great interest not only for baculoviruses but for many other DNA and RNA viruses. In this study, we elucidate this aspect of virus replication using baculovirus as an example system and both experimental and modeling studies. The existing mathematical models for the virus replication process consider DIPs as a lumped quantity and do not consider the genome length distribution of the DIPs. In this study, a detailed population balance model for the cell‐virus culture is presented, which predicts the genome length distribution of the DIP population along with their relative proportion. The model is simulated using the kinetic Monte Carlo algorithm, and the results agree well with the experimental results. Using this model, a practical strategy to maintain the DIP fraction to near to its maximum and minimum limits has been demonstrated.     

Structure-based Design of a Specific,Homogeneous Luminescence Enzyme Reporter Assay for SARS-CoV-2

Recombinant antibodies (Abs) against the SARS-CoV-2 virus hold promise for treatment of COVID-19 and high sensitivity and specific diagnostic assays. Here, we report engineering principles and realization of a Protein-fragment Complementation Assay (PCA) detector of SARS-CoV-2 antigen by coupling two Abs to complementary N- and C-terminal fragments of the reporter enzyme Gaussia luciferase (Gluc). Both Abs display comparably high affinities for distinct epitopes of viral Spike (S)-protein trimers. Gluc activity is reconstituted when the Abs are simultaneously bound to S-protein bringing the Ab-fused N- and C-terminal fragments close enough together (8 nm) to fold. We thus achieve high specificity both by requirement of simultaneous binding of the two Abs to the S-protein and also, in a steric configuration in which the two Gluc complementary fragments can fold and thus reconstitute catalytic activity. Gluc activity can also be reconstituted with virus-like particles that express surface S-protein with detectable signal over background within 5 min of incubation. Design principles presented here can be readily applied to develop reporters to virtually any protein with sufficient available structural details. Thus, our results present a general framework to develop reporter assays for COVID-19, and the strategy can be readily deployed in response to existing and future pathogenic threats and other diseases.     

肠出血性大肠杆菌O157∶H7 EspA和EspB蛋白的重组表达及其免疫效果

目的：通过DNA重组技术表达肠出血性大肠杆菌（EHEC）0157：H7的EspA和EspB蛋白,并分析它们的免疫保护性。方法：采用PCR技术从EHEC0157：H7基因组中扩增espA和espB基因,连接至pET-22b（4-）载体上,转化至宿主细胞大肠杆菌BL21（DE3）,经IPTG诱导表达,用亲和层析纯化目的蛋白,SDS-PAGE测定其相对分子质量,免疫小鼠分析其免疫保护性。结果：重组espA和espB基因片段的测序结果与GenBank中的相应基因序列完全一致,一致性均为100％;得到了纯度为95％以上的重组EspA和EspB蛋白,免疫小鼠所得到的抗体效价均为10^6。结论：重组EspA和EspB蛋白获得了可溶性表达,表达的蛋白具有良好的免疫保护性,为进一步制备疫苗奠定了基础。     

A new kumamolisin-like protease from Alicyclobacillus acidocaldarius: an enzyme active under extreme acidic conditions

A new serine-carboxyl proteinase, called kumamolisin-ac, was purified from the thermoacidophilic bacterium Alicyclobacillus acidocaldarius. The enzyme is a monomeric protein of 45?kDa, active over a wide temperature range (5.0–70°C) and extremely acidic pHs (1.0–4.0), showing maximal proteolytic activity at pH?2.0 and 60°C. Interestingly, kumamolisin-ac displayed a significant proteolytic activity even at 5°C, thus suggesting a sort of cold-adaptation for this enzyme. The protease was remarkably stable at high temperatures (t_1/2 at 80°C, 10?h, pH?2.0) and over a broad range of pH (2.0–7.0). Substrate analysis indicated that kumamolisin-ac was active on a variety of macromolecular substrates, such as haemoglobin, hide powder azure, and azocoll. In particular, a high specific activity was detected towards collagen. The corresponding gene was cloned, expressed and the recombinant protease, was found to be homologous to proteases of the ‘S53’ family. From the high identity with kumamolisin and kumamolisin-As, known as collagenolytic proteases, kumamolisin-ac can be considered as the third collagenolytic affiliate within the ‘S53’ family. Cleavage specificity investigation of kumamolisin-ac revealed a unique primary cleavage site in bovine insulin B-chain, whereas a broad specificity was detected using bovine α-globin as substrate. Thus, kumamolisin-ac could represent an attractive candidate for industrial-scale biopeptide production under thermoacidophilic conditions.     

The MultiBac Protein Complex Production Platform at the EMBL

Proteomics research revealed the impressive complexity of eukaryotic proteomes in unprecedented detail. It is now a commonly accepted notion that proteins in cells mostly exist not as isolated entities but exert their biological activity in association with many other proteins, in humans ten or more, forming assembly lines in the cell for most if not all vital functions.^1,2 Knowledge of the function and architecture of these multiprotein assemblies requires their provision in superior quality and sufficient quantity for detailed analysis. The paucity of many protein complexes in cells, in particular in eukaryotes, prohibits their extraction from native sources, and necessitates recombinant production. The baculovirus expression vector system (BEVS) has proven to be particularly useful for producing eukaryotic proteins, the activity of which often relies on post-translational processing that other commonly used expression systems often cannot support.³ BEVS use a recombinant baculovirus into which the gene of interest was inserted to infect insect cell cultures which in turn produce the protein of choice. MultiBac is a BEVS that has been particularly tailored for the production of eukaryotic protein complexes that contain many subunits.⁴ A vital prerequisite for efficient production of proteins and their complexes are robust protocols for all steps involved in an expression experiment that ideally can be implemented as standard operating procedures (SOPs) and followed also by non-specialist users with comparative ease. The MultiBac platform at the European Molecular Biology Laboratory (EMBL) uses SOPs for all steps involved in a multiprotein complex expression experiment, starting from insertion of the genes into an engineered baculoviral genome optimized for heterologous protein production properties to small-scale analysis of the protein specimens produced.^5-8 The platform is installed in an open-access mode at EMBL Grenoble and has supported many scientists from academia and industry to accelerate protein complex research projects.