Expression and Purification of Human Coagulation Factor X in Mammalian CHO-DG44 Cells

[Objectives]This study was conducted to obtain a Chinese hamster ovary cell line that stably expresses recombinant human coagulation factor X(rhFX),and to induce efficient expression of the target gene with different concentrations of methotrexate(MTX).[Methods]PCR was performed to obtain the rhFX gene,and a recombinant expression plasmid pOptiVEC-rhFX was constructed and subjected to double restriction endonuclease digestion and sequencing identification.CHO-DG44(DHFR-)cells were transfected by the liposome method,and the target protein was purified by affinity chromatography and detected by SDS-PAGE electrophoresis and Western blot.A cell line with efficient and stable expression of the target gene was obtained by increasing the concentration of MTX to select positive clones.[Results]PCR yielded a 1509 bp rhFX sequence,and the results of double digestion and sequencing showed that the constructed pOptiVEC-rhFX plasmid was correct.After transfection of cells,MTX significantly increased protein expression.When MTX reached 1.0μmol/L,the expression efficiency of the target protein was(9±0.27)μg/ml.The purity of the target protein purified by affinity chromatography was 93%,which could be used for subsequent experiments.The expression efficiency of rhFX in eukaryotic mammalian cells was improved by increasing MTX concentration,and an affinity chromatography purification process for the target protein was preliminarily established.[Conclusions]The results of this study provide data support for the expression and purification of rhFX,and will lay a solid foundation for the development of drugs related to rhFX.

Suppression of oscillatory congestion via trunk link bandwidth and control gain in star network

The time delay-induced instability in an Internet congestion control model is investigated. The star topology is considered, and the link bandwidth ratio and the control gain are selected as the tunable parameters for congestion suppression. The stability switch boundary is obtained by the eigenvalue analysis for the linearized system around the equilibrium. To investigate the oscillatory congestion when the equilibrium becomes unstable, the center manifold reduction and the normal form theory are used to study the periodic oscillation induced by the delay. The theoretical analysis and numerical simulation show that the ratio between bandwidths of the trunk link and the regular link,rather than these bandwidths themselves, is crucial for the stability of the congestion control system. The present results demonstrate that it is not always effective to increase the link bandwidth ratio for stabilizing the system, and for some certain delays, adjusting the control gain is more efficient.

Cdc42 deletion yielded enamel defects by disrupting mitochondria and producing reactive oxygen species in dental epithelium

Developmental defects of enamel are common due to genetic and environmental factors before and after birth.Cdc42,a Rho family small GTPase,regulates prenatal tooth development in mice.However,its role in postnatal tooth development,especially enamel formation,remains elusive.Here,we investigated Cdc42 functions in mouse enamel development and tooth repair after birth.Cdc42 showed highly dynamic temporospatial patterns in the developing incisors,with robust expression in ameloblast and odontoblast layers.Strikingly,epithelium-specific Cdc42 deletion resulted in enamel defects in incisors.Ameloblast differentiation was inhibited,and hypomineralization of enamel was observed upon epithelial Cdc42 deletion.Proteomic analysis showed that abnormal mitochondrial components,phosphotransferase activity,and ion channel regulator activity occurred in the Cdc42 mutant dental epithelium.Reactive oxygen species accumulation was detected in the mutant mice,suggesting that abnormal oxidative stress occurred after Cdc42 depletion.Moreover,Cdc42 mutant mice showed delayed tooth repair and generated less calcified enamel.Mitochondrial dysfunction and abnormal oxygen consumption were evidenced by reduced Apool and Timm8a1 expression,increased Atp5j2 levels,and reactive oxygen species overproduction in the mutant repair epithelium.Epithelium-specific Cdc42 deletion attenuated ERK1/2 signaling in the labial cervical loop.Aberrant Sox2 expression in the mutant labial cervical loop after clipping might lead to delayed tooth repair.These findings suggested that mitochondrial dysfunction,up-regulated oxidative stress,and abnormal ion channel activity may be among multiple factors responsible for the observed enamel defects in Cdc42 mutant incisors.Overall,Cdc42 exerts multidimensional and pivotal roles in enamel development and is particularly required for ameloblast differentiation and enamel matrix formation.

从江香猪TPM3基因的克隆与序列分析

[目的]对从江香猪TPM3基因进行扩增、克隆和序列分析。[方法]通过RT-PCR从猪背最长肌中扩增TPM3基因CDS区并进行克隆,利用DNAMAN和BioEdit等软件对克隆的序列进行分析。[结果]成功克隆了从江香猪TPM3基因并构建了pUCm-T-TPM3载体;获得了TPM3-1和TPM3-2两个序列,TPM3-1有两个碱基的差异,相似度达99.73%;TPM3-2有一个碱基的变化,同时插入了一段76 bp的片段,相似度达99.87%;密码子偏好性分析显示,UUG编码亮氨酸的频率为0.49%,而CUG编码亮氨酸的频率为2.89%,说明第49位碱基的转换可能提高蛋白的合成效率;RNA二级结构分析显示,碱基突变会影响TPM3基因RNA二级结构和最小自由能的变化;蛋白理化性质分析显示,氨基酸的改变对α螺旋、β折叠及转角等二级结构影响不明显。[结论]从江香猪TPM3基因的碱基突变可能影响原肌球蛋白的合成,为探究其对从江香猪肉质及种资源的开发利用提供试验依据。

Alkaline activation of endogenous latent TGFβ1 by an injectable hydrogel directs cell homing for in situ complex tissue regeneration

Utilization of the body’s regenerative potential for tissue repair is known as in situ tissue regeneration.However,the use of exogenous growth factors requires delicate control of the dose and delivery strategies and may be accompanied by safety,efficacy and cost concerns.In this study,we developed,for the first time,a biomaterial-based strategy to activate endogenous transforming growth factor beta 1(TGFβ1)under alkaline conditions for effective in situ tissue regeneration.We demonstrated that alkaline-activated TGFβ1 from blood serum,bone marrow fluids and soaking solutions of meniscus and tooth dentin was capable of increasing cell recruitment and early differentiation,implying its broad practicability.Furthermore,we engineered an injectable hydrogel(MS-Gel)consisting of gelatin microspheres for loading strong alkaline substances and a modified gelatin matrix for hydrogel click crosslinking.In vitro models showed that alkaline MS-Gel controllably and sustainably activated endogenous TGFβ1 from tooth dentin for robust bone marrow stem cell migration.More importantly,infusion of in vivo porcine prepared root canals with alkaline MS-Gel promoted significant pulp-dentin regeneration with neurovascular stroma and mineralized tissue by endogenous proliferative cells.Therefore,this work offers a new bench-to-beside translation strategy using biomaterial-activated endogenous biomolecules to achieve in situ tissue regeneration without the need for cell or protein delivery.