百日咳毒素S1亚单位突变体在大肠杆菌中的表达及纯化

目的克隆百日咳毒素S1亚单位,获得其突变体(S1/9K-129G,rS1),构建原核表达系统,并鉴定融合蛋白的特异性。方法采用PCR和重叠延伸扩增得到突变体,将其与pMD18-T连接,转化至大肠杆菌DH5α,筛选得到阳性重组克隆载体pMD18-T-rS1,经双酶切得到rS1活性结构域编码片段。将其插入pQE-30载体中,转化大肠杆菌M15株,经IPTG诱导表达。表达产物经镍离子柱纯化,并进行SDS-PAGE和Western blot鉴定。结果PCR及重叠延伸获得了约700bp的目的基因片段,并获得阳性重组表达载体克隆,表达产物为相对分子质量27000左右的蛋白,Western blot检测能与兔抗百日咳毒素多价抗血清发生反应。结论成功表达并纯化了百日咳毒素S1亚单位突变体,为研制百日咳疫苗候补抗原的相关分子免疫学研究奠定了基础。     

Gene Expression of Type VI Secretion System Associated with Environmental Survival in Acidovorax avenae subsp. avenae by Principle Component Analysis

Valine glycine repeat G (VgrG) proteins are regarded as one of two effectors of Type VI secretion system (T6SS) which is a complex multi-component secretion system. In this study, potential biological roles of T6SS structural and VgrG genes in a rice bacterial pathogen, Acidovorax avenae subsp. avenae (Aaa) RS-1, were evaluated under seven stress conditions using principle component analysis of gene expression. The results showed that growth of the pathogen was reduced by H₂O₂ and paraquat-induced oxidative stress, high salt, low temperature, and vgrG mutation, compared to the control. However, pathogen growth was unaffected by co-culture with a rice rhizobacterium Burkholderia seminalis R456. In addition, expression of 14 T6SS structural and eight vgrG genes was significantly changed under seven conditions. Among different stress conditions, high salt, and low temperature showed a higher effect on the expression of T6SS gene compared with host infection and other environmental conditions. As a first report, this study revealed an association of T6SS gene expression of the pathogen with the host infection, gene mutation, and some common environmental stresses. The results of this research can increase understanding of the biological function of T6SS in this economically-important pathogen of rice.     

Photoactivation of Cell-Free Expressed Archaerhodopsin-3 in a Model Cell Membrane

Transmembrane receptor proteins are located in the plasma membranes of biological cells where they exert important functions. Archaerhodopsin (Arch) proteins belong to a class of transmembrane receptor proteins called photoreceptors that react to light. Although the light sensitivity of proteins has been intensely investigated in recent decades, the electrophysiological properties of pore-forming Archaerhodopsin (Arch), as studied in vitro, have remained largely unknown. Here, we formed unsupported bilayers between two channels of a microfluidic chip which enabled the simultaneous optical and electrical assessment of the bilayer in real time. Using a cell-free expression system, we recombinantly produced a GFP (green fluorescent protein) labelled as a variant of Arch-3. The label enabled us to follow the synthesis of Arch-3 and its incorporation into the bilayer by fluorescence microscopy when excited by blue light. Applying a green laser for excitation, we studied the electrophysiological properties of Arch-3 in the bilayer. The current signal obtained during excitation revealed distinct steps upwards and downwards, which we interpreted as the opening or closing of Arch-3 pores. From these steps, we estimated the pore radius to be 0.3 nm. In the cell-free extract, proteins can be modified simply by changing the DNA. In the future, this will enable us to study the photoelectrical properties of modified transmembrane protein constructs with ease. Our work, thus, represents a first step in studying signaling cascades in conjunction with coupled receptor proteins.     

High Expression of LAMP3 Is a Novel Biomarker of Poor Prognosis in Patients with Esophageal Squamous Cell Carcinoma

Lysosomal-associated membrane protein 3 (LAMP3), identified as a molecular marker of mature dendritic cells, is one of the LAMP family members. Its expression was induced by hypoxia, and was associated with hypoxia mediated metastasis in breast and cervical cancers. However, epithelial expression of LAMP3 and its prognostic value in esophageal squamous cell carcinoma (ESCC) is still unknown. In the current study, mRNA expression of LAMP3 in 157 ESCC tissues and 50 adjacent normal tissues was detected by quantitative real-time PCR (qRT-PCR). LAMP3 protein expression in 46 paired cancerous and normal tissues was detected by immunohistochemistry (IHC). Then, DNA copy number was examined to observe its potential correlation with mRNA expression. The results showed that both mRNA and protein expression level of LAMP3 was significantly higher in cancerous tissues compared with normal controls (p < 0.001). LAMP3 DNA copy number was amplified in 70% of ESCC tissues and positive correlated with mRNA expression (p = 0.037). Furthermore, patients with higher LAMP3 expression had worse overall survival (HR = 1.90, 95% CI = 1.17–3.09, p = 0.010) and disease-free survival (HR = 1.80, 95% CI = 1.18–2.74, p = 0.006). In conclusion, our results suggest that epithelial LAMP3 expression is an independent prognostic biomarker for ESCC.     

Quantitative Real-Time PCR Analysis of YKL-40 and Its Comparison with Mammalian Chitinase mRNAs in Normal Human Tissues Using a Single Standard DNA

YKL-40 (YKL for the first three N-terminal residues of a 40 kDa protein) belongs to a group of human chitinase-like proteins (CLPs), which are similar to chitinases but lack chitinolytic activity. YKL-40 mRNA and its protein levels have been reported elevated in multiple disorders including asthma, cystic fibrosis, rheumatoid arthritis and malignant tumors. Here, we quantified the YKL-40 mRNA levels and compared them with chitinases and housekeeping genes in normal human tissues. To establish the quantitative real-time PCR (qPCR) system for evaluation of relative YKL-40 mRNA levels, we constructed a human standard DNA molecule by ligating cDNAs of YKL-40, two mammalian chitinases and two housekeeping genes in a one-to-one ratio. We generated cDNAs from various normal human tissues and analyzed the YKL-40 mRNA expression levels using a qPCR system with the standard DNA. We found that YKL-40 mRNA is present widely in human tissues while its expression patterns exhibit clear tissue specificity. Highest YKL-40 mRNA levels were detected in the liver, followed by kidney, trachea and lung. The levels of YKL-40 mRNA in the kidney and liver were more than 100-times higher than those of chitotriosidase mRNA. Our study provides for the first time a comprehensive analysis of the relative expression levels of YKL-40 mRNA versus mammalian chitinases in normal human tissues.