蛋白质免疫印迹技术在水产品中的应用

蛋白质免疫印迹技术作为一门新兴的技术,可对待测样品中目的蛋白质进行定性定量分析,在蛋白质组学研究中得到广泛应用,也为探究水产品中蛋白质组成及变化规律研究带来了新的途径。本文在介绍蛋白质免疫印迹技术原理及特点的基础上,总结了近年来该技术在水产品加工、贮藏、品质鉴别及质量安全方面的应用研究进展,探讨了蛋白质免疫印迹技术在蛋白质组学、多组学技术联用等研究水产品中蛋白质变化的应用前景并进行了展望,以期为水产品中品质与安全领域的蛋白质免疫印迹技术相关研究提供参考与借鉴。     

成渝城镇密集区建设与西部大开发

成渝城镇密集区是我国五大城镇密集区中唯一位于西部的地区。西部大开发战略已开始启动,本文分析了成渝城镇密集区面临的历史机遇,成渝城镇密集区的特征和建设成渝城镇密集区的意义,最后对成渝城镇密集区的建设提出了建议。     

Gel-Based Proteomic Identification of Suprabasin as a Potential New Candidate Biomarker in Endometrial Cancer

Endometrial cancer (EC) is the most frequent gynaecologic cancer in postmenopausal women. We used 2D-DIGE and mass spectrometry to identify candidate biomarkers in endometrial cancer, analysing the serum protein contents of 10 patients versus 10 control subjects. Using gel-based proteomics, we identified 24 candidate biomarkers, considering only spots with a fold change in volume percentage ≥ 1.5 or intensity change ≤ 0.6, which were significantly different between cases and controls (p < 0.05). We used Western blotting analysis both in the serum and tissue of 43 patients for data validation. Among the identified proteins, we selected Suprabasin (SBSN), an oncogene previously associated with poor prognosis in different cancers. SBSN principal isoforms were subjected to Western blotting analysis in serum and surgery-excised tissue: both isoforms were downregulated in the tissue. However, in serum, isoform 1 was upregulated, while isoform 2 was downregulated. Data-mining on the TCGA and GTEx projects, using the GEPIA2.0 interface, indicated a diminished SBSN expression in the Uterine Corpus Endometrial Cancer (UCEC) database compared to normal tissue, confirming proteomic results. These results suggest that SBSN, specifically isoform 2, in tissue or serum, could be a potential novel biomarker in endometrial cancer.     

人甲胎蛋白AFP在大肠杆菌中表达和鉴定

本研究通过应用基因工程技术,重组构建编码人全长甲胎蛋白AFP基因的原核表达载体pET22b-AFP,并将其转化到大肠杆菌宿主菌BL21（DE3）中进行诱导表达目的蛋白AFP。采用PCR法扩增编码AFP蛋白的cDNA序列片段,并将其克隆到含有His-tag的pET22b原核表达载体上;重组质粒经双酶切及测序鉴定正确后转化到大肠杆菌BL21(DE3)中进行IPTG诱导表达;对诱导过程中不同浓度的IPTG和不同的温度进行优化;选定最佳的优化条件进行诱导表达目的蛋白;SDS-PAGE电泳和Western Blot法鉴定表达产物。结果表明,通过PCR扩增技术获得了编码AFP蛋白的基因片段;重组质粒经双酶切和基因测序等方法鉴定后,确认了重组质粒已经构建成功;表达产物通过利用SDS-PAGE和Western Blot法检测到在66 kDa附近出现条带,与预期值相符,表明重组AFP蛋白已成功表达。     

Cellular assessment of the extract of bambangan (Mangifera pajang) as a potential cytoprotective agent for the human hepatocellular HepG2 cell line

This study was conducted to investigate the potential of bambangan (Mangifera pajang) fruit extracts in the protection against oxidative damage caused by tert-butyl hydroperoxide in the human hepatocellular HepG2 cell line. Proteins which might be involved in the cytoprotective mechanism were investigated using western blotting technique. Quercetin was used as a positive control. The results showed that only the kernel extract of M. pajang and quercetin displayed cytoprotective activity in HepG2 cells, with EC₅₀ values of 1.2 and 5.3 μg/ml, respectively. Expression of quinone reductase, glutathione reductase and methionine sulfoxide reductase A proteins were significantly up-regulated by quercetin, suggesting their involvement in the cytoprotective activity of quercetin. However, expressions of only glutathione reductase and methionine sulfoxide reductase A proteins were significantly up-regulated by the kernel extract, again suggesting their involvement in the cytoprotective activity of bambangan kernel extract. Future study is needed to investigate the involvement of other cytoprotective proteins in the cytoprotection mechanism.     

Ca2+、ATP、ADP和AMP对鸭胸肉中肌动球蛋白解离的影响

为了解促进肌动球蛋白解离的因素,从鸭胸肉中提取肌动球蛋白,研究Ca2+、三磷酸腺苷（adenosinetriphosphate,ATP）及其降解产物对肌动球蛋白的解离效果。通过蛋白质免疫印迹技术测定肌动蛋白含量的变化来研究肌动球蛋白的解离情况。研究发现,7.5～64 mmol/L Ca2+对肌动球蛋白的解离无促进作用（P＞0.05）,而Ca2+浓度升高到200 mmol/L时,能显著促进肌动球蛋白的解离（P＜0.05）;单独的ATP对肌动球蛋白的解离无促进作用（P＞0.05）;Ca2+和ATP共同作用于肌动球蛋白后,可显著促进肌动球蛋白的解离（P＜0.05）;二磷酸腺苷（adenosine diphosphate,ADP）、一磷酸腺苷（adenosine monophosphate,AMP）均可显著促进肌动球蛋白的解离（P＜0.05）,且不同浓度处理组间无显著性差异（P＞0.05）。因此,可以推断ADP、AMP以及Ca2+和ATP的共同作用对肌动球蛋白的解离有促进作用。     

文蛤特异性过敏原免疫识别的初步研究

目的：贝类是人类最优质的食用蛋白资源之一,也是联合国粮农组织公布的八大类过敏食物之一,贝类过敏反应严重影响着过敏人群的身体健康和生活质量,为此开展贝类过敏原的识别检测研究很有必要。方法：通过问卷调查初步了解食物过敏现状,获取自诉贝类过敏患者血清和正常人阴性对照血清,采用特异性IgE 检测试剂盒筛选贝类过敏血清,应用组织捣碎提取蛋白、聚丙烯酰胺凝胶电泳和免疫印迹等实验方法研究文蛤特异性过敏原。结果：文蛤肌肉主要蛋白的相对分子量约为200kD 以上、200、90、80、70、46、36 和24kD,其中能被贝类过敏血清特异性识别的90kD 组分约占文蛤肌肉总蛋白的10%～20%,且主要为盐溶性蛋白。结论：文蛤的特异性过敏原为90kD 蛋白组分。     

铜绿微囊藻生物钟蛋白的表达纯化与抗体制备

为了获得融合表达的铜绿微囊藻(Microcystic aeruginosa)生物钟蛋白KaiA、KaiB、KaiC并制备其相应的多克隆抗体,将kaiA、kaiB、kaiC基因分别克隆到原核表达质粒pET-His中.重组质粒pET-His-KaiA,pET-His-KaiB和pET-His-KaiC经酶切和测序鉴定后,分别转化E.coli BL21(DE3)进行融合表达.经SDS-PAGE分析可知,融合表达的KaiA、KaiB和KaiC蛋白表达量可分别达到菌体总蛋白的25%、40%和20%.经亲和层析后融合蛋白KaiA和KaiB的纯度分别达95%和92%,而KaiC经胶回收纯化后纯度也可达93%.将纯化后的三种Kai蛋白作为抗原分别免疫小鼠制备多克隆抗体,经ELISA检测抗体滴度表明,制备的抗KaiA、抗KaiB和抗KaiC的多克隆抗体效价高,分别可达到1:50000、1:60000和1:100000.Western blotting结果表明:获得的多克隆抗体具有较高的效价,抗体能识别相应的Kai蛋白,具有较高的特异性,能用于铜绿微囊藻生物钟蛋白KaiA、KaiB和KaiC的表达节律检测.