赖型钩体重组质粒pDJH_2亚克隆及其在大肠杆菌中的高效表达

为了探讨ｐＤＪＨ２的１．９ｋｂ０１７株钩体ＤＮＡ片段可否作为钩体重组疫苗广谱保护性抗原的候选，采用重组ＤＮＡ技术，将ｐＤＪＨ２的１．９ｋｂＤＮＡ片段分别与ｐＴ７－７，ｐＲＳＥＴ系列重组，转化入大肠杆菌进行亚克隆，ＩＰＴＧ诱导表达。结果显示：阳性亚克隆ｐＤＪｔ．ｐＤＪｒＢ１能在大肠杆菌中高效表达；对其进行ＳＤＳ－ＰＡＧＥ分析，可见６８ｋｄ和２３ｋｄ处出现新带，与钩体０１７株外膜同分子量蛋白带位置一致，免疫印迹（特异性抗０１７株外膜抗血清）在６８ｋｄ和２３ｋｄ处分别有印迹；用ｐＤＪｔ亚克隆重组质粒主动免疫豚鼠，可抵抗强毒力株攻击，表现出免疫保护作用。本实验结果提示：（１）ｐＤＪＨ２１．９ｋｂ重组ＤＮＡ片段能以不同质粒为载体，在不同大肠杆菌株中高效表达；（２）该重组ＤＮＡ片段表达产物——６８ｋｄ和２３ｋｄ蛋白，可能是赖型钩体０１７株外膜的保护性抗原。     

自身抗原Ro 52 kd多肽分子cDNA表达克隆的构建及其融合蛋白在大肠杆菌中的表达

采用ＰＣＲ法克隆自身抗原Ｒｏ５２ｋｄ多肽分子的ｃＤＮＡ，定向插入表达载体ＰＧＥＸ－４Ｔ－１，并导入大肠杆菌中表达重组融合蛋白，经免疫印迹法表明，重组融合蛋白具有Ｒｏ５２ｋｄ的抗原性。这为今后对Ｒｏ５２ｋｄ抗原表位的精确定位，分析特定抗原表位与疾病的相关性及制备重组抗原用于临床检测奠定了基础。     

Rat olfactory neurons express a 200 kDa neurofilament

Neurofilament expression in peripheral olfactory neurons of adult rats was investigated by immunoblotting and immunohistochemistry using monoclonal antibodies specific for each of the 3 neurofilament proteins. Immunoblotting analysis of olfactory epithelium extracts demonstrated the presence of only the 200 kDa (NFH) polypeptide; the 68 kDa (NFL) and 160 kDa (NFM) neurofilaments were not detected. Similarly, no immunoreactivity was observed in tissue sections using the NFL and NFM antibodies. In contrast, when sections were probed with the antibody to NFH, immunoreactivity was localized primarily in the dendritic knobs and near the cell bodies of the receptor cells.     

Hypoxia-induced ABR change and heat shock protein expression in the pontine auditory pathway of young rabbits

The auditory brainstem response (ABR) was compared with the immunohistochemical expression of heat shock protein (HSP-72) and microtubule-associated protein 2 (MAP-2) of the brainstem auditory pathway in young rabbits subjected to hypoxic stress. Severe hypoxia for 2 h produced significant prolongation and decreased amplitude of the later component of ABR. HSP-72 expression was distinctly increased in the cochlear nucleus, but there was less induction in the inferior colliculus under severe hypoxia. MAP-2 immunostaining of neuropiles in the inferior collicular nucleus was decreased slightly after severe-long hypoxia, but cytoplasmic staining did not change. The present ABR change, which was produced by brainstem hypoxia-ischemia and acidosis, may be due to the neural cytoarchitectural derangement and less induction of stress proteins in the upper brainstem.     

RH株弓形虫表面抗原p22基因与p30基因联合表达后的免疫活性

目的:研究弓形虫表面抗原p22基因与p30基因的联合表达。方法:用RT-PCR及PCR方法获得p22和p30基因,联合构建在表达载体中,经酶切与测序鉴定后,用IPTG对工程菌进行诱导表达,表达产物行SDS-PAGE蛋白凝胶电泳及Western-blot免疫印迹检测。结果:经RT-PCR可得到长约438bp的p22外显子基因片段,蛋白电泳结果显示,阳性重组菌在66.5Kda位置上明显多一条带,此条带可与p22抗体结合并使免疫印迹显示阳性结果。结论:弓形虫表面抗原p22基因的有效基因片段与p30基因片段联合表达后,在融合蛋白中仍具有免疫原性。     

Expression levels of hepatic cytochrome P450 enzymes in Aldh2-deficient mice following ethanol exposure: a pilot study

We determined expression levels of hepatic cytochrome P450 (CYP) 2E1, 2B1/2 and 4B1 enzymes in aldehyde dehydrogenase 2 (Aldh2) +/+ and Aldh2 –/– mice by immunoblotting assay following subchronic ethanol exposure for eight days. Using ethanol exposure, the protein expression levels of CYP2E1, 2B1/2 and 4B1 in Aldh2 +/+ mice were increased by factors of 2.61, 1.88 and 2.01 compared with Aldh2 +/+ mice that were not exposed to ethanol, respectively. On the other hand, in the Aldh2 –/– mice, CYP2E1, 2B1/2 and 4B1 protein expression levels after ethanol treatment were shown to be 1.99, 1.05 and 1.33 greater than those of Aldh2 –/– control mice, respectively. We also found an interesting fact in the present study; the Aldh2 –/– mice were shown to have higher CYP2E1 (by a factor of 4.19), 2B1/2 (by a factor of 2.89) and 4B1 (by a factor of 1.53) protein expression levels than Aldh2 +/+ mice despite the lack of ethanol treatment. These results suggest that CYP2E1, 2B1/2 and 4B1 play some role in ethanol metabolism and that Aldh2-deficient individuals may have higher levels of CYP2E1, 2B1/2 and 4B1 enzymes compared to Aldh2 wild-type individuals.     

Phosphorylation-status of phospholamban and calsequestrin modifies their affinity towards commonly used antibodies

Phospholamban (PLB) and calsequestrin (CSQ) play important roles in sarcoplasmic reticulum Ca(2+) transport and storage in cardiac muscle. Specific antibodies have been frequently used to quantitate CSQ and PLB protein levels. Here we demonstrate that two of the commonly available anti-PLB antibodies, anti-PLB-2D12 and anti-PLB-A1, show lower reactivity to phosphorylated than dephosphorylated PLB. A custom anti-PLB antibody, generated using a peptide corresponding to amino acids 2-14, is not affected by the phosphorylation state of PLB. In contrast, anti-CSQ reacts less with dephosphorylated CSQ than with phosphorylated CSQ. All three commercially available antibodies tested in this study have been widely used to quantify PLB and CSQ expression, and the results are integrated in many publications. Our studies reveal that the phosphorylation status of PLB and CSQ can affect antibody reactivity and may lead to over- or underestimation of the relative protein content and erroneous interpretation of data.     

High-affinity IgE receptor-beta chain expression in human mast cells

The high-affinity IgE receptor (FcepsilonRI)-beta gene is one of the atopy-associated genes, but its biological significance is largely unknown. In this study, we generated the anti-FcepsilonRI-beta chain antibody to clarify beta-chain protein expression in human mast cells. The FcepsilonRI-beta antibody showed specific binding to a 27 kDa protein with Western blotting and membrane bound immunostaining using cultured mast cells. Monomeric IgE sensitization increased beta-chain expression as well as mature alpha-chain expression in mast cells. Upregulation of beta-chain expression with monomeric IgE treatment suggests possible roles of FcepsilonRI-beta protein as an atopy-related molecule.     

二种丙型肝炎病毒抗体确证试剂盒检测性能的比较

目的 应用丙型肝炎病毒抗体确证试验检测肝病患者血清抗HCV,进一步确认HCV感染.方法 对北京万泰生物药业股份有限公司初步研制重组免疫印迹法检测抗HCV试剂(简称CWT)与CHIRON RIBA HCV 3.0 Strip Immunoblot Assay进行比较,采用477份血清标本进行检测分析(慢性丙型肝炎病毒感染者血清350份、非甲非戊型肝炎患者血清7份、对照组采用乙型肝炎患者血清30份、戊肝肝炎患者血清30份及正常献血人员血清60份).结果 120份对照组非丙型肝炎患者血清均为抗HCV阴性;350份慢性丙型肝炎病毒感染者血清,国产试剂检出阳性341份,9份不确定;CHIRON RIBA HCV 3.0 SIA试剂检出阳性343份,不确定7份.7份非甲非戊型肝炎患者血清两种试剂检测均为阳性2份,阴性4份,不确定结果1份.两种试剂比较,检测结果一致率为99.16%(473/477),两者有很强的一致性(Kappa=0.98).结论 两种试剂对丙型肝炎病毒抗体的检测方法具有高度的一致性.特别是对于非甲-非戊型肝炎患者中的HCV感染者更有一定的诊断意义.

Abstract：

Objective To detect anti-HCV in serum of hepatic disease patients by performing the confirmatory test, and further to confirm HCV infection. Methods Two recombinant immunoblot assays (CWT and CHIRON RIBA HCV 3.0 Strip Immunoblot Assay) were used respectively to detect anti-HCV in 477 human serum samples, which comprised 350 HCV-infected patients' specimens, 7 none-A none-E hepatitis specimens, 30 HBV-infected patients' specimens, 30 hepatitis E virus infected patients'specimens, and 60 specimens drawn from blood donors. The latter three groups served as controls. Results A total of 120 control non-HCV-infected patients' specimens were negative when tested by both assays. Among 350 HCV-infected patients, 341 were positive and 9 were indeterminated by CWT assay; 343 were positive and 7 were indeterminated by CHIRON RIBA HCV 3. 0 SIA. Seven none-A none-E hepatitis specimens tested by both assays turned out to be 2 positive, 4 negative and 1 indeterminate. The consistency rate of these two assays was 99. 16% (Kappa=0.98). Conclusion CWT assay is highly coherent with CHIRON RIBA HCV 3.0 SIA assay in the methodology of anti-HCV antibody detection, which can be applied in the determination of HCV infection among none-A none-E hepatitis patients.     

人黑色素瘤A375细胞中B7-H4表达的实验研究

目的:探讨B7-H4在人黑色素瘤A375细胞中表达及临床意义。方法:体外培养人黑色素瘤A375细胞,倒置显微镜观察细胞形态;采用RT-PCR和Western blot检测B7-H4 mRNA的转录水平及蛋白的表达水平。结果:B7-H4 mRNA和蛋白在A375细胞中表达均增高。结论:B7-H4在人黑色素瘤A375细胞中表达升高,可为黑色素瘤的诊断和治疗提供参考。