肺结核病患者PBMC膜白介素-2受体(CD25)表达及其临床意义

目的 :探讨外周血单个核细胞 (PBMC)膜白介素 - 2受体 m IL- 2 R,(CD2 5 )表达在肺结核病鉴别诊断中的应用价值。方法 :用生物素 -链霉亲和素 (BSA)法检测肺结核、支气管肺炎患者 T细胞亚群及植物血凝素 (PHA)诱导前后 CD2 5表达水平。结果 :支气管肺炎患者 CD+ 3、CD+ 4、CD+ 8水平分别为 (6 2 .32±6 .34) %、(47.5 2± 7.16 ) %、(32 .12± 6 .5 5 ) % ,CD+ 4/ CD+ 8比值为 1.5 2± 0 .4 3,PHA诱导前后 CD2 5水平分别为 (4.5 6± 1.5 2 ) %、(35 .12± 7.2 1) %。空洞型肺结核 CD+ 3、CD+ 4、CD+ 8、CD+ 4/ CD+ 8水平分别为 (41.13±5 .2 5 ) %、(43.38± 5 .15 ) %、(36 .2 5± 3.4 6 ) %和 1.15± 0 .2 1,非空洞型肺结核 CD+ 3、CD+ 4、CD+ 8、CD+ 4/ CD+ 8水平分别为 (46 .2 9± 5 .6 0 ) %、(47.2 1± 4 .86 ) %、(32 .36± 4 .0 3) %、1.4 6± 0 .2 5 ,相互比较 CD+ 3、CD+ 3/CD+ 8差异均有显著性 (P<0 .0 1和 P<0 .0 5 )。空洞型肺结核与非空洞型肺结核患者 PHA诱导前后 CD2 5水平分别为 (2 .13± 1.14 ) %、(2 7.2 5± 3.5 0 ) %和 (3.4 3± 1.35 ) %、(31.14± 4 .11) % ,两者相比差异均有显著性 (P<0 .0 1)。结论 :肺结核病患者体内存在明显的细胞免疫功能紊乱 ,主要表现为 CD2 5表达水平降     

In vivo biotinylation of recombinant beta-glucosidase enables simultaneous purification and immobilization on streptavidin coated magnetic particles

Beta-glucosidase from Bacillus licheniformis was in vivo biotinylated in Escherichia coli and subsequently immobilized directly from cell lysate on streptavidin coated magnetic particles. In vivo biotinylation was mediated by fusing the Biotin Acceptor Peptide to the C-terminal of beta-glucosidase and co-expressing the BirA biotin ligase. The approach enabled simultaneous purification and immobilization of the enzyme from crude cell lysate on magnetic particles because of the high affinity and strong interaction between biotin and streptavidin. After immobilization of the biotinylated beta-glucosidase the specific activity (using p-nitrophenyl-β-d-glucopyranoside as substrate) was increased 6.5 fold (compared to cell lysate). Immobilization of the enzyme resulted in improved thermal stability compared to free enzyme; after 2 h of incubation (at 50 °C) the residual enzyme activity of immobilized and free beta-glucosidase was 67 and 13%, respectively. The recyclability of immobilized beta-glucosidase was examined and it was observed that the enzyme could be recycled at least 9 times and retain 89% of its initial activity.     

Androgenic control of antagglutinin secretion in the boar epididymal epithelium

Summary Antagglutinin, a specific protein synthesized by the boar epididymis, was secreted by the principal cells of the initial segment, the caput and the corpus, but was not detectable in the caudal cells. Castration completely abolished the synthesis and secretion of antagglutinin in all epididymal cells. Androgen replacement suggests that the epithelial cells from different segments have differential regulatory mechanisms. The proximal zone appeared refractory to exogenous testosterone; the median zone was a typical androgen-dependent region; and the caudal cells, where an unusual secretion of antagglutinin was detected, revealed still a different reaction pattern. It is postulated that these latter cells depend not solely on androgen but also or exclusively on other factors. Our results, which demonstrate a primary role of the Golgi complex in the secretory process in the epididymal cells, also suggest that the apical smooth endoplasmic reticulum may be implicated in the intracellular transport of glycoproteins to the cell surface.     

An electrochemical enzyme bioaffinity electrode based on biotin-streptavidin conjunction and bienzyme substrate recycling for amplification

A signal amplificatory electrochemical immunoassay with biotin-streptavidin conjunction and multienzymatic-based substrate recycling was developed in this work. Biotinylated secondary antibody (bio-IgG) was preliminarily assembled onto the immunosensor interface based on the sandwich format. Streptavidin was then loaded based on biotin-streptavidin conjunction. Owing to four identical binding sites of streptavidin to biotin, amounts of biotinylated alkaline phosphatase (bio-AP) were attached, and this improved the catalytic performance of the proposed immunosensor. Under the enzyme catalysis of AP, the electroinactive p-aminophenylphosphate (PAPP) substrate was rapidly hydrolyzed into the electroactive p-aminophenol (PAP) product, which next oxidized at the electrode surface into p-quinoneimine (PQI). In the presence of diaphorase (DI), PQI was reduced back to PAP, leading to a reversible cycle of PAP. Then the oxidized state of DI was regenerated into its reduced native state by its natural substrate, nicotinamide adenine dinucleotide (NADH). With the several amplification factors mentioned above, a wider linear ranged from 10⁻¹⁴ to 10⁻⁵ g ml⁻¹ was acquired with a relatively low detection limit of 3.5 × 10⁻⁵ g ml⁻¹ for human IgG. In addition, the nonspecific adsorption of proposed immunosensor was also investigated here.     

Immunocytochemical localization of antagglutinin in the boar epididymis

Summary Antagglutinin, a specific protein synthesized by the boar epididymis, was localized by the biotin-streptavidin method in all the principal cells of the caput and corpus epididymidis as well as in the lumen of this organ. Intracellular staining, which was first detected in the initial segment, appeared stronger in the distal caput and in the corpus but diminished and disappeared in the caudal epididymal cells. In all the principal cells, a consistent reaction product was localized in the large Golgi complex. Only slight and diffuse immunoreactive material was detected in the cytoplasm, except in the middle caput where the heterogeneous reactive granules appeared to be intracellular sites of degradation of this protein. In the lumen, the intensity of reaction increased from the caput to the cauda. Antagglutinin appeared strongly associated with the luminal surfaces, especially around and between the stereocilia. However, the spermatozoa also exhibited a distinct pattern of immunostaining. The results are discussed in relation to protein secretion in the epididymis and to the role of antagglutinin in the gamete-interaction process.     

应用纳米金探针检测HSV-2的目视化基因芯片

本实验建立了一种应用金标链霉亲和素探针的目视化高灵敏度检测单纯疱疹病毒2型(HerpesSimplexVirus-2,HSV-2)的基因芯片。该芯片以HSV-2DNA聚合酶的高保守区为靶序列,设计HSV-2特异性引物和探针,通过PCR反应使扩增产物标记上生物素;氨基修饰的探针固定在活化的玻片上,与生物素标记的扩增产物杂交;利用生物素与链酶亲和素高亲合力的特性,加入纳米金标记的链酶亲和素后形成生物素-链酶亲和素-纳米金生物反应放大系统;银染反应后,达到目视化检测HSV-2效果。该HSV-2检测基因芯片能目视化检测出100fmol/L的HSV-2扩增产物,具有灵敏度高,低成本的特点,通过临床标本验证表明该芯片具有较高的准确性。