T7 RNA多聚酶基因的克隆、序列测定及其在E.coli中的表达

从BL21(DE3)E．coli菌株中以PCR的方法扩增得到了与T7RNA多聚酶(T7RNApolymerase，T7pol)基因大小一致的DNA片断。将PCR产物纯化后直接克隆到pGEM—T载体中，经酶切鉴定和DNA序列分析表明克隆得到了正确的T7pol基因。将T7pol基因亚克隆入pET-28b( )中，构建得到原核表达质粒pET28T7。该质粒的BL21(DE3)pLysS转化菌在IPTG的诱导下可表达约98800的蛋白，这与T7pol的相对分子质量一致。将该质粒转化DH5α、JMl09、HBl01、BL21(DE3)和BL21(DE3)pLysS等5种不同的宿主菌，仅有转化T7pol酶活性受到抑制的宿主菌BL21(DE3)pLysS才能得到转化子，而其余4种T7T7pol酶活性不受抑制的E．coli宿主菌不能得到转化子。pET28T7原核表达质粒这种仅能在T7pol酶活性受到抑制的宿主菌中才能存活的现象说明本试验所克隆的T7pol基因能正确表达出具有RNA转录酶活性的蛋白。     

Development of Immune Response to Experimental Bovine Trichophyton vervucosum Infection

Abstract— Cell mediated and humoral immune responses to experimental Trichophyton verrucosum infection were assessed by sequential cutaneous biopsies, antibody assessments and microscopic monitoring of fungal presence. Histopathologic examination showed the accrual of lymphocytes and other inflammatory cells in the dermis of infected sites. Immunoperoxidase staining of frozen sections with monoclonal antibody preparations revealed an influx of macrophages, BoCD4+ and B0CD8+ lymphocytes and γδ T cells from the 5th day to the 33rd day of infection. A moderate influx of B cells was observed. Protein G-colloidal gold staining revealed the presence of immunoglobulins in the dermis and superficial epidermal layers. Trichophyton specific serum antibodies appeared between days 33 and 55. Microscopic assessment of infected tissues revealed an increase in T, verrucosum elements (mycelium and ectothrix spores) from days 19 to 55. Fungal elements in infected areas did not decrease until after both humoral and cell mediated elements of the immune response were established. These responses imply a combination of cell mediated and humoral events were associated with T. verrucosum immunity and clearance in the calf. Résumé— Le réponse immunitaire humorale et cellulaire a une infection expérimentale àT. verrucosum a été appréciée par des biospsies cutanées successives, des dosages d'anticorps et la recherche microscopique de champignons. L'examen histopathologique a montré un afflux de lymphocytes et d'autres cellules inflammatoires dane le derme des sites infectés. Les marquages en immunopéroxydase par un anticorps monoclonal de coupes congelées a montré un influx de macrophages, lymphocytes BoCD4+ et BoCD8+ et des cellules T γδ, du 5e au 33e jour de l'infection. Un marquage par une protéine G—or colloidal a révélé d'immunogolglobulines dans le derme et les couches supéerficielles de l'épiderme. Les anticorps spécifiques de Trichophyton sont apparus entre 33 et 55 jours. L'examen microscopique des tissus infectés a révélé une augmentation du nombre d'éléments de T. verrucosum (mycelium et spores ectothrix) du 19e au 55e jours. Les éléments fongiques dans les zones infectées n'ont pas diminué avant que les réponses humorales et cellulaires ne solent établies. Ces résponses impliquent qu'une coopération des réponses humorales et cellulaires étaient associées dans l'immunité et la défense contre T. verrucosum. Zusammenfassung— Die zellvermittelten und humoralen Immunantworten auf die experimentelle Infektion mit T. verrucosum wurden durch eine Serie von Hautbiopsien, Antikörperuntersuchungen und mikroskopischer Untersuchung auf das Vorhandensein von Pilzen ausgewertet. Die histopathologische Untersuchung zeigte eine Ansammlung von Lymphozyten und anderen Entzündungszellen in der Dermis der infizierten Stellen. Die Immunperoxidasefärbung der Gefrierschnitte mit monoklonalen Antikörperzubereitungen zeigte einen Influx von Makrophagen, BoCd4-und BoCD8-Lymphozyten und gamma-delta-T-Zellen vom 5. bis zum 33. Tag der Infektion. Es wurde auch ein mäßiger Influx von B-Zellen beobachtet. Die Protein-G-kolloidale Goldfärbung zeigte die Anwesenheit von Immunglobulinen in der Dermis und den oberflächlichen epidermalen Schichten. Trichophyton-spezifische Serumantikörper traten zwischen Tag 33 und 55 auf. Die mikroskopische Untersuchung infizierter Gewebe zeigte eine Zunahme von T. verrucosum-Bestandteilen (Myzel und exktothrixe Sporen) vom Tag 19 bis 55. Pilzteile in infizierten Bereichen verminderten sich weder, nachdem humorale, noch nachdem zellvermittelte Elemente der Immunantwort auftraten. Diese Reaktionen deuten an, daß eine Kombination von zellvermittelten und humoralen Vorgängen im Zusammenhang mit T. verrucosum-Immumtät und Abheilung beim Kalb vorliegt. Resumen Por medio de biposias cutáneas secuenciales, medida de anticuerpos y exámen microscópico de presencia de hongos, se estudió la respuesta inmunitaria de tipo humoral y celular producida por la infección experimental con T. verrucosum. El exámen histopatológico reveló la presencia de agregación de linfocitos y otras células inflamatorias procedentes de la dermis de los puntos infectados. La tintura por medio de inmunoperoxidasa de las secciones congeladas, con preparaciones monoclonales de anticuerpos, demostró un aflujo de macrófagos BoCD4 + y BoCD8 + linfocitos y linfocitos, Tαδ, desde el quinto hasta el día 33 la infección. También se observó un aflugo moderado de linfocitos B. La tintura aúrica de proteina coloidal G reveló la presencia de inmunoglobulinas en al dermis y capas superficiales de la epidermis. Los anticuerpos específicos para la especie Trichophyton aparecieron entre los días 33 y 55. El exámen microscópico de los tejidos afectados demostró un incremento, de los elementos füngicos T. verrucosum (micelio y esporas exótricas) desde los días 19 al 55. Los elementos fúngicos en áreas infectadas no disminuyeron hasta después del establecimiento de ambos tipos de respuesta inmunitaria, humoral y celular. Estas respuestas implican que la combinación de ciertos fenómenos de inmunidad celular y humoral, están relacionados con la desaparición y la inmunidad de la infección producia por T. verrucosum en el ternero.     

Rapid Screening for Aluminum Tolerance in Cereals by Use of the Chlorophyll Fluorescence Test

The effects of aluminum on the photosynthetic apparatus were examined in cereals grown in nutrient solutions (pH 4.5) at two Al levels (0 and 148 μM). The chlorophyll fluorescence kinetics results confirmed that the soft wheat ‘BHG’ cultivar has the potential for growth on acid soils while triticale cultivars ‘Niovi’ and ‘Dada’ appeared to be relatively tolerant. The percentage decrease in F_v/F_m of the less tolerant cultivars after Al-treatment indicated a decrease in the efficiency of the primary photochemistry of PS II, while the decrease in the ratio F_V/F_osuggested that exposure of the cultivars ‘Dio’ and ‘Appulo E’ to aluminum caused injury to the thylakoid structure. The percentage fluctuations of the ratio F_v/F_mwere shown to correlate very closely with the assessment of injury as evaluated by the relative top fresh weight. Measurements of chlorophyll fluorescence in vivo could be used to monitor injury caused by “Al-stress”, and thus they may serve as a rapid screening test for Al tolerance.     

牛流行热病毒灭活疫苗免疫后牛外周血淋巴细胞亚类分布的研究

对牛流行热病毒灭活疫苗免疫并攻每后，牛外周血淋巴细胞亚类的变化进行了研究。结果表明，灭活苗免疫后，牛的CD4^ 显著升高，可能与参与辅助B淋巴细胞合成抗体有关，攻毒后，γδT细胞均显著上升，免疫组在攻毒后3周仍保持在相当的高水平，IL－2Rα阳性细胞在攻毒后高热期也有显著升高，而CD8^ 细胞没有明显变化。     

Cytochemical properties of chicken blood cells resembling both thrombocytes and lymphocytes

Generally accepted criteria were used to identify typical nucleated thrombocytes and typical small lymphocytes in chicken-blood smears subjected to modified-Wright staining. Other cells, here referred to as "intermediate cells," were difficult to classify because in some aspects they resembled thrombocytes while they also had features typical of small lymphocytes. The "intermediate cells" had small, round or oval nuclei with coarsely condensed chromatin, characteristic of both thrombocytes and small lymphocytes. In addition, "intermediate cells" had moderately abundant cytoplasmic volumes, typical of thrombocytes but blue cytoplasm lacking both granules and vacuoles, which is characteristic of small lymphocytes. It made little difference to the thrombocyte count whether these cells were classified as thrombocytes or small lymphocytes; however, this decision made a substantial difference to the lymphocyte count in some chicken-blood smears. Most "intermediate cells" (351 of 410 cells examined) were nonfluorescent after treatment with formaldehyde gas. Furthermore, most "intermediate cells" failed to acquire characteristic pigments when subjected to either Grimelius staining (179 of 204 cells examined) or periodic acid-Schiff staining (173 of 206 cells examined). Typical small lymphocytes reacted in the same way, failing to fluoresce after gaseous formaldehyde treatment (65 of 65 cells examined) and failing to react during Grimelius staining (41 of 44 cells examined) or periodic acid-Schiff staining (21 of 21 cells examined). In contrast, almost all typical thrombocytes became fluorescent in response to gaseous formaldehyde (709 of 718 cells examined) and gave positive reactions when subjected to Grimelius staining (381 of 382 cells examined) or periodic acid-Schiff staining (322 of 326 cells examined). These findings suggested that "intermediate cells" should be classified as lymphocytes in differential cell counts.     

Identification of feline blood B and T lymphocytes by cell electrophoresis.

The electrokinetic properties of feline blood lymphocytes isolated by centrifugation over Ficoll-Isopaque gradient were investigated. A biphasic electrophoretic mobility (EPM) distribution was regularly observed with a low-mobility (LM) population (mean EPM: 0.82) accounting for 32% of blood lymphocytes and a high-mobility (HM) population (mean EPM: 1.09) representing 68% of blood lymphocytes. Following fractionation on nylon-wool columns, lymphocytes with B-cell properties (64% sIg⁺; 9% guinea pig erythrocytes (GPE)-rosette⁺, PHA and Con A unresponsive) were enriched in the adherent fraction and belonged mainly (78%) to the LM population. In contrast, lymphocytes with T properties (5% sIg⁺, 42% GPE-rosette⁺, PHA and Con A responsive) were recovered in the effluent fraction and comprised 84% of HM elements.Thus, in cat blood, LM lymphocytes are likely to represent in majority B cells and HM lymphocytes T cells. This indicates that cell electrophoresis provides an interesting mean for differentiating B and T cells in the cat.     

Analysis of the distribution and clonality of TCR Vβ T cells in cord blood

AIM:To investigate the distribution and clonality of TCR Vβ subfamily T cells in cord blood. METHODS:The CDR3 of TCR Vβ 24 subfamily genes were amplified in mononuclear cells from 13 cases of cord blood. To observe the usage of TCR Vβ repertoire, the PCR products were further labeled with fluorescent and analyzed by genescan technique for the CDR3 size, to evaluate clonality of the detectable TCR Vβ T cells. Peripheral bloods from 10 cases of normal individuals and T cell line Molt-4 and Jurkat served as controls. RESULTS:Only 38.78%±16.26% of 24 Vβ subfamily T cell were selectively expressed in cord blood, predominantly in Vβ 3, 5, 8, 9 and 13, whereas all 24 Vβ subfamilies could be detected in T cells from peripheral blood of normal individuals. Genescan analysis showed that all PCR products of TCR Vβ subfamilies from cord blood or normal individual peripheral blood displayed multi-peaks. CONCLUSION:Some TCR Vβ subfamily T cells were absent in cord blood. All TCR Vβ subfamily T cells in cord blood displayed polyclonality.     

中药成分对雏鸡外周血T淋巴细胞转化的影响

38 0只雏鸡随机分为 19组 ,于 11日龄试验组分别肌肉注射高、低剂量的当归多糖、黄芪多糖、板蓝根多糖、淫羊藿多糖、蜂胶多糖、淫羊藿黄酮、蜂胶黄酮、黄芪皂甙和人参皂甙等 9种中药成分 ,对照组注射同量生理盐水 ,每天 1次 ,连续 3d,于 14日龄用新城疫 系弱毒疫苗首免 ,2 8日龄二免。分别于 35、4 2、4 9和 5 6日龄心脏采血 ,监测 T淋巴细胞转化的动态变化。结果表明 ,所有中药成分均能显著促进 T淋巴细胞转化 ,并有一定的量效和时效关系