日本囊对虾组织蛋白酶B基因的原核表达及纯化

采用原核表达的方法得到日本囊对虾(Marsupenaeus japonicus)组织蛋白酶B基因的重组蛋白。以日本囊对虾卵巢组织为试验材料,采用双标签(GST和His)的方法,用带有EcoRⅠ和NotⅠ酶切位点以及6×His-tag的特异引物扩增Cathepsin B的开放阅读框,并连接至表达质粒pGEX-4T-2中。将重组表达质粒导入大肠杆菌BL21中,在30℃条件下,用终浓度为1mmol/L的IPTG(isopropylβ-D-1-thiogalactopyranoside)诱导5h得到最佳诱导量,His亲和柱进行纯化,纯化蛋白依次于8、6、4、2、1、0 mol/L尿素缓冲液中梯度透析,得到复性后可溶于水的重组蛋白,经SDS-PAGE检测,得到单一条带,其分子质量约为63ku。取冻干后的蛋白制备多克隆抗体,抗体效价达50 000,经Western blot检测,同样可在63ku处得到该条带,表明制备的组织蛋白酶B(CB)多克隆抗体具有特异性,该抗体可特异识别CB蛋白。     

大片吸虫成虫cDNA文库的构建和组织蛋白酶L1基因的克隆

目的大片吸虫cDNA文库的构建及组织蛋白酶L1(Fasciola gigantica Cathepsin L1,FgCL1)基因的克隆。方法提取大片吸虫成虫总RNA,运用SMART技术构建大片吸虫cDNA文库。根据文献设计并合成PCR引物,从上述文库中克隆大片吸虫FgCL1基因,定向克隆至原核表达载体pET28a中。结果文库容量为2.08×106pfu/mL,重组率为98%。扩增后的文库滴度为6.23×109 pfu/mL,插入片段平均大小约为1 000 bp。应用两个已知基因(FgCL1 FgGST)从文库中成功钓取到相应的全长基因。将含有FgCL1的阳性克隆测序,用Genebank Blast进行序列比较,证实为FgCL1基因。结论成功构建了cDNA文库,提供筛选大片吸虫基因资源;从该文库中克隆出FgCL1,为进一步表达该基因,研制诊断试剂盒创造了条件。     

鸭肉中组织蛋白酶B的特性研究

从樱桃谷肉鸭腿肌中提取组织蛋白酶B,研究了pH值、温度、蛋白酶激活剂和抑制剂对其酶活的影响。结果表明:组织蛋白酶B的最适反应pH值为5.5,最适反应温度为40℃,在30℃下和pH 5.0～5.5范围内有较好的稳定性。激活剂二硫苏糖醇(DTT)和L-半胱氨酸(L-cys-teine)对组织蛋白酶B有一定的激活作用;抑制剂E-64对组织蛋白酶B有明显抑制作用;金属蛋白酶抑制剂已二胺四乙酸(EDTA)和丝氨酸蛋白酶抑制剂苯甲基磺酸氟(PMSF)对组织蛋白酶B无抑制作用。     

Gene structure of carp Cyprinus carpio cathepsin B

ABSTRACT:   The 2474 nucleotides of carp cathepsin B gene (corresponding to the part of open reading frame and 3'non-coding region of cDNA) have been determined by polymerase chain reaction cloning, which was organized into nine exons and eight introns. The boundary sequences of the exon-intron junctions conformed to the GT/AG consensus rule. One polyadenylation signal sequece of AATAAA was found in the 3'non-coding region. The sizes of the determined introns were approximately 100-200 bp in length (varied from 71 bp-239 bp), which were significantly shorter than those of mouse cathepsin B gene.     

Localization of cathepsin K and tartrate-resistant acid phosphatase in synovium and cranial cruciate ligament in dogs with cruciate disease

OBJECTIVE: To localize cathepsin K and tartrate-resistant acid phosphatase (TRAP) in synovium and cranial cruciate ligament (CCL) of dogs with cruciate disease. ANIMALS: Dogs (n=15) with cruciate disease and ruptured CCL, and 12 dogs with intact CCL. METHODS: Synovium and CCL were examined histologically and cells containing cathepsin K or TRAP were identified immunohistochemically and histochemically, respectively. RESULTS: Increased cellular localization of cathepsin K and TRAP was detected in synovium and ruptured CCL in dogs with cruciate disease, when compared with tissues from dogs with intact CCL. Inflammation of synovium with TRAP+ macrophage-like cells was seen in 73% of dogs with CCL disease, but was not seen in dogs with intact CCL. The presence of cathepsin K and TRAP protein in synovium and CCL tissues was significantly correlated in dogs with CCL rupture. CONCLUSION: Inflammation of the epiligament of ruptured CCL with cathepsin K+ and TRAP+ macrophage-like cells forms part of a similar, more generalized chronic inflammatory change within the periarticular tissues of the stifle of a large proportion of dogs with CCL rupture. CLINICAL RELEVANCE: Production of matrix-degrading enzymes by the synovium may induce progressive pathologic rupture of the CCL. Therefore, these collagenolytic pathways may offer a novel target for medical therapy of joint inflammation in canine patients with cruciate disease.     

Muscle Quality and Proteolytic Enzymes of Farmed Atlantic Cod (Gadus morhua) During Storage: Effects of Pre-Slaughter Handling and Increased Storage Temperature

ABSTRACT

In the present experiment, the characteristics of minimally handled, directly slaughtered farmed Atlantic cod were compared to cod subjected to 2 h preslaughter handling stress. The results strongly indicate that increasing the storage temperature from 0 to 5°C had a larger negative impact on the quality characteristics than the handling stress applied. Further, there may be interaction effects between handling, storage temperature, and storage time.     

日本囊对虾卵母细胞发育过程中组织蛋白酶C比活性测定

组织蛋白酶C是一种溶酶样半胱氨酸蛋白酶,近年我们报道了组织蛋白酶C的表达与日本囊对虾(Marsupenaeus japonicus) 卵母细胞最后成熟阶段密切相关,本研究对卵母细胞发育主要不同时相特别是卵母细胞最后生理成熟过程进行了连续观察,测定了组织蛋白酶C在卵母细胞发育过程中的比活性。结果表明,日本囊对虾卵母细胞发育可划分为9个时相,即卵原细胞时相、核仁时相、周边核仁时相、初级卵黄发生时相、次级卵黄发生时相、皮质棒早期时相、皮质棒中期时相、皮质棒晚期时相、排卵期时相,生发泡的破裂是在排卵之前就已经发生;免疫印迹实验结果显示组织蛋白酶C蛋白只在卵母细胞最后成熟阶段即皮质棒发生时期表达,但酶活性测定结果表明组织蛋白酶C比活力在即皮质棒发生前后没有变化,酶活性较低,以上数据提示组织蛋白酶C可能与受精时皮质棒的释放以及受精卵外胶膜的形成有关。