链激酶

The recombinant mouse enterokinase light chain could be well expressed in Ecoli, but most of them were inclusion.

利用pET32a/BL21(DE3)表达系统成功地在大肠杆菌中表达了重组鼠源性肠激酶轻链，表达量约占大肠杆菌BL21(DE3)总蛋白的30%，但多以包涵体的形式存在。

Recombinant mouse enterokinase can be expressed in Ecoli in inclusion manner so that the prokaryotic expression system for recombinant enterokinase with native conformation needs to be optimized.

鼠源性肠激酶轻链在原核细胞中多以包涵体形式表达，天然构象重组肠激酶的获得还需对表达系统进行优化。

Methods The cDNA coding sequencing of enterokinase light chain was amplified by RT-PCR from mouse dodecadactylon mucosa and cloned into pET32a expression plasmid. The recombinant enterokinase light chain was expressed in BL21(DE3) and purified with Ni-affinity chromatography.

采用RT-PCR从C57BL/6J小鼠的十二指肠肠系膜黏膜组织中钓取肠激酶轻链的cDNA，将其克隆入pET32a原核表达载体中，并在大肠杆菌BL21(DE3)中进行表达，然后以镍亲和层析法对表达产物进行纯化。

The objective of the study was to obtain the gene of bovine enterokinase light chain,which would be used in the cleavage and purification of fusion proteins.The fragment of bovine enterokinase light chain cDNA was obtained by RT-PCR from a sold bovine′s duodenal mucosa, then cloned into the pUCmT cloning vector and sequenced.Compared with the sequence deposited in GenBank,the cloned gene sequence is correct.

为克隆表达牛肠激酶轻链编码基因，以期应用于融合蛋白的切割与纯化，从市售北方黄牛十二指肠组织中提取总RNA，以RT-PCR方法扩增其cDNA片段，将此片段克隆于pUCmT载体中，经过特异性限制性内切酶酶切分析片段正确后，进行全序列分析。

The protein encoded by this sequence was deduced to be consisted with 213 amino acids, signal peptide sequence from 1 to 17 position, and transmembrane regions from 171 to 190 position, whose grand average of hydropathicity is computed to be 0.031, secondary structure is composed by alpha helix (57.28%), extended strand (6.57%) and random coil (36.15%), subcellular localization was cytoplasmic by CELLO v.2.5. There are some function sites predicted by PROSCAN software, such as one N-glycosylation site (151-154aa), one protein kinase C phosphorylation site (193-195aa), two casein kinase II phosphorylation sites (155-158aa and 173-176aa), and one N-myristoylation site (97-102aa).

推测编码蛋白由213个氨基酸组成，信号肽序列位于1-17aa，亲水性指数为0.031，跨膜区域位于171-190aa，二级结构由-螺旋（57.28%）、延伸主链（6.57%）和无规卷曲（36.15%）组成；亚细胞定位于细胞质，含有N-糖基化位点1个(151-154aa)，蛋白激酶C磷酸化位点1个(193-195aa)，酪蛋白激酶Ⅱ磷酸化位点2个(155-158aa and 173-176aa)，N端酰基化位点1个(97-102aa)。

OrLPT1 ORF encods a polypeptide with 535 amino acid residues (molecular mass of 57.6KDaltons), which shares more than 64% amino acid identity with some other high-affinity Pi transporters of plants. Hydropathy analysis of the deduced polypeptides showed that the OrLPT1 is highly hydrophobic and contains 12 putative membrane-spanning regions.

OrLPT1 ORF 编码长为535 个氨基酸(分子量57.6 KDaltons)的蛋白，与其他植物物种磷酸盐转运蛋白的同源性在64%以上；多肽链的疏水性分析表明，OrLPT1 具有12 个疏水的跨膜区及高亲和力磷酸盐转运蛋白中保守的3 个磷酸化作用位点：蛋白激酶C作用位点、N端糖基化部位和酪蛋白激酶II 作用部位； 3。

Glioma is still one of refractory disease in the neurosurgical field; the development of new primary and adjuvant treatment is vital. Recently, the gene therapy of glioma is developed rapidly and there are many methods about the gene therapy that include: suicide gene therapy, immunologic gene therapy, drug resistangce gene therapy, angiostatin gene therapy and so on. The sucide gene therapy is the most potential approach of antitumer, these nonmammalian genes encode enzyme that convert nontoxic prodrugs into highly toxic metablites. Cells transfected with suicide genes are targeted for specific negative selection, witch can be induced by administrtion of the corresponding produg. Among the enzyme/produg combinations, two of the best characterized system are herpes simplex virus thymidine kinase /ganciclovir and Escherichia coli cytosine deaminase /5-flourocytosine (5-FC). The formor can convert the antiviral nucleoside analogs acyclovir , ganciclovir to their nucleoside monophosphate derivatives, the monophosphate forms are subsequently phosphorylated by endogenus cellular kinases to triphosphates, these molecules are potent inhibitors of DNA synthesis.

近年来脑胶质瘤的基因治疗发展迅速，应运而生的方法有自杀基因、免疫基因、多药耐药基因以及抗血管生成基因等，其中自杀基因被认为是最有前景的基因治疗方法，它又称病毒介导的酶／药物前体疗法，是利用转基因技术将哺乳动物细胞中所不含有的自杀基因转入到哺乳动物肿瘤细胞中，该基因表达的产物可将无毒的药物前体转化为毒性药物，从而选择性杀伤该肿瘤细胞，常用的自杀基因有单纯疱疹病毒-胸苷激酶基因和大肠杆菌胞嘧啶脱氨酶基因，前者催化无毒性抗病毒核苷类似物如丙氧鸟苷、无环鸟苷等成为单磷酸核苷衍生物，然后在内源性细胞激酶作用下转化为具有明显毒性的三磷酸核苷，作为DNA合成链的终止剂和DNA合成酶的抑制剂，干扰细胞DNA的合成；后者编码的胞嘧啶脱氨酶可催化5-氟胞嘧啶（5-FC）脱氨成为5-氟尿嘧啶（5-FU），然后代谢为有毒性的5-氟尿嘧啶-5′三磷酸（5-FUTP）和5-氟-2′脱氧尿嘧啶-5′磷酸（5-FdUTP），5-FUTP通过与UTP竞争性结合而抑制mRNA和tRNA的合成，5-FdUTP则作用于胸苷合成酶，导致TMP衰竭而阻止DNA的合成，最终诱导肿瘤细胞凋亡。

Finally confirmed that,The thighbone and the osteoporosis morbidity is possibly connected protein 6, respectively are: The lactoferrin light chain, membrane association protein A3, the enolase, ATP gather the enzyme, the acetyl coenzyme A reductases, the myo calcium protein; The lumbar vertebra and the osteoporosis morbidity is possibly connected protein 5, respectively are: The actin, the keratin, the enolase, ATP gather the enzyme, the myosin; The thighbone and the strong bone valuable curative effect is possibly connected protein 9, respectively are: The enolase, ATP gather the enzyme, the myo-calcium protein, the creatine activating enzyme isozyme, the phosphoglyceric acid change flavor the enzyme, the myosin, the lactoferrin light chain, the pyruvic acid activating enzyme isozyme, the crown protein; The lumbar vertebra and the strong bone valuable curative effect are possibly connected protein 8, respectively are: The carbonic anhydrase, the actin, the αB-crystal protein, 3-phospho-glycerol aldehyde oxidase, the serum albumin, ATP gather the enzyme, the myosin, the enolase.

最后确认：股骨与骨质疏松发病可能相关的蛋白6个，分别为：乳铁蛋白轻链、膜联蛋白A3、烯醇化酶、ATP合酶、乙酰辅酶A还原酶、肌钙蛋白；腰椎与骨质疏松发病可能相关的蛋白5个，分别为：肌动蛋白、角蛋白、烯醇化酶、ATP合酶、肌球蛋白；股骨与强骨宝疗效可能相关的蛋白9个，分别为：烯醇化酶、ATP合酶、肌钙蛋白、肌酸激酶同工酶、磷酸甘油酸变味酶、肌球蛋白、乳铁蛋白轻链、丙酮酸激酶同工酶、冠蛋白；腰椎与强骨宝疗效可能相关的蛋白8个，分别为：碳酸酐酶、肌动蛋白、αB-晶体蛋白、3-磷酸甘油醛脱氢酶、血清白蛋白、ATP合酶、肌球蛋白、烯醇化酶。

Prourokinase is not only a single-chain precursor of urokinase , but also one of the two human plasminogen activators.

尿激酶原是人机体内仅有的两种内源性纤溶酶原激活剂之一，为双链尿激酶的酶原前体。

Mechanical strains also regulated the protein and mRNA expression of several differentiation markers, as well as the activation of extracellular signal-regulated kinases, p38 MAP kinase and protein kinase B in a frequency-dependent manner. Furthermore, the inhibition of p38 pathway could block the strain-frequency induced the phenotype modulation of VSMCs, neither ERKs nor Akt. Frequency of mechanical strain, not conditioned medium, regulated the phenotype of VSMCs in a frequency-dependent manner. Rho-GDI alpha was suppressed by the mechanical strain at 1Hz.

采用免疫细胞化学法检测VSMCs形态和排列的变化；RT-PCR和Western blotting检测表型标志分子α-肌动蛋白、肌球蛋白重链(SM1/2)、肌动蛋白相关蛋白SM22α和调宁蛋白(h1-calponin)的mRNA和蛋白水平的变化；抑制剂或RNA干扰阻断可能的信号调节分子的活性或表达，包括p38、细胞外信号调节激酶1/(2extracellular signal-regulated kinases， ERK1/2)、蛋白激酶B和Rho-鸟苷酸解离抑制因子(Rho-guanine nucleotide dissociation inhibitor， Rho-GDI alpha)，研究了不同频率张应变对VSMCs表型转化的影响及其调节机制。

And Pharaoh spoke to Joseph, saying, Your father and your brothers have come to you.

47：5 法老对约瑟说，你父亲和你弟兄们到你这里来了。

Additionally, the approximate flattening of surface strip using lines linking midpoints on perpendicular lines between geodesic curves and the unconditional extreme value method are discussed.

提出了用测地线方程、曲面上两点间短程线来计算膜结构曲面测地线的方法，同时，采用测地线间垂线的中点连线和用无约束极值法进行空间条状曲面近似展开的分析。