酶蛋白

Thus, it is important to enucleate the catalytic centre and mechanism of transferrins as a phosphatase, which may have a potential significant in the protein phosphorylation process.

据文献报导，传铁蛋白家族中的某些乳传铁蛋白和铁结合蛋白具有磷酸酶的功能，因此，研究传铁蛋白磷酸酶学功能位点和作用机制并进一步确定它们在磷酸化过程的作用具有重要意义。

With the aid of PDQuest 2-DE software, 11 significant protein dots were found out and then 9 of them are pick out to go through peptide mass fingerprinting based on MALDI TOF MS and NCBI database searching . In all, 5 proteins were identified: Ribulose bisphosphate carboxylase , Nucleic acid-binding protein , Ribosomal protection tetracycline resistance protein , hypothetical protein FG04726.1 [Gibberella zeae PH-1], nitric oxide synthase .

应用PMF技术对这11个具有显著表达差异蛋白点中的9个进行鉴定，得到了5个质谱阳性鉴定结果，其中2个蛋白分别为玉米种属的1，5-二磷酸核酮糖羧化酶／加氧酶和叶绿体的核酸结合蛋白；其它的3个蛋白在玉米蛋白质数据库中未见报道，但与其它物种基因编码的蛋白质具有高度的序列同源性。

Sequences analysis indicated 26 unique cDNA sequences that encoded putative proteins showing similarities to HL35 antigen U, hlim3, heat shock protein, NADH dehydrogenase subunit 1 and calreticulin of H. longicornis, disulfide isomerase of Ambiyomma variegatum and Ixodes scapularis.

结果表明：在长角血蜱雌蜱唾液腺cDNA表达文库中获得26个长角血蜱免疫相关cDNA序列，其编码蛋白与长角血蜱HL35、黏附素hlim3假定蛋白、热休克蛋白、NADH脱氢酶、钙网蛋白以及杂色花蜱和肩突硬蜱的二硫异构酶等具有同源性。

The results showed that expression plasmid pET22b-lysB was constructed successfully. Highly purified recombination protein was obtained 33.2 mg from 1 L LB culture medium. A screening for His-LysB activity on esterase and lipase substrates confirmed the lipolytic activity. With p-nitrophenyl butyrate as substrate, the thermal stability of the enzyme was poor when the temperature was above 30oC. The enzyme exhibited higher stability at pH 5.0–9.5. The optimum temperature and pH for the lipolytic activity of His-LysB were 23oC and 7.5 respectively. Under the optimum conditions, the specific activity of His-LysB was 1.3 U/mg. Zn2+, Cu2+, Mg2+, Mn2+and phenylmethane sulfonyl fruoride severely inhibited the lipolytic activity of His-LysB.

结果表明：成功构建了pET22b-lysB表达载体，并从1 L的LB培养物中获得了33.2 mg高纯度重组蛋白；His-LysB具有分解脂肪的能力，属于脂肪酶；生物化学特性分析表明：丁酸对硝基苯为水解底物，His-Lys热稳定性不佳，30℃以下比较稳定，随着温度的升高，稳定性逐渐降低；该蛋白具有较高的pH值适应性，pH 5.0~9.5范围内稳定性较高；在23℃和pH 7.5时酶活力最高，其比酶活为1.3 U/mg；金属离子Zn2+、Cu2+、Mg2+、Mn2+和苯甲基磺酰氟抑制剂对酶活具有强烈的抑制作用。

Detect with Western Blot protein of MAb counterpoise form and the natural phosphoric acid is enzymatic reactivity in the cell, obtain 6 individual plant to be able to be stabilized in all secrete the individual plant of cross tumour cell that fights phosphoric acid enzymatic MAb, go enzymaticly to study double particularity phosphoric acid further phosphation action mechanism and its activation formerly in mitosis way of enzymatic signal of albumen phosphoric acid turns guide provide strong tool.

用Western blot检测mAb对重组蛋白和细胞中天然磷酸酶的反应性，共获得6株可稳定分泌抗磷酸酶mAb的杂交瘤细胞株，为进一步探究双特异性磷酸酶的去磷酸化功能机理以及其在有丝分裂原激活蛋白磷酸酶信号途径中的信号转导提供强有力的工具。

One of them was identified as aldehyde dehydrogenase 2, and the others were both identified as mouse RIKEN cDNA 2900053E13, suggesting that the protein might be post translationally modified.

通过对差异蛋白的胶内酶解和高效液相色谱／离子阱质谱鉴定，发现其中一个蛋白点为乙醛脱氢酶2（aldehyde dehydrogenase 2），另外两个差异点鉴定结果为同一个蛋白RIKEN cDNA 2900053E13，提示该蛋白在正常和心肌缺血心肌的线粒体内修饰状态发生了变化。

Enzyme activity assay demonstrated that the Trx-BTGase expressed in E. coli could be functional in polymerizing other proteins, and no influence on the biological activities of BTGase to polymerize BSA was found in case of the thrombin cleavage of this fusion protein.

酶活性分析表明表达的Trx-BTGase融合蛋白具有交联蛋白的活性，并发现Trx-BTGase融合蛋白和经凝血酶酶切后得到的BTGase单体都能催化牛血清白蛋白的聚合反应。

We also strongly suggest that the recently reported chaperone acitivity of periplasmic PDI-ase and PPIase assayed by traditional substrate should be reexamined using periplasmic proteins as substrate due to the significant difference between those two classes of proteins, the possible chaperone fuctions of PDIase and PPIase in vivo should be reappraised.

同时，由于本实验结果显示膜间质蛋白与常用的分子伴侣测活底物蛋白在聚集行为上存在极显著的区别，我们建议对最近报导的膜间质蛋白二硫键异构酶和脯氨酸顺反异构酶分子伴侣活性以膜间质蛋白为底物再进行验证，对其在膜间质是否发挥分子伴侣功能重新慎重考虑。

The PCR products were examined by agarose gel electrophoresis. The target gene fragments were purified by gel extraction kit and ligated to cloning vector pMD18-T. The recombinant vectors were transformed into host strain E. coli K802 by lithium chloride method, screened and identified with PCR and restrictive enzymatic digestion. Their sequences were confirmed by DNA sequencing.(2) sTWEAK1 gene was subcloned into expression vector pProEx HTb and transformed into E. coli BL21. sTWEAK2 gene was subcloned into expression vector pMAL-C2x and transformed into E. coli TB1. The recombinant vectors were screened and identified with PCR and restrictive enzymatic digestion. The recombinant fusion proteins were induced to express with IPTG, detected by coomassie brilliant blue-stained SDS-polyacrylamide gel electrophoresis , and confirmed by Western blot analysis.(3) The sTWEAK1 fusion protein was purified with Ni-NTA Spin Kit.(4) The biological activity was assayed on transformed and tumor cells by microplate photometer after crystal violet or sulfur rodamine B staining.(5) The contents of IL-8 in the supernatant of 1990 cell cultures were determined by ELISA.(6) The morphological changes of the sensitive cells were observed by light and transmission electron microscopies.(7) The cell cycle and apoptotic rate were assayed by flow cytometry in 1990 and M85 cells.(8) The effect of fusion proteins on induction of NF-κB in 1990 and LOVO cells was detected with Dual-Luciferase Reporter Assay system.(9) The TWEAK gene was subcloned into Adeno-X Viral DNA with pShuttle vector and transfected into HEK293 cells by lipofectamine method.

(1)本研究用RT-PCR方法，从人组织细胞总RNA中扩增可溶性TWEAK胞外区（sTWEAK1和sTWEAK2）的cDNA序列及全长编码序列，用琼脂糖凝胶电泳分析PCR产物，胶回收目的基因片段，连接到pMD18-T克隆载体中，转化大肠杆菌K802，PCR和酶切筛选阳性克隆，全自动DNA测序验证序列；(2)sTWEAK1和sTWEAK2分别亚克隆到pProEx HTb和pMAL-C2x表达载体中，分别转化大肠杆菌BL21和TB1，PCR筛选和酶切鉴定，阳性克隆用IPTG诱导表达，表达产物用SDS-PAGE分析和Western blot验证融合蛋白；(3)用NTA-Ni Spin试剂盒初步分离纯化sTWEAK1融合蛋白；(4)用体外培养的肿瘤细胞和正常对表达产物进行活性检测，贴壁细胞用结晶紫染色法，悬浮细胞用磺酰罗丹明B染色法，酶标仪检测OD值；(5)敏感细胞用ELISA法检测细胞培养上清中IL-8的含量；(6)用光镜和电镜观察敏感细胞死亡和细胞凋亡情况；(7)用流式细胞仪分析表达产物对敏感细胞凋亡率和细胞周期的影响；(8)用双荧光素酶报告基因检测法，测定表达产物对敏感细胞NF-κB的影响；(9)用pShuttle穿梭质粒将TWEAK重组到腺病毒载体上，用脂质体转染法转染HEK293细胞，PCR鉴定重组质粒。

As hollow fiber membranes thickness are different, yet urea clearance rate in simulation solution is relatively high, which can reach more than 64% and little difference among them; the BSA have a relatively high rejection, which can be achieved over 98%. There is a certain difference in the Lysozyme clearance rate, the smaller thickness, the higher the rate of removal, and the removal rate of 10μm thickness of the membrane is 29%. The clearance rates of urea were very high using different fiber diameter dialyzers, at around 80%. Urea clearance ratecan reach to the highest 88.6% using 0.26mm fiber diameter dialyzer. The clearance rates of Lysozyme vary greatly, the smaller the diameter, the higher clearance rate, and the highest removal rate of lysozyme is 62.79% using 0.26mm fiber diameter dialyzer. However, there is little impact on the rejection of BSA, which can reach 98%.

不同壁厚中空纤维膜透析器对模拟液中尿素的清除率均可达到64％以上，且差异不大，对牛血清白蛋白的截留率均可达到98％以上，对溶菌酶的清除率有一定的差异，壁厚越小清除率越高，壁厚为10μm的膜对溶菌酶的清除率为29％；不同纤维内径透析器对尿素的清除率均在80％左右，纤维内径为0.26mm的透析器对尿素的清除率最高，达到88.6％，对溶菌酶的清除率差别很大，内径越小，清除率越高，内径为0.26mm的透析器对溶菌酶的清除率达到62.8％，对牛血清白蛋白的截留影响很小，截留率都能达到98％以上。

Do you know, i need you to come back

你知道吗，我需要你回来

Yang yinshu、Wang xiangsheng、Li decang,The first discovery of haemaphysalis conicinna.

1〕 杨银书，王祥生，李德昌。安徽省首次发现嗜群血蜱。