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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。

Objective To evaluate Borna disease virusFluorescent-Quantitation Real Time PCR kit and study the preliminary application results of the kit.

目的 评价博尔纳病病毒(Borna disease virus， BDV)实时荧光定量PCR试剂盒的各项指标，并了解其实际应用效果。

Important things that should always be a part of your repair kit are spoke tool, spanners, screwdriver, pump, Allen key, puncture repair kit, a bottom bracket tool as well as some lubricant.

重要的事情，应该永远是部分的修理工具的发言工具，扳手，螺丝刀，泵，阿伦关键，穿刺修理包，一中轴工具，以及一些润滑剂。

Must have items for short riding trips include wrenches for all the nuts and bolts on the bike, tire levers, pump, patch kit/tubes, chain tool, crescent wrench, zip ties, duct tape and a small first aid kit.

项目必须有短扳手骑马旅行包括所有的具体细节在自行车，轮胎杠杆，泵，补丁包/管，链条工具，月牙扳手，拉鍊关系，胶带和一个小急救箱。

This kit, which includes the plastic index throttling plate and mounting hardware may be thought of as a replacement handle kit, because it is the same subassembly that is mounted to the basic valve when it is ordered with a lever handle.

这种组件，包括塑料刻度节流板和安装五金件可能被视为是一种替代手柄组件，因为它与在订购配置杠杆手柄的基本型阀门上安装的手柄是相同的总成。

House reef diving is available on demand and the staff even transport your kit to and from your entry/exit point for you - after struggling in and out of freezing scottish lochs with enough kit to challenge an olympic weightlifter this is diver heaven.

需要时屋礁潜水也提供，员工甚至把你潜水的成套用品来来回回地为你送到你的潜入点和潜出点--在冰冷的苏格兰湖泊里带着足够的成套用品努力地潜入潜出让你可以挑战奥林匹克举重者，这是潜水者的天堂。

House reef diving is available on demand and the staff even transport your kit to and from your entry/exit point for you - after struggling in and out of freezing scottish lochs with enough kit to challenge an olympic weightlifter this is diver heaven.

若要求去岛屿周围水下潜水也是可以的，工作人员甚至会在进水口或出水口为你准备好潜水装备，与戴着沉重（堪比奥运举重运动员举重）的潜水装备在苏格兰冰冷的海湾潜泳相比，这里就是潜水天堂。

House reef diving is available on demand and the staff even transport your kit to and from your entry/exit point for you - after struggling in and out of freezing scottish lochs with enough kit to challenge an olympic weightlifter this is diver heaven.

如果需要，也可以进行岛下水景潜游。工作人员会在下水处或出水口为你准备好潜泳用具。与在苏格兰冰冷的海湾，自带沉重的潜水用具（像是挑战奥林匹克举重冠军一样）相比，这里真是潜水者的天堂。

After the analysis, I use MICROSOFT Corporation VISUAL the BASIC development kit, uses each kind of object-oriented development kit which it provides, the data window this can in particular convenient and the succinct operation database intellectualized object, first in a short time establishes the system application prototype, then, carries on the demand iteration to the initial prototype system, revises and the improvement unce

经过分析，我使用MICROSOFT公司的VISUAL BASIC开发工具，利用其提供的各种面向对象的开发工具，尤其是数据窗口这一能方便而简洁操纵数据库的智能化对象，首先在短时间内建立系统应用原型，然后，对初始原型系统进行需求迭代，不断修正和改进，直到形成用户满意的可行系统。

When you first go to Hale End, they give you the tracksuit and your kit and you're so excited you want to sleep in the kit!

当你第一次来到Hale End的时候，他们会发给你一套训练服，还有装备，那时候的感觉太激动了，恨不得穿着训练服睡觉！

With Death guitarist Schuldiner adopting vocal duties, the band made a major impact on the scene.

随着死亡的吉他手Schuldiner接受主唱的职务，乐队在现实中树立了重要的影响。

But he could still end up breakfasting on Swiss-government issue muesli because all six are accused of nicking around 45 million pounds they should have paid to FIFA.

不过他最后仍有可能沦为瑞士政府&议事餐桌&上的一道早餐，因为这所有六个人都被指控把本应支付给国际足联的大约4500万英镑骗了个精光。