再连接

Methods: total rna was isolated from hepatocellular carcinoma cell line hepg2. subsequently, a 1kb fragment of hccr 2 encoding region was amplified by rt pcr and cloned into promega teasy vector. after confirmed by dna sequencing, the full length encoding region of hccr 2 was amplified by pcr and cloned into pires2 egfp. the recombinant eukaryotic expression vector was confimed by dna sequencing.

从人肝癌细胞株hepg2提取rna，利用rt pcr方法，先克隆出一包括hccr 2编码区的长约1 kb的片段，并将其与t载体连接并转化，测序证实无误后，以hccr 2/t载体为模板，再通过pcr克隆出hccr 2的编码区序列，将其连接到pires2 egfp真核表达载体，并通过测序获得证实。

METHODS: Total RNA was isolated from hepatocellular carcinoma cell line HepG2. Subsequently, a 1kb fragment of HCCR2 encoding region was amplified by RTPCR and cloned into Promega TEasy vector. After confirmed by DNA sequencing, the fulllength encoding region of HCCR2 was amplified by PCR and cloned into pIRES2EGFP. The recombinant eukaryotic expression vector was confimed by DNA sequencing.

从人肝癌细胞株HepG2提取RNA，利用RTPCR方法，先克隆出一包括HCCR2编码区的长约1 kb的片段，并将其与T载体连接并转化，测序证实无误后，以HCCR2/T载体为模板，再通过PCR克隆出HCCR2的编码区序列，将其连接到pIRES2EGFP真核表达载体，并通过测序获得证实。

A wind- and motor-powered land vehicle with a fore-and-aft longitudinal axis comprising a body supported by wheels, at least one of said wheels fixed to rotate in a plane parallel to the fore-and-aft axis of said vehicle, an airfoil system comprised of two vertically extending airfoils rigidly mounted on said body in an arrangement having bilateral symmetry about a vertical plane through said fore-and-aft axis, said airfoil system providing a forward propelling force of said vehicle when the airfoil system is subjected to certain cross wind conditions, and self-contained motor propulsion means on said vehicle for providing acceleration from a stationary position and for propelling the vehicle either independently or in combination with propelling forces generated by wind action on said airfoil system, each airfoil having a plurality of airfoil sections telescopically fitted together, the lower airfoil section being rigidly mounted in fixed relationship to said body, said airfoil system including a retracting mechanism for lowering upper airfoil sections of said airfoil system when desired and for subsequently extending said airfoil system, said retracting mechanism including a plurality of linkages interconnecting said sections and an actuator means operatively connected between said body and to said linkages for lowering and extending each airfoil, and an intermediate airfoil extending between the upper sections of each of said airfoils so as to provide an inverted U-shaped airfoil.

风力和电动机驱动的陆地车辆与前列和尾部长轴组成的一个机构的支持，车轮，至少有一说，车轮固定在一个旋转平面平行的前列和前后轴的说，车辆，翼型系统组成的两个垂直扩展翼型硬性安装在说，机构在安排后，双边对称约一垂直面说，通过国内外和前后轴，表示翼型系统提供了一个向前推进的力量说，车辆时，翼型系统是受到某些横风条件下，和自我载电机推进的手段就表示，车辆提供加速从一个固定的立场和为推进车辆单独或结合，与推进力量所产生的风的行动表示翼型系统，每个机翼有一个多元化翼型第telescopically装在一起，较低的翼型节被硬性安装在固定的关系，说身体，说翼型系统包括一个撤消的机制，降低上翼型章节说，翼型系统时，理想的和其后再将说，翼型制度，说收回机制包括多元化的联系互连说，部分和一个致动器的手段手术之间的连接说，身体和说，联系降低，并延长每翼型，及一个中间翼型延长之间的路段上的每说翼型，以便提供一个倒U -形翼型。

A contemporary twist on the aristocratic oil portrait above the mantel, the artwork consists of a motion-activated video camera affixed to a large flat-panel television and a computer, tracking and displaying the movements of whatever passes by: underdressed masters of the house, delivery people, ex-wives.

这件艺术品是把壁炉架上方兼具贵族气息与自恋倾向的油画肖像进行具当代精神的改造，它包含一台靠感应动作启动的摄影机，摄影机再与大型平面电视及电脑连接，能追踪并显示任何通过镜头前方的物体，例如衣著随便的屋主、送货的人、诸位前妻。

Why is it the Warlock talent Soul Link provides 20% mitigation from all sources of damage, 5% more damage, and can be additionally talented to provide an undispellable 70 resistance to all magic schools, or, 5% more damage and 35 resistance to all magic schools, but the Priest's Shadowform talent only provides 15% mitigation from PHYSICAL damage and 15% more SHADOW damage, and completely shuts down over 50% of the Priest's abilities

为什么术士的灵魂连接天赋（31点）能提供20%全伤害减免，5%全法伤提高，并且假如天赋支持，能在这基础上再提供70点不能驱散的抗性，或者5%额外的伤害和35点抗性；然而牧师的暗影形态（31点天赋）只能提供15%的&物理&伤害减免，和15%的&暗影&伤害提高，并且使得牧师一半的法术都无法使用？

Why is it the Warlock talent Soul Link provides 20% mitigation from all sources of damage, 5% more damage, and can be additionally talented to provide an undispellable 70 resistance to all magic schools, or, 5% more damage and 35 resistance to all magic schools, but the Priest's Shadowform talent only provides 15% mitigation from PHYSICAL damage and 15% more SHADOW damage, and completely shuts down over 50% of the Priest's abilities

为什么术士的灵魂连接天赋（31点）能提供20%全伤害减免，5%全法伤提高，并且如果天赋支持，能在这基础上再提供70点不能驱散的抗性，或者5%额外的伤害和35点抗性；然而牧师的暗影形态（31点天赋）只能提供15%的&物理&伤害减免，和15%的&暗影&伤害提高，并且使得牧师一半的法术都无法使用？

Why is it the Warlock talent Soul Link provides 20% mitigation from all sources of damage, 5% more damage, and can be additionally talented to provide an undispellable 70 resistance to all magic schools, or, 5% more damage and 35 resistance to all magic schools, but the Priests Shadowform talent only provides 15% mitigation from PHYSICAL damage and 15% more SHADOW damage, and completely shuts down over 50% of the Priests abilities

为什么术士的灵魂连接天赋（31点）能提供20%全伤害减免，5%全法伤提高，并且如果天赋支持，能在这基础上再提供70点不能驱散的抗性，或者5%额外的伤害和35点抗性；然而牧师的暗影形态（31点天赋）只能提供15%的&物理&伤害减免，和15%的&暗影&伤害提高，并且使得牧师一半的法术都无法使用？

Why is it the Warlock talent Soul Link provides 20% mitigation from all sources of damage, 5% more damage, and can be additionally talented to provide an undispellable 70 resistance to all magic schools, or, 5% more damage and 35 resistance to all magic schools, but the Priest's Shadowform talent only provides 15% mitigation from PHYSICAL damage and 15% more SHADOW damage, and completel恶意广告huts down over 50% of the Priest's abilities

为什么术士的灵魂连接天赋（31点）能提供20%全伤害减免，5%全法伤提高，并且如果天赋支持，能在这基础上再提供70点不能驱散的抗性，或者5%额外的伤害和35点抗性；然而牧师的暗影形态（31点天赋）只能提供15%的&物理&伤害减免，和15%的&暗影&伤害提高，并且使得牧师一半的法术都无法使用？

Chicken IL-15(ChIL-15), Which was discovered in 2001, plays a main role in activating NK cells and CD8+ memory T cells, and may therefore have potential as a vaccine adjuvant.According to published ChIL-15 gene sequence, a pair of primers were designed and synthesized to clone ChIL-15 cDNA from ConA-activated chicken splenocytes by RT-PCR. Recombinant plasmid pUC19ChIL-15 carrying the ChIL-15 gene was constructed. The gene encoding mature ChIL-15 (mChIL-15) was amplified from pUC19ChIL-15 by PCR and cloned into pMD 18-T vector. After digested with Kpn I /Pst I , the mChIL-15 gene was subcloned into prokaryotic expression vector pPRoEX橦Ta and transformed into E.coli DH5a .The transformant was induced by IPTG, and the recombinant ChIL-15(rChIL-15) was expressed as a fusion protein with a histidine hexamer tag at the N-terminal end of the protein. Following expression, the protein was purified by ProBond?Nickel-Chelating Resin. The uninduced and induced protein lysates and the purified protein were separated by SDS-PAGE and transferred onto nitrocellulose membrane to identify rChIL-15 by Western blot. The rChIL-15 was used to immune the guinea pig to obtain rChIL-15 polyclonal antibody.

参考已发表的ChIL-15基因序列，设计合成引物，应用RT-PCR技术从刀豆球蛋白A活化的白来航鸡脾淋巴细胞中克隆ChIL-15 cDNA，将其与SmalⅠ酶切处理的pUC19载体连接，构建重组质粒pUC19ChIL-15；用PCR技术从pUC19ChIL-15中扩增出去信号肽的成熟ChIL-15(mChIL-15)基因，与pMD 18-T载体相连构建重组质粒pMDChIL-15；然后用KpnⅠ／PstⅠ双酶切下mChIL-15基因片段，定向亚克隆到经同样酶切处理的带有6个组氨酸标签的表达载体pP_RoEX~HTa中，构建重组质粒pP_RoChIL-15，对其测序确认读框正确后，转入大肠杆菌DH5α中诱导表达并进行纯化，用重组ChIL-15(rChIL-15)免疫豚鼠，制备多克隆抗体；再用HindⅢ／PstⅠ从质粒pP_RoChIL-15上切下mChIL-15基因，定向亚克隆到经同样酶切处理的杆状病毒转移载体pMelBacB中，经酶切、PCR和序列测定鉴定后，与杆状病毒线形化DNABac-N-Blue~(TM DNA共转染Sf9昆虫细胞，经三轮蚀斑纯化，构建重组杆状病毒rBac-ChIL-15，用该重组病毒感染处于对数生长期的Sf9细胞，按不同的时间收取样品，离心后对其上清和沉淀进行SDS-PAGE和Western blot分析。

This connection, now re-established, can never be taken from me, and this is one of the greatest gifts.

这一连接，现在得到重建而永不再从我身上被拿掉，而这就是对我的最好礼物之一。

For a big chunk of credit-card losses; the number of filings (and thus charge-off rates) would be rising again, whether

年美国个人破产法的一个改动使得破产登记急速下降，而后引起了信用卡大规模的亏损。

Eph. 4:23 And that you be renewed in the spirit of your mind

弗四23 而在你们心思的灵里得以更新