查询词典 come out into the open

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。

And load up the cabin with rats and snakes and so on, for company for Jim; and then you kept Tom here so long with the butter in his hat that you come near spiling the whole business, because the men come before we was out of the cabin, and we had to rush, and they heard us and let drive at us, and I got my share, and we dodged out of the path and let them go by, and when the dogs come they warn't interested in us, but went for the most noise, and we got our canoe, and made for the raft, and was all safe, and Jim was a free man, and we done it all by ourselves, and WASN'T it bully, Aunty!

还有你把汤姆拖住了老半天，害得他帽子里那块黄油都化掉了，差点儿把整个儿这回事给弄糟了，因为那些人在我们从小屋里出来以前就来到了，因此我们不得不急着冲出去。他们一听到我们的声响便追赶我们，我就中了这一枪。我们闪开了小道，让他们过去。那些狗呢，它们追了上来，可对我们没有兴趣，光知道往最热闹的地方跑。我们找到了独木船，划出去找木筏子，终于一切平安无事，杰姆也成了自由人。凡此种种，都是我们自个儿干出来的，难道不是棒极了么，姨妈？

Swallow go and can come back again, willow dried up can lives again, peachblossom gone but can come out again, who the f*k can tell me , why out f**king days gone but never come back.

燕子去了有再回来的时候，杨柳枯了有再青的时候。桃花谢了有再开的时候，谁能告诉我，为什么我们的日子一去不复返。

I, Shannon Blood Sausage. Stand-pig small intestine washed first with warm water to soak rice for half an hour, picked up the rice, adding blood, mix with the rice and put into a large stone or blood beater, then add green onion, salt, has been Net diesel refining pig, star anise powder mix, wash the pig has been poured into the small intestine, ligated at both ends into the pot, cook the back, pay attention to the fire can not be too fast and can not boil water, to keep turning in order to avoid open rupture Blood Sausage, and come to naught, cooked to salvage, you can cut consumption, increase can also be cooked pan fried dishes, the use of soy sauce, vinegar, monosodium glutamate pair of points is expected, it is delicious, Miao can not be made.

一、香血肠。将猪小肠洗净备用，先用温水将大米浸泡半个小时，捞起白米，加入猪血，与米搅匀，放入大石磨或打浆机磨成血米浆，再加入葱花、盐、已炼的猪网油渣、八角粉等拌匀，灌入已洗净的猪小肠中，结扎两端，放入锅内，慢火煮熟，注意火不能太猛，水不能烧开，要经常翻动，以免血肠破裂开，而前功尽弃，煮熟后捞起，即可切块食用，也可用菜锅加熟油煎后，用酱油、醋、味精配成的点料，更是味香可口，妙不可言。

Put Twisted Sisters on MTV, yeah[01:46.40]Those were the days[01:49.49]I'll come back[01:50.75]To Iron Maiden and 7-Up[01:53.99]Bananarama and Breakfast Club[01:57.35]Come back[01:59.37]Come back[02:00.93]I'll come back[02:03.77]When Huey Lewis was the news[02:07.46]And we went flying with Tom Cruise[02:11.26]When shoulder pads and looking tough, ooh-oh-oh[02:15.26]Was the dirty David Hasselhoff[02:18.64]Too many men with heavy makeup on[02:22.34]Too many poodles with guitars[02:25.97]Thanks for all the joy you've given us[02:29.81]You will always be our superstars[02:35.28]Back to the 80s[02:36.86]Back to Soap[02:38.99]Back to Rocky and Cherry Coke, yeah[02:42.62]Those were the days[02:45.10]And we go[02:46.47]Back to the 80s[02:47.94]Say 'Hey there, Mister T.

无论里根是演员还是总统，[04：35.09]都在80年代留下了无法抹灭的印记。[04：39.15]satellite："卫星"，[04：40.81]在歌词中指的是"orbital satellites轨道卫星"，[04：44.80]这暗指里根时代的 star wars "星球大战"计划，[04：49.21]此计划在克林顿被停止执行）[04：53.80]skinny leather ties：是80年代在欧美国家风行的装束，[04：57.75]男孩们都喜欢戴着细条长的皮质领带，[05：00.89]尤其受到乐队的乐手们的宠爱。

Come on, come on Give me everything I deserve all I can get, you aint seen no-nothing yet I'm gonna grab the brassy golden ring Come on, come on I want everything I know it's gonna happen...

来吧，来吧给我的一切我应该所有我可以，你是不是没有看到，但没有我一定抓住黄铜金戒指来吧，来吧我想要的一切我知道一定发生过。。。

Come back come back come back come back to meeeee, Punisher

回来回来回来回到我的身边，冤家

When motion is towards the location of the addressee,come is much preferable to go in English while both laiand quare common in use in Chinese.When motion is towards the location of the addressee at a past time,laimust be used in Chinese if the addressee is there at the time of utterance while qumust be used if the addressee is not there at the time of utterance.By contrast,come is used in English either when the addressee is there or when he is not there at the speaking time,while go is only used when the addressee is not there at the time of utterance. When both the speaker and the addressee serve as the figure,which is indicated by the phrases like with me/us/you in English and the phrases like he ni,he nimen,gen wo,gen womenin Chinese,come is much preferred to go for the figure"s motion,while in the same context,quis used for the figure"s motion.What"s more,come is more frequently used to indicate the figure"s movement towards a place where neither the speaker nor the addressee is located at the time of utterance,while quis often used in Chinese in the same case.

当移动体朝向听话人所在方位移动时，英语多用come，汉语可用"来/去"；当移动体在一个过去的时间朝向听话人所在方位移动时，汉语可用"来"表示听话人在说话时间仍在位移参照点，用"去"表示听话人在说话时间已不在位移参照点，而英语在两种情况下都可用come，用go则表示听话人在说话时间已不在位移参照点；当说话人和听话人一起作为移动体而句中主语只是说话人或听话人一方时，英语多用come，汉语多用"去"。

You could be my brand new game You begin and roll the dice I would smile and break the ice Tell me how could I resist when you're my favorite kiss Kissy kissy makes me happy Honey honey sweet as candy Kissy kissy take my hand and come along with me Kissy kissy makes me happy Honey honey sweet as candy Kissy kissy love is grand with you my sugar bee See the moon and see the sun, shining down on everyone They know how in love we are, I heard them tell it to a star There's no mountain, there's no sea that could keep your love away from me No matter what the distance is, you're still my favorite kiss Kissy kissy makes me happy Honey honey sweet as candy Kissy kissy take my hand and come along with me Kissy kissy makes me happy Honey honey sweet as candy Kissy kissy love is grand with you my sugar bee La la la, tell me what your secret is La la la, why won't you let me know La la la, nothing makes me feel like this La la la, cause you're my favorite kiss Kissy kissy makes me happy Honey honey sweet as candy Kissy kissy take my hand and come along with me Kissy kissy makes me happy Honey honey sweet as candy Kissy kissy love is grand with you my sugar bee Kissy kissy makes me happy Honey honey sweet as candy Kissy kissy take my hand and come along with me Kissy kissy makes me happy Honey honey sweet as candy Kissy kissy love is grand with you my sugar bee La la la, tell me what your secret is La la la, why won't you let me know La la la, nothing makes me feel like this La la la, cause you're my favorite kiss

您能是我全新的游戏您开始和滚动模子我会微笑和会打破僵局告诉我怎样能我抵抗当您是我喜爱的亲吻 Kissy kissy 牌子我愉快蜂蜜蜂蜜甜点作为糖果 Kissy kissy 作为我的手和来与我一起 Kissy kissy 牌子我愉快蜂蜜蜂蜜甜点作为糖果 Kissy kissy 爱是盛大的与您我的糖蜂看见月亮和看见太阳，发光下来在大家他们知道怎么在爱我们是，我听见他们告诉它对星没有山，没有能保持您的爱去从我的海不管距离是，您仍然是我喜爱的亲吻 Kissy kissy 牌子我愉快蜂蜜蜂蜜甜点作为糖果 Kissy kissy 作为我的手和来与我一起 Kissy kissy 牌子我愉快蜂蜜蜂蜜甜点作为糖果 Kissy kissy 爱是盛大的与您我的糖蜂 La la la，告诉我是什么您的秘密 La la la，为什么不会将您告诉我 La la la，没什么做我感受象这样 La la la，导致您是我喜爱的亲吻 Kissy kissy 牌子我愉快蜂蜜蜂蜜甜点作为糖果 Kissy kissy 作为我的手和来与我一起 Kissy kissy 牌子我愉快蜂蜜蜂蜜甜点作为糖果 Kissy kissy 爱是盛大的与您我的糖蜂 Kissy kissy 牌子我愉快蜂蜜蜂蜜甜点作为糖果 Kissy kissy 作为我的手和来与我一起 Kissy kissy 牌子我愉快蜂蜜蜂蜜甜点作为糖果 Kissy kissy 爱是盛大的与您我的糖蜂 La la la，告诉我是什么您的秘密 La la la，为什么不会将您告诉我 La la la，没什么做我感受象这样 La la la，导致您是我喜爱的亲吻

Beyonce knowles唱的啊 You need to stop playing round with all them clowns and the wangstas Good girls gotta get down with the gangstas Go head girl put some back and some neck up on it While I stand up in the background and check up on it Ooh Boy you looking like you what you see Won't you come over check up on it,(I'm gonna let you work up on it) Ladies let em check up on it,(watch it while he check up on it) Dip it, pop it, twirk it, stop it, check on me tonight If you got it, flaunt it, boy I know you want it While I turn around you watch me check up on it Oohhh you watchin me shake it, I see it in ya face 'n' Ya can't take it, it's blazin, you watch me in amazement You can look at it, as long as you don't grab it If you don't go braggin, I'm a let you have it You think that I am teasin, but I ain't got no reason I'm sure that I can please ya, but first I gotta read ya Ooh Boy you looking like you like what you see Won't you come over check up on it (I'm gonna let you work up on it) Ladies let em check up on it (watch it while he check up on it) Dip it, pop it, twirk it, stop it, check on me tonight (x2) I can tell you wanna taste it, but I'm gone make you chase it Got to be patient, I like my men patient More patience, you take might get you in more places You can't be abrasive, has to know to pace it If I let you get up on it, you gotta make a promise That you gon put it on me, like no ones put it on me Don't bore me, just show me, all men talk but don't please I can be a tease, but I really wanna please you Ooh Boy you lookin like you like what you see Won't you come over check up on it,(I'm gonna let you work up on it) Ladies let em check up on it,(watch it while he check up on it) Dip it, pop it, twirk it, stop it, check on me tonight (x2) I'm checking on you boo, do what chu do And while dance I'ma glance at this beautiful view I'm keep my hands in my pants, I need to glue em w/ glue I'm in a trance all eyes on you and your crew Me and my mans don't dance, but to feel ya'll bump and grind If won't hurt if you gone try one time They all hot, but let me see this ones mine Its slim thug and DC outta H town Ohh Boy you looking like you like what you see Won't you come over check up on it,(I'm gonna let you work up on it) Ladies let em check up on it,(watch it while he check up on it) Dip it, pop it, twirk it, stop it, check on me tonight x4

slimthug，你必须停止玩回合与所有这些小丑与wangstas好女孩gottagetdown 随着gangstas走头女孩把一些背部和颈部一些比它在我站起背景及检查它oohboy你看你喜欢什么，你会看到你不来对清理出来，(我gonna让你工作起来就)让各位em察看着它，沾，流行资讯，twirk它，阻止它， checkonme今晚如果你有它，炫耀它，男孩我知道你要它当我转身，你看我检查它oohhh你watchin 我摇，我看它在亚面子'n'ya不能把它，它的blazin，你看我一无所有你可以看看，只要你不抓这个问题，如果你不去braggin，我是一个让你如果你觉得我teasin，但我是不会有任何理由，我相信我能请娅但是，我首先gotta阅读娅ooh男孩找你喜欢你喜欢你见到不会你来超过清理它(我gonna让你工作起来就)让各位em检查它沾，流行资讯，twirk它，阻止它， checkonmetonight(x2)，我可以告诉你想品尝，但我已离开人世，使你追逐它必须有耐心，我喜欢我的男病人更耐心，你也许可以得到你的地方越来越多，你不能磨料已经知道它的速度，如果我让你起床，那 yougotta做出承诺，你gonputitonme，不一样的说得对我并不负有我，只告诉我，所有男人说话，但不讨人喜欢，我可以戏耍，但是我真的想请你oohboylookin你喜欢你喜欢你见到不会你来对清理出来，(我gonna让你工作起来就)让各位em察看着它，沾，流行资讯，twirk它，阻止它， checkonmetonight(x2)我查你boo，做什么不楚而舞i'ma一眼这个美丽的看法，我把我的手在我的裤子，我需要胶水emw/胶水我在发呆都看你和你的船员和我我mans不跳舞，但感觉ya'll碰及研磨如果不会伤害如果你去尝试一次他们都热，但让我看到这样的矿井其slimthug直流outtah镇ohhboy你看喜欢你喜欢你见到不会你来清理，那(我gonna让你工作起来就)让各位em察看着它，沾，流行资讯，twirk它，阻止它， checkonme今晚x4

But this is impossible, as long as it is engaging in a market economy, there are risks in any operation.

但是，这是不可能的，只要是搞市场经济，是有风险的任何行动。

We're on the same wavelength.

我们是同道中人。