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慢病毒載體構(gòu)建,,包裝,,純化服務(wù)|實(shí)驗(yàn)技術(shù)服務(wù)

時(shí)間:2015/8/1閱讀:247
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關(guān)鍵詞:慢病毒載體構(gòu)建,包裝,,純化服務(wù)|實(shí)驗(yàn)技術(shù)服務(wù)
簡介:世界*品牌慢病毒載體構(gòu)建,,包裝,純化服務(wù)|實(shí)驗(yàn)技術(shù)服務(wù)原裝,,質(zhì)量保證,*,。
慢病毒載體構(gòu)建,包裝,,純化服務(wù)|實(shí)驗(yàn)技術(shù)服務(wù)——pCzn1質(zhì)粒+Arctic Express宿主菌低溫表達(dá)體系,。
我們具備豐富的蛋白表達(dá)設(shè)計(jì)經(jīng)驗(yàn)及實(shí)驗(yàn)操作技巧,承諾客戶不成功不收費(fèi),。我們所有的實(shí)驗(yàn)數(shù)據(jù)真實(shí)可信,,我們提供原始的表達(dá)菌株和克隆質(zhì)粒,,客戶可以依據(jù)我們的實(shí)驗(yàn)報(bào)告重復(fù)我們的實(shí)驗(yàn)結(jié)果。
產(chǎn)品品牌:   http://www.,。,。com/goodsid/fenleiyi/2868603/1.html    
測序?qū)嶒?yàn)流程:
慢病毒是逆轉(zhuǎn)錄病毒的一種,具有逆轉(zhuǎn)錄病毒的基本結(jié)構(gòu)和特性:整合入宿主細(xì)胞基因組,,長期表達(dá),,可用于轉(zhuǎn)基因動物制備;但也有不同于逆轉(zhuǎn)錄病毒的組份和特性:比逆轉(zhuǎn)錄病毒具有更高的滴度,,不僅可以感染分裂期細(xì)胞,,更重要的,還能感染非分裂期的細(xì)胞,。利用慢病毒載體,,可以研究啟動子的調(diào)控、過表達(dá)特定基因或沉默特定基因,。
(一) 實(shí)驗(yàn)流程(1和2為并列步驟)
1.慢病毒過表達(dá)質(zhì)粒載體的構(gòu)建
設(shè)計(jì)上下游特異性擴(kuò)增引物,,同時(shí)引入酶切位點(diǎn),PCR(采用高保真KOD酶,,3K內(nèi)突變率為0%)從模板中(CDNA質(zhì)?;蛘呶膸欤┱{(diào)取目的基因CDS區(qū)(coding sequence)連入T載體。將CDS區(qū)從T載體上切下,,裝入慢病毒過表達(dá)質(zhì)粒載體,。
2.慢病毒干擾質(zhì)粒載體的構(gòu)建
合成siRNA對應(yīng)的DNA頸環(huán)結(jié)構(gòu),退火后連入慢病毒干擾質(zhì)粒載體
3. 慢病毒載體的包裝與濃縮純化
制備慢病毒穿梭質(zhì)粒及其輔助包裝原件載體質(zhì)粒,,三種質(zhì)粒載體分別進(jìn)行高純度無內(nèi)毒素抽提,,共轉(zhuǎn)染293T細(xì)胞,轉(zhuǎn)染后6 h 更換為*培養(yǎng)基,,培養(yǎng)24和48h后,,分別收集富含慢病毒顆粒的細(xì)胞上清液,病毒上清液通過超離心濃縮病毒,。
(二) 實(shí)驗(yàn)材料
2.1慢病毒載體,、包裝細(xì)胞和菌株
該病毒包裝系統(tǒng)為三質(zhì)粒系統(tǒng),組成為pspax2, pMD2G, pLVX-IRES-ZsGreen1/pLVX-shRNA2,。其中質(zhì)粒上的ZsGreen1表達(dá)框能表達(dá)綠色熒光蛋白(GFP),。
慢病毒載體構(gòu)建包裝實(shí)驗(yàn)流程如下:
1.根據(jù)目的基因相關(guān)信息(序列,基因序列號等),,構(gòu)建含有外源基因或序列的重組載體,;
2.對于測序正確的重組質(zhì)粒,提取和純化高質(zhì)量(不含內(nèi)毒素)的重組質(zhì)粒,;
3.使用重組載體和慢病毒包裝質(zhì)粒共轉(zhuǎn)染293T細(xì)胞,,進(jìn)行病毒包裝并收集上清液;
4.通過超濾和超速離心濃縮和純化病毒,;
5.用病毒液感染293T細(xì)胞,,測定病毒滴度;

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