山东医药
山東醫藥
산동의약
SHANDONG MEDICAL JOURNAL
2014年
14期
11-14
,共4页
张丽%贾雄飞%牛华%郑瑞%张望龙%毛小琴
張麗%賈雄飛%牛華%鄭瑞%張望龍%毛小琴
장려%가웅비%우화%정서%장망룡%모소금
蛋白激酶C受体1%过表达载体%干扰载体%RNA干扰
蛋白激酶C受體1%過錶達載體%榦擾載體%RNA榦擾
단백격매C수체1%과표체재체%간우재체%RNA간우
receptor for activated C-kinase 1%over-expression vector%interference vector%RNA interference
目的:构建并鉴定蛋白激酶C受体1( RACK1)过表达及干扰真核表达载体。方法采用逆转录聚合酶链反应( RT-PCR)从人肝癌细胞系Huh-7.5.1中扩增RACK1全长cDNA系列,用巢式PCR扩增对应的CDS,并将其克隆到PIRES2-EGFP,PCR鉴定后进行测序分析。以人RACK1基因为靶基因,设计合成两对短发夹结构的互补DNA序列和一对阴性对照序列,退火后与酶切后的载体RNAi-Ready pSIREN-RetroQ-ZsGreen进行连接,并行酶切分析和测序鉴定。将构建好的过表达载体和干扰载体用脂质体转染HUVEC细胞系。结果两对引物PCR扩增的序列大小与预期结果一致,PIRES2-EGFP/RACK1载体954个碱基成功插入到预计位点,序列完全一致。 RNA干扰载体pRetroQ/RACK1-1、pRetroQ/RACK1-2和pRetroQ/HK插入序列均与预计相符。荧光显微镜观察,转染的HUVEC细胞均表达EGFP和GFP。结论成功构建RACK1过表达载体及其RNA干扰载体,并可被成功导入HU-VEC细胞系。
目的:構建併鑒定蛋白激酶C受體1( RACK1)過錶達及榦擾真覈錶達載體。方法採用逆轉錄聚閤酶鏈反應( RT-PCR)從人肝癌細胞繫Huh-7.5.1中擴增RACK1全長cDNA繫列,用巢式PCR擴增對應的CDS,併將其剋隆到PIRES2-EGFP,PCR鑒定後進行測序分析。以人RACK1基因為靶基因,設計閤成兩對短髮夾結構的互補DNA序列和一對陰性對照序列,退火後與酶切後的載體RNAi-Ready pSIREN-RetroQ-ZsGreen進行連接,併行酶切分析和測序鑒定。將構建好的過錶達載體和榦擾載體用脂質體轉染HUVEC細胞繫。結果兩對引物PCR擴增的序列大小與預期結果一緻,PIRES2-EGFP/RACK1載體954箇堿基成功插入到預計位點,序列完全一緻。 RNA榦擾載體pRetroQ/RACK1-1、pRetroQ/RACK1-2和pRetroQ/HK插入序列均與預計相符。熒光顯微鏡觀察,轉染的HUVEC細胞均錶達EGFP和GFP。結論成功構建RACK1過錶達載體及其RNA榦擾載體,併可被成功導入HU-VEC細胞繫。
목적:구건병감정단백격매C수체1( RACK1)과표체급간우진핵표체재체。방법채용역전록취합매련반응( RT-PCR)종인간암세포계Huh-7.5.1중확증RACK1전장cDNA계렬,용소식PCR확증대응적CDS,병장기극륭도PIRES2-EGFP,PCR감정후진행측서분석。이인RACK1기인위파기인,설계합성량대단발협결구적호보DNA서렬화일대음성대조서렬,퇴화후여매절후적재체RNAi-Ready pSIREN-RetroQ-ZsGreen진행련접,병행매절분석화측서감정。장구건호적과표체재체화간우재체용지질체전염HUVEC세포계。결과량대인물PCR확증적서렬대소여예기결과일치,PIRES2-EGFP/RACK1재체954개감기성공삽입도예계위점,서렬완전일치。 RNA간우재체pRetroQ/RACK1-1、pRetroQ/RACK1-2화pRetroQ/HK삽입서렬균여예계상부。형광현미경관찰,전염적HUVEC세포균표체EGFP화GFP。결론성공구건RACK1과표체재체급기RNA간우재체,병가피성공도입HU-VEC세포계。
Objective To construct and identify the over-expression and interference eukaryotic expression vectors of receptor for activated C-kinase 1 (RACK1).Methods The RACK1 gene full-length reading frame was amplified from the human hepatoma cell line Huh-7.5.1 by RT-PCR.Then we amplified the corresponding CDS by nested PCR and cloned it into PIRES2-EGFP.The monoclone was identified by PCR and DNA sequencing .At the same time, we designed and syn-thesized complementary DNA sequences of 2 pairs of short hairpin structure and a pair of negative control sequence with hu-man RACK1 gene.After annealing, they were linked into restriction enzyme digested RNAi-Ready pSIREN-RetroQ-Zs-Green vector , and then identified by enzyme digestion analysis and DNA sequencing .The constructed over-expression vec-tor and interference vectors were transfected into HUVEC cell line by liposome .Results The size of two pairs of primers amplified by PCR sequences was completely true .A total of 954 nt oligonucleotides were successfully inserted into the vec-tor PIRES2-EGFP.The inserted sequences of RNA interference vectors pRetroQ /RACK1-1, pRetroQ/RACK1-2 and pRet-roQ/HK were consistent with our expectations .The EGFP and GFP could be seen in the transfected HUVEC cell line by fluorescence microscopy .Conclusion The over-expression vector and siRNA expression vectors of RACK 1 are successful-ly constructed , and can be introduced into HUVEC cell line .