生物技术通讯
生物技術通訊
생물기술통신
LETTERS IN BIOTECHNOLOGY
2013年
1期
57-60
,共4页
郝春秋%彭梅娟%谢玉梅%周云%魏欣%马力%王素娜%李瑞娟%张岩%白雪帆%贾战生
郝春鞦%彭梅娟%謝玉梅%週雲%魏訢%馬力%王素娜%李瑞娟%張巖%白雪帆%賈戰生
학춘추%팽매연%사옥매%주운%위흔%마력%왕소나%리서연%장암%백설범%가전생
结缔组织生长因子%RNA干扰%腺病毒载体
結締組織生長因子%RNA榦擾%腺病毒載體
결체조직생장인자%RNA간우%선병독재체
connective tissue growth factor%RNA interference%adenovirus vector
目的:构建能介导结缔组织生长因子(CTGF)基因RNA干扰的复制缺陷型腺病毒表达载体.方法:以大鼠CTGF基因为靶序列,设计并合成含编码短发夹RNA序列的寡核苷酸,构建腺病毒穿梭质粒p-shuttle-CTGF,酶切及测序分析正确后,与腺病毒骨架质粒pAdEasy-1共转染AD-293细胞,进行病毒包装,得到腺病毒载体Ad.H1-CTGF,用该载体感染HSC-T6细胞,观察其对CTGF基因表达抑制的效果.结果:构建的腺病毒穿梭质粒p-shuttle-CTGF经酶切、测序分析证实正确;包装的病毒载体滴度为4×1010 PFU/mL,感染HSC-T6细胞后,Western印迹证实CTGF表达显著减少.结论:构建的腺病毒载体Ad.H1-CTGF可有效抑制HSC-T6中CTGF的表达,为抗纤维化研究提供了有力的工具.
目的:構建能介導結締組織生長因子(CTGF)基因RNA榦擾的複製缺陷型腺病毒錶達載體.方法:以大鼠CTGF基因為靶序列,設計併閤成含編碼短髮夾RNA序列的寡覈苷痠,構建腺病毒穿梭質粒p-shuttle-CTGF,酶切及測序分析正確後,與腺病毒骨架質粒pAdEasy-1共轉染AD-293細胞,進行病毒包裝,得到腺病毒載體Ad.H1-CTGF,用該載體感染HSC-T6細胞,觀察其對CTGF基因錶達抑製的效果.結果:構建的腺病毒穿梭質粒p-shuttle-CTGF經酶切、測序分析證實正確;包裝的病毒載體滴度為4×1010 PFU/mL,感染HSC-T6細胞後,Western印跡證實CTGF錶達顯著減少.結論:構建的腺病毒載體Ad.H1-CTGF可有效抑製HSC-T6中CTGF的錶達,為抗纖維化研究提供瞭有力的工具.
목적:구건능개도결체조직생장인자(CTGF)기인RNA간우적복제결함형선병독표체재체.방법:이대서CTGF기인위파서렬,설계병합성함편마단발협RNA서렬적과핵감산,구건선병독천사질립p-shuttle-CTGF,매절급측서분석정학후,여선병독골가질립pAdEasy-1공전염AD-293세포,진행병독포장,득도선병독재체Ad.H1-CTGF,용해재체감염HSC-T6세포,관찰기대CTGF기인표체억제적효과.결과:구건적선병독천사질립p-shuttle-CTGF경매절、측서분석증실정학;포장적병독재체적도위4×1010 PFU/mL,감염HSC-T6세포후,Western인적증실CTGF표체현저감소.결론:구건적선병독재체Ad.H1-CTGF가유효억제HSC-T6중CTGF적표체,위항섬유화연구제공료유력적공구.
Objective: To construct a replication-deficient recombinant adenovirus vector expressing small interfer?ence RNA(siRNA) against the connective tissue growth factor(CTGF) gene. Methods: The siRNA sequence target?ing CTGF mRNA was synthesized and shuttle plasmid p-shuttle-CTGF was constructed, which was co-transfected into AD-293 cells with the bone plasmid pAdEasy-1 to obtain the homologous recombination adenovirus vector Ad.H1-CTGF. The concentration of CTGF protein in HSC-T6 cells infected with adenovirus vector Ad.H1-CTGF was determined by Wetsern blot. Results: The p-shuttle-CTGF was identified by endonuclease and sequencing. The adenovirus vector Ad.H1-CTGF was successfully constructed with the titre of 4×1010 PFU/mL by purification. The concentration of CTGF protein in HSC-T6 cells was decreased observably after Ad.H1-CTGF infected by Western blot. Conclusion: The constructed Ad.H1-CTGF can effectively inhit the expression of CTGF in HSC-T6 cells, which provided a powerful tool for anti-fibrosis study.
