膽囊癌hTR錘頭狀核酶基因真核表達載體的構建_《中國普外基礎與臨床雜志》

作者：

靳斌 ¹ , 姜希宏 ² , 王學浩 ¹ , 張峰 ¹ , 王偉 ³ , 劉賢錫 ³

1.南京醫科大學第一附屬醫院肝臟移植中心（南京210029）;;
2.山東大學齊魯醫院（濟南250012）;;
3.山東大學分子生物學實驗中心（濟南250012）;

關鍵詞：

膽囊癌端粒酶核酶基因表達

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目的用反轉錄PCR法，調取膽囊癌細胞內的端粒酶RNA(hTR)基因，并針對其序列設計錘頭狀核酶切割基因序列，并將其構建入真核表達載體pTriEx4內。
方法根據hTR cDNA序列，設計引物，經RT-PCR法從體外培養的膽囊癌細胞內調取hTR模板區基因，根據其測序結果，合成錘頭狀核酶基因，與經相應酶切的真核表達載體連接，酶切鑒定重組體的正確性。
結果經RT-PCR從細胞內調出68 bp序列，與hTR cDNA比較，與模板區域堿基序列一致。核酶基因重組子經酶切鑒定序列正確。
結論 RT-PCR法可調出膽囊癌hTR基因有效片段；成功構建了針對hTR模板區的核酶基因的真核表達載體，為膽囊癌的基因治療奠定了基礎。

引用本文： 靳斌,姜希宏,王學浩,張峰,王偉,劉賢錫. 膽囊癌hTR錘頭狀核酶基因真核表達載體的構建. 中國普外基礎與臨床雜志, 2004, 11(5): 436-439. doi: 復制

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2. Li QX, Robbins JM, Welch PJ, et al. A novel functional genomics approach identifies mTERT as a suppressor of fibroblast transformation [Ｊ]. Nucleic Acids Res, 2000; 28(13)∶2605.
3. Kanazawa Y, Ohkawa K, Ueda K, et al. Hammerhead ribozymemediated inhibition of telomerase activity in extracts of human hepatocellular carcinoma cells [Ｊ]. Biochem Biophys Res Commun, 1996; 225(2)∶570.
4. DeYoung M, Siwkowski AM, Lian Y, et al. Catalytic properties of hairpin ribozymes derived from Chicory yellow mottle virus and arabis mosaic virus satellite RNAs [Ｊ]. Biochemistry, 1995; 34(48)∶15785.
5. Feng J, Funk WD, Wang SS, et al. The RNA component of human telomerase [Ｊ]. Science, 1995; 269(5228)∶1236.
6. Greider CW. Telomerase activity, cell proliferation, and cancer [Ｊ]. Proc Natl Acad Sci USA, 1998; 95(1)∶90.
7. Yokoyama Y, Takahashi Y, Shinohara A, et al. Attenuation of telomerase activity by a hammerhead ribozyme targeting the template region of telomerase RNA in endometrial carcinoma cells [Ｊ]. Cancer Res, 1998; 58(23)∶5406.
8. Cech TR. Ribozymes, the first 20 years [Ｊ]. Biochem Soc Trans, 2002; 30(6)∶1162.

《中國普外基礎與臨床雜志》

膽囊癌hTR錘頭狀核酶基因真核表達載體的構建

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《中國普外基礎與臨床雜志》

膽囊癌hTR錘頭狀核酶基因真核表達載體的構建

摘要 全文 圖表 視頻 參考文獻 施引文獻 補充材料

Format

Content

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