【摘要】： 目的：獲得人β-防御素-2(hBD-2)基因并構建其真核表達載體，轉染小鼠纖維母細胞(L929)，檢測重組hBD-2表達載體在L929細胞的表達，初步測定hBD-2的體外抗菌活性，為hBD-2基因轉染和表達的研究奠定基礎。 方法：根據(jù)Genebank中hBD-2cDNA核苷酸序列設計PCR引物。采用RT-PCR方法從人宮頸癌組織獲取人β-防御素-2基因，然后進行克隆，構建真核表達載體pCAGG-hBD-2，，酶切鑒定并測序。用磷酸鈣共沉淀法將pCAGG-hBD-2導入L929細胞，用RT-PCR法檢測hBD-2基因mRNA的表達，用Western-blot法鑒定hBD-2基因的蛋白表達，最后采用Kirby-Bauer紙片擴散法檢測重組hBD-2的抗菌活性。 結果： 1．RT-PCR擴增出約300bp的片段，測序分析結果表明與Genebank中登錄的hBD-2cDNA序列一致，該片段為hBD-2編碼全序列。 2．所構建的真核表達載體pCAGG-hBD-2經酶切鑒定并測序，結果表明hBD-2基因與表達載體連接方向正確，可以進行轉染。 3．以pCAGG-hBD-2轉染后L929細胞的總RNA為模板，進行RT-PCR反應，可擴增出特異性條帶；Western blot檢測呈陽性。表明我們構建的表達載體pCAGG-hBD-2轉染L929細胞后，可正確表達hBD-2。 4．K-B紙片法檢測結果顯示轉染pCAGG-hBD-2細胞的上清液對金黃色葡萄球菌(ATCC 25923)、銅綠假單胞菌(ATCC 27853)有明顯的殺滅效應。 結論：成功構建了hBD-2的真核表達載體pCAGG-hBD-2，轉染pCAGG-hBD-2的小鼠纖維母細胞能高效表達hBD-2，轉染細胞的上清液對金黃色葡萄球菌(ATCC 25923)、銅綠假單胞菌(ATCC 27853)有殺滅作用。
[Abstract]:Aim: to obtain human 尾 -defensin -2 (hBD-2) gene and construct its eukaryotic expression vector, transfect it into mouse fibroblast cells (L929), detect the expression of recombinant hBD-2 expression vector in L929 cells, and preliminarily determine the antibacterial activity of hBD-2 in vitro. To lay a foundation for the study of hBD-2 gene transfection and expression. Methods: PCR primers were designed according to hBD-2cDNA nucleotide sequence in Genebank. Human 尾 -defensin 2 gene was obtained from human cervical cancer tissue by RT-PCR method, and then cloned. The eukaryotic expression vector pCAGG-hBD-2, was digested and sequenced. PCAGG-hBD-2 was introduced into L929 cells by calcium phosphate coprecipitation method. The expression of hBD-2 gene mRNA was detected by RT-PCR method, the protein expression of hBD-2 gene was identified by Western-blot method, and the antibacterial activity of recombinant hBD-2 was detected by Kirby-Bauer disk diffusion method. Results: the fragments of about 300bp were amplified by 1.RT-PCR, and the results of sequencing showed that they were consistent with the hBD-2cDNA sequences registered in Genebank. 2. The constructed eukaryotic expression vector pCAGG-hBD-2 was identified by restriction endonuclease digestion and sequenced. The results showed that the hBD-2 gene was connected to the expression vector in the correct direction. Using the total RNA of L929 cells transfected with pCAGG-hBD-2 as template, the specific bands could be amplified by RT-PCR reaction and detected positive by Western blot. The results showed that the expression vector pCAGG-hBD-2 was transfected into L929 cells. The results showed that the supernatant of transfected pCAGG-hBD-2 cells had obvious killing effect on Staphylococcus aureus (ATCC 25923) and Pseudomonas aeruginosa (ATCC 27853). Conclusion: the mouse fibroblasts transfected with hBD-2 eukaryotic expression vector pCAGG-hBD-2, can efficiently express the supernatant of hBD-2, transfected cells, which can kill Staphylococcus aureus (ATCC 25923) and Pseudomonas aeruginosa (ATCC 27853).
【學位授予單位】：蘭州大學
【學位級別】：碩士
【學位授予年份】：2007
【分類號】：R392

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相關期刊論文 前2條

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