人β-防御素-2基因在小鼠纤维母细胞中的表达及抗菌活性的检测
发布时间：2018-09-15 05:53
【摘要】： 目的：获得人β-防御素-2(hBD-2)基因并构建其真核表达载体，转染小鼠纤维母细胞(L929)，检测重组hBD-2表达载体在L929细胞的表达，初步测定hBD-2的体外抗菌活性，为hBD-2基因转染和表达的研究奠定基础。 方法：根据Genebank中hBD-2cDNA核苷酸序列设计PCR引物。采用RT-PCR方法从人宫颈癌组织获取人β-防御素-2基因，然后进行克隆，构建真核表达载体pCAGG-hBD-2，，酶切鉴定并测序。用磷酸钙共沉淀法将pCAGG-hBD-2导入L929细胞，用RT-PCR法检测hBD-2基因mRNA的表达，用Western-blot法鉴定hBD-2基因的蛋白表达，最后采用Kirby-Bauer纸片扩散法检测重组hBD-2的抗菌活性。 结果： 1．RT-PCR扩增出约300bp的片段，测序分析结果表明与Genebank中登录的hBD-2cDNA序列一致，该片段为hBD-2编码全序列。 2．所构建的真核表达载体pCAGG-hBD-2经酶切鉴定并测序，结果表明hBD-2基因与表达载体连接方向正确，可以进行转染。 3．以pCAGG-hBD-2转染后L929细胞的总RNA为模板，进行RT-PCR反应，可扩增出特异性条带；Western blot检测呈阳性。表明我们构建的表达载体pCAGG-hBD-2转染L929细胞后，可正确表达hBD-2。 4．K-B纸片法检测结果显示转染pCAGG-hBD-2细胞的上清液对金黄色葡萄球菌(ATCC 25923)、铜绿假单胞菌(ATCC 27853)有明显的杀灭效应。 结论：成功构建了hBD-2的真核表达载体pCAGG-hBD-2，转染pCAGG-hBD-2的小鼠纤维母细胞能高效表达hBD-2，转染细胞的上清液对金黄色葡萄球菌(ATCC 25923)、铜绿假单胞菌(ATCC 27853)有杀灭作用。
[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).
【学位授予单位】：兰州大学
【学位级别】：硕士
【学位授予年份】：2007
【分类号】：R392
本文编号：2243970
[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).
【学位授予单位】：兰州大学
【学位级别】：硕士
【学位授予年份】：2007
【分类号】：R392
【参考文献】
相关期刊论文 前2条
1 李春丽,何国庆,崔淑贞;人β防御素3基因的合成及其克隆[J];河南农业大学学报;2005年03期
2 雷撼;钱桂生;梁永杰;黄桂君;吴国明;;人β防御素-2基因在SPC细胞中的转染表达[J];同济大学学报(医学版);2006年01期
本文编号：2243970
本文链接：https://www.wllwen.com/yixuelunwen/binglixuelunwen/2243970.html
最近更新
教材专著
