Hela细胞转染实验

1.真核表达载体构建 csrB : 碱基数:369 bp(Hfq依赖性)2883155..2883523 gctagcGTCGACAGGGAGTCAGACAA CGAAGTGAACATCAGGATGATGACAC TTCTGCAGGACACACCAGGATGGTGTTTCAGGGAAAGGCTTCTGGATGAA GCGAAGAGGATGACGCAGGACGCGTTAAAGGACACCTCCAGGATGGAGAA TGAGAACCGGTCAGGATGATTCGGTGGGTCAGGAAGGCCAGGGACACTTC AGGATGAAGTATCACATCGGGGTGGTGTGAGCAGGAAGCAATAGTTCAGG ATGAACGATTGGCCGCAAGGCCAGAGGAAAAGTTGTCAAGGATGAGCAGG GAGCAACAAAAGTAGCTGGAATGCTGCGAAACGAACCGGGAGCACTGTG AATA CAGTGCTCCCTTTTTTTATTtgtaca 引物:上游csrB -F:gctagc GTCGACAGGGAGTCAGACAA NheI酶切位点 下游csrB -R:tgtaca AATAAAAAAAGGGAGCACTGBsrGΙ酶切位点 oxyS:碱基数:110 bp(Hfq依赖性) GAAACGGAGCGGCACCTCTTTTAACCCTTGAAGTCACTGCCCGTTTCGAGA GTTTCTCAACTCGAATAAC TAAAGCCAAC GTGAACTTTT GCGGATCTCC AGGATCCGCT dsrA: 碱基数:87 bp(Hfq依赖性) AAATCCCGACCCTGAGGGTGTCGGGATGAAACTTGCTTAAGCAAGAAGCA CTTAAAAAATTCGTTACACCAGGAAATCTGATGTGTT MicF: 碱基数:93 bp2334487..2334578 GCTATCATCATTAACTTTATTTATTACCGTCATTCATTTCTGAATGTCTGTTTA CCCCTATTTCAACCGGATGCCTCGCATTCGGTTTTTTT gctagcAAGATGTTTCATTTATCGCC ATAGATGTTTCAAAATGTAAATACAAG GGAACTTTTTAAGATTATTGCGGAATGGCGAAATAAGCACCTAACATCAA GCAATAATAATTCAAGGTTAAAATCAATAACTTATTCTTAAGTATTTGACA GCACTGAATGTCAAAACAAAACCTTCACTCGCAACTAGAATAACTCCC GC TATCATCATTAACTTTATTTATTACCGTCATTCATTTCTGAATGTCTGTTTA CCCCTATTTCAACCGGATGCCTCGCATTCGGTTTTTTT ACCCTTCTTTACAC ACTTTTCATTATTCTGTGCTACCACAGAAAAACTATAACGCTTGTTAACTA TTTCACAAATAATTAACATCAGCATAATTTCCAGCAATCTTTGTTTATTTG CAATTAATTTCGTTGGGCTTTT TGTAGGTTATTTGTACAGC 引物:上游MicF -F:gctagc AAGATGTTTCATTTATCGCC NheI酶切位点 下游MicF -R:tgtaca GCTGTACAAATAACCTACA BsrGΙ酶切位点 表达载体:PEGFPN1 卡那霉素抗性,4.7kb, 选取NheΙ、BsrGΙ两个酶切位点 sRNA 真核表达质粒 1、扩增MicF、CsrB全长片段,分别利用引物两对引物扩增,添加酶切位点。PCR产物回收后与PGM-T载体连接后,化学转化至DH5α感受态细胞,测序验证 2. 测序正确后,载体命名分别为PMD18-Micf和PMD18-CsrB,用限制性内切酶NheΙ、BsrGΙ双酶切,然后与pEGFPN1载体连接,连接产物转化至DH5α感受态细胞,构建pEGFPN1-MicF和pEGFPN1-CsrB菌株,测序验证 3. 提取pEGFPN1-Micf和pEGFPN1--CsrB菌株的质粒,转化DH5α感受态细胞中,涂布含有卡那霉素抗性的LB平板,筛选阳性克隆真核表达质粒大肠杆菌 4. lipofectamine+2000试剂盒转染Hela细胞 5.细胞活性检测,脂多糖刺激免疫相关基因表达检测。 lipofectamine+2000试剂盒转染效率 参考文献:lipofectamine+2000转染Neuro-2a细胞实验条件优化 转染质粒(ug):脂质体体积(ul)=1:3(2.5ug:8ul)空质粒效率监测 6孔板覆盖率80% 转染时间48h(预实验观察72h内) 实验结果: 1.引物: 上游csrB -F:gctagc GTCGACAGGGAGTCAGACAA NheI酶切位点下游csrB -R:tgtaca AATAAAAAAAGGGAGCACTG BsrGΙ酶切位点上游MicF -F:gctagc AAGATGTTTCATTTATCGCC NheI酶切位点 下游MicF -R:tgtaca GCTGTACAAATAACCTACA BsrGΙ酶切位点 退火温度:60℃ 2.T载体构建 CSRB: GACAATGACATGATTACGAATTCGAGCTCGGTACCCGGGGATCCTCTAGAG ATTTGTACAAATAAAAAAAGGGAGCACTGTATTCACAGCGTTCCCGGTTCG TTTCACAGCATTCCAGCTACTTTTGTTGCTCCCTGCTCATCCTTGACAACTT TTCCTCTGGCCTTGCGGCCAATCGTTCATCCTGAACTATTGCTTCCTGCTCA CACCACCCCGATGTGATACTTCATCCTGAAGTGTCCCTGGCCTTCCTGACCC ACCGAATCATCCTGACCGGTTCTCATTCTCCATCCTGGAGGTGTCCTTTAAC GCGTCCTGCGTCATCCTCTTCGCTTCATACAGAAGCCTTTCCCTGAAACACC ATCCTGGTGTGTCCTGCAGAAGTGTCATCATCCTGATGTTCACTTCGTTGTC TGACTCCCTGTCGACGCTAGCAATCGTCGACCTGCAGGCATGCAAGCTTGG CACTGGCCGTCGTTTTACAACGTCGTGACTGGGAAAACCCTGGCGTTACCC AACTTAATCGCCTTGCAGCACATCCCCCTTTCGCCAGCTGGCGTAATAGCG AAGAGGCCCGCACCGATCGCCCTTCCCAACAGTTGCGCAGCCTGAATGGC GAATGGCGCCTGATGCGGTATTTTCTCCTTACGCATCTGTGCGGTATTTCAC ACCGCATATGGTGCACTCTCAGTACAATCTGCTCTGATGCCGCATAGTTAAG CCAGCCCCGACACCCGCCAACACCCGCTGACGCGCCCTGACGGGCTTGTC TGCTCCCGGCATCCGCTTACAGACAAGCTGTGACCGTCTCCGGGAGCTGCA TGTGTCAGAGGTTTTCACCGTCATCACCGAAACGCGCGAGACGAAAGGGC CTCGTGATACGCCTATTTTTATAGGTTAATGTCATGATAATAATGGTTTCTTAG ACGTCAGTGGCACTTTTCGGGGAAATGTGCGCGGAACCCCTATTTGTTTAA TTTTTCTATAC MicF: GACCTTGGACATGATTACGATTCGAGCTCGGTACCCGGGGATCCTCTAGAG ATTGCTAGCAAGATGTTTCATTTATCGCCATAGATGTTTCAAAATGTAAATAC AAGGGAACTTTTTAAGATTATTGCGGAATGGCGAAATAAGCACCTAACATC AAGCAATAATAATTCAAGGTTAAAATCAATAACTTATTCTTAAGTATTTGACA GCACTGAATGTCAAAACAAAACCTTCACTCGCAACTAGAATAACTCCCGCT ATCATCATTAACTTTATTTATTACCGTCATTCATTTCTGAATGTCTGTTTACCC CTATTTCAACCGGATGCCTCGCATTCGGTTTTTTTACCCTTCTTTACACACTT TTCATTATTCTGTGCTACCACAGAAAAACTATAACGCTTGTTAACTATTTCAC AAATAATTAACATCAGCATAATTTCCAGCAATCTTTGTTTATTTGCAATTAAT TTCGTTGGGCTTTTTGTAGGTTATTTGTACAGCAAAATGGCGCTACATGTAA TCGTCGACCTGCAGGCATGCAAGCTTGGCACTGGCCGTCGTTTTACAACGT CGTGACTGGGAAAACCCTGGCGTTACCCAACTTAATCGCCTTGCAGCACAT CCCCCTTTCGCCAGCTGGCGTAATAGCGAAGAGGCCCGCACCGATCGCCCT TCCCAACAGTTGCGCAGCCTGAATGGCGAATGGCGCCTGATGCGGTATTTT CTCCTTACGCATCTGTGCGGTATTTCACACCGCATATGGTGCACTCTCAGTA CAATCTGCTCTGATGCCGCATAGTTAAGCCAGCCCCGACACCCGCCAACAC CCGCTGACGCGCCCTGACGGGCTTGTCTGCTCCCGGCATCCGCTTACAGAC AAGCTGTGACCGTCTCCGGGAG 3.质粒提取: PEGFPN1:2000ng/ul 40ul PMD18-T-MicF: 350ng/ul 50ul PMD18-T-CsrB: 2000ng/ul 40ul 4. PEGFPN1脂质体转染预实验 (质粒3ug,脂质体量6,9,12,15ul,6孔板转染12小时) 5. 双酶切结果 6 9 12 15 M PEGFPN1 PMD18-T-MicF PMD18-T-CsrB 250bp 500bp 片段回收: PEGFPN1载体大片段:300ng/ul eGFP :155ng/ul Micf: 60ng/ul CSRB:177ng/ul 6.重组载体连接 连接条件:目的片段载体浓度比例分别:1:4-6 连接前目的片段载体45℃,3分钟后,冰浴1分钟,20ul体系 16℃连接5小时 4℃过夜 转化前45℃3分钟后,冰浴1分钟 大肠杆菌感受态混入冰浴30min,42℃热激60-90s 加入无抗生素培养基37℃50min 全部涂板(kan抗性)。 7.挑取单菌落测序 CSRB: CCATGGGATATATAGCAGAGCTGGTTTAGTGACCGTCAGATCCGCTAGCGTCGACAGGGA GTCAGACAACGAAGTGAACATCAGGATGATGACACTTCTGCAGGACACACCAGGATGGT GTTTCAGGGAAAGGCTTCTGTATGAAGCGAAGAGGATGACGCAGGACGCGTTAAAGGA CACCTCCAGGATGGAGAATGAGAACCGGTCAGGATGATTCGGTGGGTCAGGAAGGCCA GGGACACTTCAGGATGAAGTATCACATCGGGGTGGTGTGAGCAGGAAGCAATAGTTCAG GATGAACGATTGGCCGCAAGGCCAGAGGAAAAGTTGTCAAGGATGAGCAGGGAGCAA CAAAAGTAGCTGGAATGCTGTGAAACGAACCGGGAACGCTGTGAATACAGTGCTCCCTT TTTTTATTTGTACAAGTAAAGCGGCCGCGACTCTAGATCATAATCAGCCATACCACATTTGT AGAGGTTTTACTTGCTTTAAAAAACCTCCCACACCTCCCCCTGAACCTGAAACATAAAATG AATGCAATTGTTGTTGTTAACTTGTTTATTGCAGCTTATAATGGTTACAAATAAAGCAATAG CATCACAAATTTCACAAATAAAGCATTTTTTTCACTGCATTCTAGTTGTGGTTTGTCCAAAC TCATCAATGTATCTTAAGGCGTAAATTGTAAGCGTTAATATTTTGTTAAAATTCGCGTTAAAT TTTTGTTAAATCAGCTCATTTTTTAACCAATAGGCCGAAATCGGCAAAATCCCTTATAAATC AAAAGAATAGACCGAGATAGGGTTGAGTGTTGTTCCAGTTTGGAACAAGAGTCCACTATT AAAGAACGTGGACTCCAACGTCAAAGGGCGAAAAACCGTCTATCAGGGCGATGGCCCA CTACGTGAACCATCACCCTAATCAAGTTTTTTGGGGTCGAGGTGCCGTA 8.转染后Qpcr检测表达: Qpcr引物 CSRB-F:ACAACGAAGTGAACATCAGGAT CSRB-R : CGCAGCATTCCAGCTACTT 9.细胞增殖情况(CCK8) 转染24小时后 450nm 吸光度 117933.4877125614.8148 114044.4444116881.4815