【文章內(nèi)容簡介】 計與退火 根據(jù)siRNA設(shè)計原則[34]，根據(jù)MRP1靶序列，設(shè)計合成四對互補反向重復脫氧核糖核酸序列，中間間隔9nt莖環(huán)序列(TTCAAGAGA)，5′端帶有BamHⅠ酶切位點，3′端帶有HindⅢ酶切位點，用BLAST進行同源性分析，確定與其他基因無同源性。shRNA的DNA模板由上海生工合成，單鏈干涉片段退火后形成雙鏈。干涉靶點在mrp1基因mRNA全序列中的位置如下(粗體代表靶序列)：1 ccaggcggcg ttgcggcccc ggccccggct ccctgcgccg ccgccgccgc cgccgccgcc61 gccgccgccg ccgccgccag cgctagcgcc agcagccggg cccgatcacc cgccgcccgg121 tgcccgccgc cgcccgcgcc agcaaccggg cccgatcacc cgccgcccgg tgcccgccgc181 cgcccgcgcc accggcatgg cgctccgggg cttctgcagc gccgatggct ccgacccgct241 ctgggactgg aatgtcacgt ggaataccag caaccccgac ttcaccaagt gctttcagaa301 cacggtcctc gtgtgggtgc cttgttttta cctctgggcc tgtttcccct tctacttcct361 ctatctctcc cgacatgacc gaggctacat tcagatgaca cctctcaaca aaaccaaaac421 tgccttggga tttttgctgt ggatcgtctg ctgggcagac ctcttctact ctttctggga481 aagaagtcgg ggcatattcc tggccccagt gtttctggtc agcccaactc tcttgggcat541 caccacgctg cttgctacct ttttaattca gctggagagg aggaagggag ttcagtcttc601 agggatcatg ctcactttct ggctggtagc cctagtgtgt gccctagcca tcctgagatc661 caaaattatg acagccttaa aagaggatgc ccaggtggac ctgtttcgtg acatcacttt721 ctacgtctac ttttccctct tactcattca gctcgtcttg tcctgtttct cagatcgctc781 acccctgttc tcggaaacca tccacgaccc taatccctgc ccagagtcca gcgcttcctt841 cctgtcgagg atcaccttct ggtggatcac agggttgatt gtccggggct accgccagcc901 cctggagggc agtgacctct ggtccttaaa caaggaggac acgtcggaac aagtcgtgcc961 tgttttggta aagaactgga agaaggaatg cgccaagact aggaagcagc cggtgaaggt1021 tgtgtactcc tccaaggatc ctgcccagcc gaaagagagt tccaaggtgg atgcgaatga1081 ggaggtggag gctttgatcg tcaagtcccc acagaaggag tggaacccct ctctgtttaa1141 ggtgttatac aagacctttg ggccctactt cctcatgagc ttcttcttca aggccatcca1201 cgacctgatg atgttttccg ggccgcagat cttaaagttg ctcatcaagt tcgtgaatga1261 cacgaaggcc ccagactggc agggctactt ctacaccgtg ctgctgtttg tcactgcctg1321 cctgcagacc ctcgtgctgc accagtactt ccacatctgc ttcgtcagtg gcatgaggat1381 caagaccgct gtcattgggg ctgtctatcg gaaggccctg gtgatcacca attcagccag1441 aaaatcctcc acggtcgggg agattgtcaa cctcatgtct gtggacgctc agaggttcat1501 ggacttggcc acgtacatta acatgatctg gtcagccccc ctgcaagtca tccttgctct1561 ctacctcctg tggctgaatc tgggcccttc cgtcctggct ggagtggcgg tgatggtcct1621 catggtgccc gtcaatgctg tgatggcgat gaagaccaag acgtatcagg tggcccacat1681 gaagagcaaa gacaatcgga tcaagctgat gaacgaaatt ctcaatggga tcaaagtgct1741 aaagctttat gcctgggagc tggcattcaa ggacaaggtg ctggccatca ggcaggagga1801 gctgaaggtg ctgaagaagt ctgcctacct gtcagccgtg ggcaccttca cctgggtctg1861 cacgcccttt ctggtggcct tgtgcacatt tgccgtctac gtgaccattg acgagaacaa1921 catcctggat gcccagacag ccttcgtgtc tttggccttg ttcaacatcc tccggtttcc1981 cctgaacatt ctccccatgg tcatcagcag catcgtgcag gcgagtgtct ccctcaaacg2041 cctgaggatc tttctctccc atgaggagct ggaacctgac agcatcgagc gacggcctgt2101 caaagacggc gggggcacga acagcatcac cgtgaggaat gccacattca cctgggccag2161 gagcgaccct cccacactga atggcatcac cttctccatc cccgaaggtg ctttggtggc2221 cgtggtgggc caggtgggct gcggaaagtc gtccctgctc tcagccctct tggctgagat2281 ggacaaagtg gaggggcacg tggctatcaa gggctccgtg gcctatgtgc cacagcaggc2341 ctggattcag aatgattctc tccgagaaaa catccttttt ggatgtcagc tggaggaacc2401 atattacagg tccgtgatac aggcctgtgc cctcctccca gacctggaaa tcctgcccag2461 tggggatcgg acagagattg gcgagaaggg cgtgaacctg tctgggggcc agaagcagcg2521 cgtgagcctg gcccgggccg tgtactccaa cgctgacatt tacctcttcg atgatcccct2581 ctcagcagtg gatgcccatg tgggaaaaca catctttgaa aatgtgattg gccccaaggg2641 gatgctgaag aacaagacgc ggatcttggt cacgcacagc atgagctact tgccgcaggt2701 ggacgtcatc atcgtcatga gtggcggcaa gatctctgag atgggctcct accaggagct2761 gctggctcga gacggcgcct tcgctgagtt cctgcgtacc tatgccagca cagagcagga2821 gcaggatgca gaggagaacg gggtcacggg cgtcagcggt ccagggaagg aagcaaagca2881 aatggagaat ggcatgctgg tgacggacag tgcagggaag caactgcaga gacagctcag2941 cagctcctcc tcctatagtg gggacatcag caggcaccac aacagcaccg cagaactgca3001 gaaagctgag gccaagaagg aggagacctg gaagctgatg gaggctgaca aggcgcagac3061 agggcaggtc aagctttccg tgtactggga ctacatgaag gccatcggac tcttcatctc3121 cttcctcagc atcttccttt tcatgtgtaa ccatgtgtcc gcgctggctt ccaactattg3181 gctcagcctc tggactgatg accccatcgt caacgggact caggagcaca cgaaatagtccg3241 gctgagcgtc tatggagccc tgggcatttc acaagggatc gccgtgtttg gctactcc3301 ggccgtgtcc atcgggggga tcttggcttc ccgctgtctg cacgtggacc tgctgcacag3361 catcctgcgg tcacccatga gcttctttga gcggaccccc agtgggaacc tggtgaaccg3421 cttctccaag gagctggaca cagtggactc catgatcccg gaggtcatca agatgttcat3481 gggctccctg ttcaacgtca ttggtgcctg catcgttatc ctgctggcca cgcccatcgc3541 cgccatcatc atcccgcccc ttggcctcat ctacttcttc gtccagaggt tctacgtggc3601 ttcctcccgg cagctgaagc gcctcgagtc ggtcagccgc tccccggtct attcccattt3661 caacgagacc ttgctggggg tcagcgtcat tcgagccttc gaggagcagg agcgcttcat3721 ccaccagagt gacctgaagg tggacgagaa ccagaaggcc tattacccca gcatcgtggc3781 caacaggtgg ctggccgtgc ggctggagtg tgtgggcaac tgcatcgttc tgtttgctgc3841 cctgtttgcg gtgatctcca ggcacagcct cagtgctggc ttggtgggcc tctcagtgtc3901 ttactcattg caggtcacca cgtacttgaa ctggctggtt cggatgtcat ctgaaatgga3961 aaccaacatc gtggccgtgg agaggctcaa ggagtattca gagactgaga aggaggcgcc4021 ctggcaaatc caggagacag ctccgcccag cagctggccc caggtgggcc gagtggaatt4081 ccggaactac tgcctgcgct accgagagga cctggacttc gttctcaggc acatcaatgt4141 cacgatcaat gggggagaaa aggtcggcat cgtggggcgg acgggagctg ggaagtcgtc4201 cctgaccctg ggcttatttc ggatcaacga gtctgccgaa ggagagatca tcatcgatgg4261 catcaacatc gccaagatcg gcctgcacga cctccgcttc aagatcacca tcatccccca4321 ggaccctgtt ttgttttcgg gttccctccg aatgaacctg gacccattca gccagtactc4381 ggatgaagaa gtctggacgt ccctggagct ggcccacctg aaggacttcg tgtcagccct4441 tcctgacaag ctagaccatg aatgtgcaga aggcggggag aacctcagtg t