pBiFC-VN173

Objective: To construct pBIFC-VN173-CXCR4 and pBIFC-VC155-NT21MP eukaryotic expression plasmids and to investigate the interaction of chemokine receptor 4 (CXCR4) and viral macrophage inflammatory protein-II(vMIP-II) N terminal 21 peptides (NT21MP) in living cells. Methods: DNA fragment encoding NT21MP was chemically synthesized and inserted into BiFC eukaryotic expression vector pBIFC-VC155. The full length of CXCR4 DNA fragment was amplified by RT-PCR from SKBR (3) cells and inserted into BiFC eukaryotic expression plasmid pBIFC-VN173. Two recombinant vectors were identified by restriction enzyme digestion and DNA sequencing. The recombinant vectors were cotransfected into Africa green monkey kidney fibroblast COS-7 cells by using Lipofectamine 2000. The interaction of NT21MP and CXCR4 was detected by bimolecular fluorescence complementation (BiFC) assay. Results: The restriction enzyme digestion and DNA sequences and open read frames of two vectors were consistent with experiment design. The BiFC plasmids were successfully cotransfected into the target cells and expressed. The strong BiFC signals were detected in pBIFC-VN173-CXCR4 and pBIFC-VC155-NT21MP cotransfected cells and the fluorescence signal was located in the cytoplasm. Conclusion: The eukaryotic expression plasmids for BiFC assay are successfully constructed. The interaction of NT21MP and CXCR4 in living cells can be detected by using this technology.

0001 CCATTCGCCA TTCAGGCTGC GCAACTGTTG GGAAGGGCGA TCGGTGCGGG CCTCTTCGCT 
0061 ATTACGCCAG CTGGCGAAAG GGGGATGTGC TGCAAGGCGA TTAAGTTGGG TAACGCCAGG 
0121 GTTTTCCCAG TCACGACGTT GTAAAACGAC GGCCAGTGCC AAGCTGATCT ATACATTGAA 
0181 TCAATATTGG CAATTAGCCA TATTAGTCAT TGGTTATATA GCATAAATCA ATATTGGCTA 
0241 TTGGCCATTG CATACGTTGT ATCTATATCA TAATATGTAC ATTTATATTG GCTCATGTCC 
0301 AATATGACCG CCATGTTGAC ATTGATTATT GACTAGTTAT TAATAGTAAT CAATTACGGG 
0361 GTCATTAGTT CATAGCCCAT ATATGGAGTT CCGCGTTACA TAACTTACGG TAAATGGCCC 
0421 GCCTGGCTGA CCGCCCAACG ACCCCCGCCC ATTGACGTCA ATAATGACGT ATGTTCCCAT 
0481 AGTAACGCCA ATAGGGACTT TCCATTGACG TCAATGGGTG GAGTATTTAC GGTAAACTGC 
0541 CCACTTGGCA GTACATCAAG TGTATCATAT GCCAAGTCCG CCCCCTATTG ACGTCAATGA 
0601 CGGTAAATGG CCCGCCTGGC ATTATGCCCA GTACATGACC TTACGGGACT TTCCTACTTG 
0661 GCAGTACATC TACGTATTAG TCATCGCTAT TACCATGGTG ATGCGGTTTT GGCAGTACAC 
0721 CAATGGGCGT GGATAGCGGT TTGACTCACG GGGATTTCCA AGTCTCCACC CCATTGACGT 
0781 CAATGGGAGT TTGTTTTGGC ACCAAAATCA ACGGGACTTT CCAAAATGTC GTAATAACCC 
0841 CGCCCCGTTG ACGCAAATGG GCGGTAGGCG TGTACGGTGG GAGGTCTATA TAAGCAGAGC 
0901 TCGTTTAGTG AACCGTCAGA ATTGATCTAC CATGGACTAC AAAGACGATG ACGACAAGCT 
0961 TGCGGCCGCG AATTCATCGA TAGATCTGAT ATCGGTACCA GTCGACTCTA GAAGATCCAT 
1021 CGCCACCATG GTGAGCAAGG GCGAGGAGCT GTTCACCGGG GTGGTGCCCA TCCTGGTCGA 
1081 GCTGGACGGC GACGTAAACG GCCACAAGTT CAGCGTGTCC GGCGAGGGCG AGGGCGATGC 
1141 CACCTACGGC AAGCTGACCC TGAAGCTGAT CTGCACCACC GGCAAGCTGC CCGTGCCCTG 
1201 GCCCACCCTC GTGACCACCC TGGGCTACGG CCTGCAGTGC TTCGCCCGCT ACCCCGACCA 
1261 CATGAAGCAG CACGACTTCT TCAAGTCCGC CATGCCCGAA GGCTACGTCC AGGAGCGCAC 
1321 CATCTTCTTC AAGGACGACG GCAACTACAA GACCCGCGCC GAGGTGAAGT TCGAGGGCGA 
1381 CACCCTGGTG AACCGCATCG AGCTGAAGGG CATCGACTTC AAGGAGGACG GCAACATCCT 
1441 GGGGCACAAG CTGGAGTACA ACTACAACAG CCACAACGTC TATATCACCG CCGACAAGCA 
1501 GAAGAACGGC ATCAAGGCCA ACTTCAAGAT CCGCCACAAC ATCGAGTAGG GATCCCGGGT 
1561 GGCATCCCTG TGACCCCTCC CCAGTGCCTC TCCTGGCCCT GGAAGTTGCC ACTCCAGTGC 
1621 CCACCAGCCT TGTCCTAATA AAATTAAGTT GCATCATTTT GTCTGACTAG GTGTCCTTCT 
1681 ATAATATTAT GGGGTGGAGG GGGGTGGTAT GGAGCAAGGG GCAAGTTGGG AAGACAACCT 
1741 GTAGGGCCTG CGGGGTCTAT TGGGAACCAA GCTGGAGTGC AGTGGCACAA TCTTGGCTCA 
1801 CTGCAATCTC CGCCTCCTGG GTTCAAGCGA TTCTCCTGCC TCAGCCTCCC GAGTTGTTGG 
1861 GATTCCAGGC ATGCATGACC AGGCTCAGCT AATTTTTGTT TTTTTGGTAG AGACGGGGTT 
1921 TCACCATATT GGCCAGGCTG GTCTCCAACT CCTAATCTCA GGTGATCTAC CCACCTTGGC 
1981 CTCCCAAATT GCTGGGATTA CAGGCGTGAA CCACTGCTCC CTTCCCTGTC CTTCTGATTT 
2041 TAAAATAACT ATACCAGCAG GAGGACGTCC AGACACAGCA TAGGCTACCT GGCCATGCCC 
2101 AACCGGTGGG ACATTTGAGT TGCTTGCTTG GCACTGTCCT CTCATGCGTT GGGTCCACTC 
2161 AGTAGATGCC TGTTGAATTG GGTACGCGGC CAGCTTGGCT GTGGAATGTG TGTCAGTTAG 
2221 GGTGTGGAAA GTCCCCAGGC TCCCCAGCAG GCAGAAGTAT GCAAAGCATG CATCTCAATT 
2281 AGTCAGCAAC CAGGTGTGGA AAGTCCCCAG GCTCCCCAGC AGGCAGAAGT ATGCAAAGCA 
2341 TGCATCTCAA TTAGTCAGCA ACCATAGTCC CGCCCCTAAC TCCGCCCATC CCGCCCCTAA 
2401 CTCCGCCCAG TTCCGCCCAT TCTCCGCCCC ATGGCTGACT AATTTTTTTT ATTTATGCAG 
2461 AGGCCGAGGC CGCCTCGGCC TCTGAGCTAT TCCAGAAGTA GTGAGGAGGC TTTTTTGGAG 
2521 GCCTAGGCTT TTGCAAAAAG CTCCTCGAGG AACTGAAAAA CCAGAAAGTT AATTCCCTAT 
2581 AGTGAGTCGT ATTAAATTCG TAATCATGTC ATAGCTGTTT CCTGTGTGAA ATTGTTATCC 
2641 GCTCACAATT CCACACAACA TACGAGCCGG AAGCATAAAG TGTAAAGCCT GGGGTGCCTA 
2701 ATGAGTGAGC TAACTCACAT TAATTGCGTT GCGCTCACTG CCCGCTTTCC AGTCGGGAAA 
2761 CCTGTCGTGC CAGCTGCATT AATGAATCGG CCAACGCGCG GGGAGAGGCG GTTTGCGTAT 
2821 TGGGCGCTCT TCCGCTTCCT CGCTCACTGA CTCGCTGCGC TCGGTCGTTC GGCTGCGGCG 
2881 AGCGGTATCA GCTCACTCAA AGGCGGTAAT ACGGTTATCC ACAGAATCAG GGGATAACGC 
2941 AGGAAAGAAC ATGTGAGCAA AAGGCCAGCA AAAGGCCAGG AACCGTAAAA AGGCCGCGTT 
3001 GCTGGCGTTT TTCCATAGGC TCCGCCCCCC TGACGAGCAT CACAAAAATC GACGCTCAAG 
3061 TCAGAGGTGG CGAAACCCGA CAGGACTATA AAGATACCAG GCGTTTCCCC CTGGAAGCTC 
3121 CCTCGTGCGC TCTCCTGTTC CGACCCTGCC GCTTACCGGA TACCTGTCCG CCTTTCTCCC 
3181 TTCGGGAAGC GTGGCGCTTT CTCATAGCTC ACGCTGTAGG TATCTCAGTT CGGTGTAGGT 
3241 CGTTCGCTCC AAGCTGGGCT GTGTGCACGA ACCCCCCGTT CAGCCCGACC GCTGCGCCTT 
3301 ATCCGGTAAC TATCGTCTTG AGTCCAACCC GGTAAGACAC GACTTATCGC CACTGGCAGC 
3361 AGCCACTGGT AACAGGATTA GCAGAGCGAG GTATGTAGGC GGTGCTACAG AGTTCTTGAA 
3421 GTGGTGGCCT AACTACGGCT ACACTAGAAG AACAGTATTT GGTATCTGCG CTCTGCTGAA 
3481 GCCAGTTACC TTCGGAAAAA GAGTTGGTAG CTCTTGATCC GGCAAACAAA CCACCGCTGG 
3541 TAGCGGTGGT TTTTTTGTTT GCAAGCAGCA GATTACGCGC AGAAAAAAAG GATCTCAAGA 
3601 AGATCCTTTG ATCTTTTCTA CGGGGTCTGA CGCTCAGTGG AACGAAAACT CACGTTAAGG 
3661 GATTTTGGTC ATGAGATTAT CAAAAAGGAT CTTCACCTAG ATCCTTTTAA ATTAAAAATG 
3721 AAGTTTTAAA TCAATCTAAA GTATATATGA GTAAACTTGG TCTGACAGTT ACCAATGCTT 
3781 AATCAGTGAG GCACCTATCT CAGCGATCTG TCTATTTCGT TCATCCATAG TTGCCTGACT 
3841 CCCCGTCGTG TAGATAACTA CGATACGGGA GGGCTTACCA TCTGGCCCCA GTGCTGCAAT 
3901 GATACCGCGA GACCCACGCT CACCGGCTCC AGATTTATCA GCAATAAACC AGCCAGCCGG 
3961 AAGGGCCGAG CGCAGAAGTG GTCCTGCAAC TTTATCCGCC TCCATCCAGT CTATTAATTG 
4021 TTGCCGGGAA GCTAGAGTAA GTAGTTCGCC AGTTAATAGT TTGCGCAACG TTGTTGCCAT 
4081 TGCTACAGGC ATCGTGGTGT CACGCTCGTC GTTTGGTATG GCTTCATTCA GCTCCGGTTC 
4141 CCAACGATCA AGGCGAGTTA CATGATCCCC CATGTTGTGC AAAAAAGCGG TTAGCTCCTT 
4201 CGGTCCTCCG ATCGTTGTCA GAAGTAAGTT GGCCGCAGTG TTATCACTCA TGGTTATGGC 
4261 AGCACTGCAT AATTCTCTTA CTGTCATGCC ATCCGTAAGA TGCTTTTCTG TGACTGGTGA 
4321 GTACTCAACC AAGTCATTCT GAGAATAGTG TATGCGGCGA CCGAGTTGCT CTTGCCCGGC 
4381 GTCAATACGG GATAATACCG CGCCACATAG CAGAACTTTA AAAGTGCTCA TCATTGGAAA 
4441 ACGTTCTTCG GGGCGAAAAC TCTCAAGGAT CTTACCGCTG TTGAGATCCA GTTCGATGTA 
4501 ACCCACTCGT GCACCCAACT GATCTTCAGC ATCTTTTACT TTCACCAGCG TTTCTGGGTG 
4561 AGCAAAAACA GGAAGGCAAA ATGCCGCAAA AAAGGGAATA AGGGCGACAC GGAAATGTTG 
4621 AATACTCATA CTCTTCCTTT TTCAATATTA TTGAAGCATT TATCAGGGTT ATTGTCTCAT 
4681 GAGCGGATAC ATATTTGAAT GTATTTAGAA AAATAAACAA ATAGGGGTTC CGCGCACATT 
4741 TCCCCGAAAA GTGCCACCTG ACGCGCCCTG TAGCGGCGCA TTAAGCGCGG CGGGTGTGGT 
4801 GGTTACGCGC AGCGTGACCG CTACACTTGC CAGCGCCCTA GCGCCCGCTC CTTTCGCTTT 
4861 CTTCCCTTCC TTTCTCGCCA CGTTCGCCGG CTTTCCCCGT CAAGCTCTAA ATCGGGGGCT 
4921 CCCTTTAGGG TTCCGATTTA GTGCTTTACG GCACCTCGAC CCCAAAAAAC TTGATTAGGG 
4981 TGATGGTTCA CGTAGTGGGC CATCGCCCTG ATAGACGGTT TTTCGCCCTT TGACGTTGGA 
5041 GTCCACGTTC TTTAATAGTG GACTCTTGTT CCAAACTGGA ACAACACTCA ACCCTATCTC 
5101 GGTCTATTCT TTTGATTTAT AAGGGATTTT GCCGATTTCG GCCTATTGGT TAAAAAATGA 
5161 GCTGATTTAA CAAAAATTTA ACGCGAATTT TAACAAAATA TTAACGCTTA CAATTT