Team:NYMU-Taipei/Project/Receptor/Integrin

Motivation
Virus have a high mutation rate, so our immune system can not always detect mutated viruses. But virus capsid proteins have some specific binding proteins that viruses use to enter into human cells. There are some highly conserved regions on the virus proteins, since mutations of these regions will never be worth it for the viruses since the viruses will not be able to enter human cells and survive.

We wanted to express receptors that interact with several viruses (such as Adenovirus and coxsackievirus) originating from human cells in our ViroCatcher. In addition, we have to check the folding of target receptors, because our receptors come from human cells have to express and fold in bacteria. Eventually, to make contact with receptors of Adenovirus-like viruses and we have to anchor the receptors on the membrane of our ViroCatcher.

Goal
Use the receptors on human cells that bind to adenovirus-like viruses, and put them on our ViroCatcher to catch adenovirus-like viruses.

Receptors used to catch adenovirus and coxsackievirus
Previous studies indicates that several viruses like adenovirus and coxsackivirus interact with cellular receptors alpha-V integrin to entry into human cells. These virus capsid proteins has a conserve "RGD" region that interacts with the human &alpha;-V integrin receptor. Previous studies also shows that when virus lose the "RGD" motif, they reduce their ability of infection[1]. Not only can adenovirus and cosackievirus interact with receptor the &alpha;-V integrin, many other viruses can interact with it (See table 1)[2].

We chose to use receptor &alpha;-V integrin to catch viruses that has the "RGD" motif, so our ViroCatcher can catch several virus just by using the alpha-V integrin receptor.



Here we chose another receptor CAR (coxsackievirus and adenovirus receptor) that is used specially for catching adenovirus and coxsakievirus. Adenovirus and coxsakievirus not only bind to the &alpha;-V integrin receptor, it also binds to the CAR receptor when they infect our cells. They use their fiber proteins on their capsid to interact with the CAR receptor[3].

Previous studies also indicates that the binding affinity of the CAR receptor is better than &alpha;-V integrin, so we add the CAR receptor to ensure that our ViroCatcher can catch adenovirus and coxsackievirus[3].

We express the two receptors &alpha;-V integrin and the CAR receptor on our ViroCatcher membrane and use autotransporter system to anchor them on the membrane of our ViroCatcher. 1 atggcttttc cgccgcggcg acggctgcgc ctcggtcccc gcggcctccc gcttcttctc 61 tcgggactcc tgctacctct gtgccgcgcc ttcaacctag acgtggacag tcctgccgag 121 tactctggcc ccgagggaag ttacttcggc ttcgccgtgg atttcttcgt gcccagcgcg 181 tcttcccgga tgtttcttct cgtgggagct cccaaagcaa acaccaccca gcctgggatt 241 gtggaaggag ggcaggtcct caaatgtgac tggtcttcta cccgccggtg ccagccaatt 301 gaatttgatg caacaggcaa tagagattat gccaaggatg atccattgga atttaagtcc 361 catcagtggt ttggagcatc tgtgaggtcg aaacaggata aaattttggc ctgtgcccca 421 ttgtaccatt ggagaactga gatgaaacag gagcgagagc ctgttggaac atgctttctt 481 caagatggaa caaagactgt tgagtatgct ccatgtagat cacgtcagct tatttcggat 541 caagtggcag aaatcgtatc taaatacgac cccaatgttt acagcatcaa gtataataac 601 caattagcaa ctcggactgc acaagctatt tttgatgaca gctatttggg ttattctgtg 661 gctgtcggag atttcaatgg tgatggcata gatgactttg tttcaggagt tccaagagca 721 gcaaggactt tgggaatggt ttatatttat gatgggaaga acatgtcctc cttatacaat 781 tttactggcg agcagatggc tgcatatttc ggattttctg tagctgccac tgacattaat 841 ggagatgatt atgcagatgt gtttattgga gcacctctct tcatggatcg tggctctgat 901 ggcaaactcc aagaggtggg gcaggtctca gtgtctctac agagagcttc aggagacttc 961 cagacgacaa agctgaatgg atttgaggtc tttgcacggt ttggcagtgc catagctcct 1021 ttgggagatc tggaccagga tggtttcaat gatattgcaa ttgctgctcc atatgggggt 1081 gaagataaaa aaggaattgt ttatatcttc aatggaagat caacaggctt gaacgcagtc 1141 ccatctcaaa tccttgaagg gcagtgggct gctcgaagca tgccaccaag ctttggctat 1201 tcaatgaaag gagccacaga tatagacaaa aatggatatc cagacttaat tgtaggagct 1261 tttggtgtag atcgagctat cttatacagg gccagaccag ttatcactgt aaatgctggt 1321 cttgaagtgt accctagcat tttaaatcaa gacaataaaa cctgctcact gcctggaaca 1381 gctctcaaag tttcctgttt taatgttagg ttctgcttaa aggcagatgg caaaggagta 1441 cttcccagga aacttaattt ccaggtggaa cttcttttgg ataaactcaa gcaaaaggga 1501 gcaattcgac gagcactgtt tctctacagc aggtccccaa gtcactccaa gaacatgact 1561 atttcaaggg ggggactgat gcagtgtgag gaattgatag cgtatctgcg ggatgaatct 1621 gaatttagag acaaactcac tccaattact atttttatgg aatatcggtt ggattataga 1681 acagctgctg atacaacagg cttgcaaccc attcttaacc agttcacgcc tgctaacatt 1741 agtcgacagg ctcacattct acttgactgt ggtgaagaca atgtctgtaa acccaagctg 1801 gaagtttctg tagatagtga tcaaaagaag atctatattg gggatgacaa ccctctgaca 1861 ttgattgtta aggctcagaa tcaaggagaa ggtgcctacg aagctgagct catcgtttcc 1921 attccactgc aggctgattt catcggggtt gtccgaaaca atgaagcctt agcaagactt 1981 tcctgtgcat ttaagacaga aaaccaaact cgccaggtgg tatgtgacct tggaaaccca 2041 atgaaggctg gaactcaact cttagctggt cttcgtttca gtgtgcacca gcagtcagag 2101 atggatactt ctgtgaaatt tgacttacaa atccaaagct caaatctatt tgacaaagta 2161 agcccagttg tatctcacaa agttgatctt gctgttttag ctgcagttga gataagagga 2221 gtctcgagtc ctgatcatat ctttcttccg attccaaact gggagcacaa ggagaaccct 2281 gagactgaag aagatgttgg gccagttgtt cagcacatct atgagctgag aaacaatggt 2341 ccaagttcat tcagcaaggc aatgctccat cttcagtggc cttacaaata taataataac 2401 actctgttgt atatccttca ttatgatatt gatggaccaa tgaactgcac ttcagatatg 2461 gagatcaacc ctttgagaat taagatctca tctttgcaaa caactgaaaa gaatgacacg 2521 gttgccgggc aaggtgagcg ggaccatctc atcactaagc gggatcttgc cctcagtgaa 2581 ggagatattc acactttggg ttgtggagtt gctcagtgct tgaagattgt ctgccaagtt 2641 gggagattag acagaggaaa gagtgcaatc ttgtacgtaa agtcattact gtggactgag 2701 acttttatga ataaagaaaa tcagaatcat tcctattctc tgaagtcgtc tgcttcattt 2761 aatgtcatag agtttcctta taagaatctt ccaattgagg atatcaccaa ctccacattg 2821 gttaccacta atgtcacctg gggcattcag ccagcgccca tgcctgtgcc tgtgtgggtg 2881 atcattttag cagttctagc aggattgttg ctactggctg ttttggtatt tgtaatgtac 2941 aggatgggct tttttaaacg ggtccggcca cctcaagaag aacaagaaag ggagcagctt 3001 caacctcatg aaaatggtga aggaaactca gaaacttaa outer primer foward contain ompA sticky tag GCACTCGTCCGGACAAC(56deg, 65%, 17bp) ATGGCTTTTCCGCCG 53deg, 60%, 8+15bp outer primer reverse ctgcagcggccgctactagta TTAAGTTTCTGAGTTTCCTTCACC 55deg, 38%, 21+24bp inner primer1:55deg, 53%, 19bp CCATTCCACTaCAGGCTGA TCAGCCTGtAGTGGAATGG inner primer2:55deg, 41%, 22bp CTGTTTTAGCaGCAGTTGAGAT ATCTCAACTGCtGCTAAAACAG 1 atggcgctcc tgctgtgctt cgtgctcctg tgcggagtag tggatttcgc cagaagtttg 61 agtatcacta ctcctgaaga gatgattgaa aaagccaaag gggaaactgc ctatctgccg 121 tgcaaattta cgcttagtcc cgaagaccag ggaccgctgg acatcgagtg gctgatatca 181 ccagctgata atcagaaggt ggatcaagtg attattttat attctggaga caaaatttat 241 gatgactact atccagatct gaaaggccga gtacatttta cgagtaatga tctcaaatct 301 ggtgatgcat caataaatgt aacgaattta caactgtcag atattggcac atatcagtgc 361 aaagtgaaaa aagctcctgg tgttgcaaat aagaagattc atctggtagt tcttgttaag 421 ccttcaggtg cgagatgtta cgttgatgga tctgaagaaa ttggaagtga ctttaagata 481 aaatgtgaac caaaagaagg ttcacttcca ttacagtatg agtggcaaaa attgtctgac 541 tcacagaaaa tgcccacttc atggttagca gaaatgactt catctgttat atctgtaaaa 601 aatgcctctt ctgagtactc tgggacatac agctgtacag tcagaaacag agtgggctct 661 gatcagtgcc tgttgcgtct aaacgttgtc cctccttcaa ataaagctgg actaattgca 721 ggagccatta taggaacttt gcttgctcta gcgctcattg gtcttatcat cttttgctgt 781 cgtaaaaagc gcagagaaga aaaatatgaa aaggaagttc atcacgatat cagggaagat 841 gtgccacctc caaagagccg tacgtccact gccagaagct acatcggcag taatcattca 901 tccctggggt ccatgtctcc ttccaacatg gaaggatatt ccaagactca gtataaccaa 961 gtaccaagtg aagactttga acgcactcct cagagtccga ctctcccacc tgctaaggta 1021 gctgccccta atctaagtcg aatgggtgcg attcctgtga tgattccagc acagagcaag 1081 gatgggtcta tagtatag outer primer forward contain ompA sticky tag GCACTCGTCCGGACAAC(56deg, 65%, 17bp)ATGGCGCTCCTGCTGTGC 62deg, 67%, 18bp outer primer reverse ctgcagcggccgctactagta CTATACTATAGACCCATCCTTGCTC 55deg, 44%, 25bp
 * alpha-V integrin sequence
 * receptor CXADR(CAR) sequence