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1、 南华大学硕士学位论文粪肠球菌Ace重组蛋白的表达、纯化及黏附活性研究姓名：武楠申请学位级别：硕士专业：病原生物学指导教师：杨祚升20070501 Expression and purification of Ace recombinant protein ofEnterococcus faecalis and characterization of its adherenceAbstractObjective: This study was carried out to construct a recombinant vector containting thegene encoding A
2、ce A domain of Enterococcus faecalis (E.faecalis), then express afusion protein in E.coli M15 and purify it, and the immunocompetence of therecombinant protein was analyzed. To identify its role in adhesion of E.faecalis, andto lay the foundation for development of quick diagnostic kit applying to d
3、etectE.faecalis.Methods: The Ace A domain was amplified by PCR from the genome of E.faecalisJH2-2, subcloned into the expression vector pQE30 to generate recombinantplasmid pQE30/Ace, then expressed in E.coli M15, and analyzed by SDS-PAGEand Western blot. The fusion protein was purified with Ni-NTA
4、affinitychromatography, and the protein concentration was determined by the BCA proteinassay kit. Newzealand rabbits were immunized with the fusion protein andantibodies to rAce in sera were detected to evaluate the immunocompetence ofrAce. Then the adhesion and inhibition assay were carried out wit
5、h theFITC-labeled E.faecalis JH2-2, and the results were observed by IFM.Results: The size of PCR amplification product was about 930bp. Restriction enzymedigestion analysis and sequencing showed that the inserted target gene was ace, andit had 100% similarity with gene reported by GenBank, the resu
6、lts of BLASTconfirmed that the sequence was ace. A soluble fusion protein with molecular5 Query 301 TCGCAACCGAATGTGATAGAAACGGATTTCGGAACAGCAACGGCGACTCAACGTTTGACG 360|Sbjct 361 TCGCAACCGAATGTGATAGAAACGGATTTCGGAACAGCAACGGCGACTCAACGTTTGACG 420Query 361 ATTGAAGGAGTGACCAACACAGAGACTGGCCAAATTGAGCGAGACTATCCG
7、nnnnnnnAT 420|Sbjct 421 ATTGAAGGAGTGACCAACACAGAGACTGGCCAAATTGAGCGAGACTATCCGTTTTTTTAT 480Query 421 AAAGTAGGCGATTTGGCTGGAGAGTCAAATCAAGTACGTTGGTTTTTAAATGTGAACCTC 480|Sbjct 481 AAAGTAGGCGATTTGGCTGGAGAGTCAAATCAAGTACGTTGGTTTTTAAATGTGAACCTC 540Query 481 AATAAATCCGATGTCACAGAAGATATTTCAATTGCGGATCGACAAGGAAGTGG
8、TCAACAA 540|Sbjct 541 AATAAATCCGATGTCACAGAAGATATTTCAATTGCGGATCGACAAGGAAGTGGTCAACAA 600Query 541 TTAAATAAAGAGAGTTTTACATTTGATATTGTGAATGACAAAGAAACTAAATATATTTCA 600|Sbjct 601 TTAAATAAAGAGAGTTTTACATTTGATATTGTGAATGACAAAGAAACTAAATATATTTCA 660Query 601 CTTGCCGAGTTTGAGCAACAAGGTTATGGCAAAATTGACTTCGTAACAGATAATG
9、ACTTT 660|Sbjct 661 CTTGCCGAGTTTGAGCAACAAGGTTATGGCAAAATTGACTTCGTAACAGATAATGACTTT 720Query 661 AATTTACGTTTTTATCGGGATAAAGCACGCTTTACTTCCTTTATCGTCCGTTACACTTCG 720|Sbjct 721 AATTTACGTTTTTATCGGGATAAAGCACGCTTTACTTCCTTTATCGTCCGTTACACTTCG 780Query 721 ACAATCACGGAAGCAGGCCAACATCAAGCAACATTTGAAAATAGTTATGACATCAAT
10、TAT 780|Sbjct 781 ACAATCACGGAAGCAGGCCAACATCAAGCAACATTTGAAAATAGTTATGACATCAATTAT 840Query 781 CAACTAAACAATCAAGACGCAACGAATGnnnnnnnTACATCACAGGTTAAAAATGTTTTT 840|Sbjct 841 CAACTAAACAATCAAGACGCAACGAATGAAAAAAATACATCACAGGTTAAAAATGTTTTT 900Query 841 GTAGAAGGCGAGGCAAGCGGCAATCAAAATGTGGAAATGCCAACAGAAGAAAGTCTAGA
11、C 900|Sbjct 901 GTAGAAGGCGAGGCAAGCGGCAATCAAAATGTGGAAATGCCAACAGAAGAAAGTCTAGAC 960Query 901 ATTCCTTTAGAGACAATAGAAGAATGGGAA 930|Sbjct 961 ATTCCTTTAGAGACAATAGAAGAATGGGAA 99023 附 录（一）、pQE30/ Ace 重组质粒正向测序图：40 （二）、pQE30/ Ace 重组质粒反向测序图：41 42 ProteinJ.Infect Immun,1999;67(1):193-200.5.6.7.8.Lund B,Edlund C.
12、Bloodstream Isolates of Enterococcus faecium Enrichedwith the Enterococcal Surface Protein Gene, esp, Show Increased Adhesionto Eukaryotic CellsJ.Clin Microbiol,2003;41(11): 5183-5185.Shankar N,Lockatell CV,Baghdayan AS,et al. Role of Enterococcus faecalisSurface Protein Espinthe Pathogenesis of Ascending Urinary Tract. InfectionJ.Infect Immun,2001;69(7):4366-4372.Toledo-Arana A,Valle J,Solano C,et al. The enterococcal surface protein, Esp,is involved in Enterococcus faecalis biofilm formationJ.Appl EnvironMicrobiol,2001;67(10):45
