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Cloning and prokaryotic expression of recombinant pneumococcal PrtA protein

Lu Jie1,Wang Zhongwei2,Ji Hong2,Ma Yue1,Li Chang3,Jiang Shijun1 (1.Department of Basic Medicinet,Daqing Medical College,Daqing,Heilongjiang 163312,China; 2.College of Animal Science and Technology,Heilongjiang Bayi Agricultural University,Daqing,Heilongjiang 163319,China;3.Admission and Employment Offices,Daqing Medical College,Daqing,Heilongjiang 163312,China)  
Objective To construct a prokaryotic expression vector carrying PrtA gene,and to express recombinant pneumococcal surface proteins cell wall associated serine proteinase A(PrtA)fusion protein with activities.Methods PrtA gene specific primers were designed and synthesized according to database in GenBank.RT-PCR,amplifying target fragment was constructed by testifying different optimized condition.Then,PrtA gene was amplified by RT-PCR from total RNA and cloned into pMD18-T vector.The positive pMD18-T-PrtA plasmid was constructed by screening and identification.Then the target gene was subcloned into the downstream of pET-30a(+) vector,which was denominated as pET-30a-PrtA,and the insertion of target gene into pET-30a (+) vector was confirmed by PCR and restriction enzyme digestion.The recombinant strain E.coli BL21 (pET-30a-PrtA) was induced by 0.6 mmol/L IPTG and the recombinant PrtA protein was expressed at high level.It has been proved that the recombinant PrtA protein of Streptococcus pneumoniae(S.pneumoniae) had good reactionogenicity by western-blot.Results The PrtA gene shared 99% nucleotide homology to GenBank sequences.Reconstructed pET-30a-PrtA plasmid,being induced by IPTG,showed that different concentrations of IPTG all could induce recombinant protein expression,and when the concentration of IPTG eventually was 0.6 mmol/L,the expression of objective protein reached the highest level,accounts for about 18% of thalline total proteins.Clear specificity channel of molecular weight about 40×103 could be detected among thalline total proteins of reconstructed plasmid,when being induced by IPTG for 6 h,with monoclonal antibody labeled by Anti-His,which matched with expectation and indicated that the expressed protein was recombinant pneumococcal PrtA protein.Conclusion PrtA gene from S.pneumoniae was cloned and expressed successfully,laying bases for further research of PrtA protein to be used as immunogen for the prevention and treatment of diseases caused by S.pneumoniae.
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