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《Progress in Veterinary Medicine》 2018-05
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Prokaryotic Expression of Paenibacillus larvae Metalloprotease Gene and Preparation of Polyclonal Antibodies

GE Ting;HE Xiao-jie;Yeerbaole;Asika·Xiarefuhan;LEI Cheng-hong;WANG Zhen-bao;College of Veterinary Medicine,Xinjiang Agricultural University;Yili Vocational and Technical College;Yili Entry-Exit Inspection and Quarantine Bureau;  
The study cloned the PLMP gene from Paenibacillus larvae of honeybees,constructed the prokaryotic expression vector for expressing and purifying of recombinant protein,prepared polyclonal antibodies against recombinant proteins in male rabbits,so as to lay a foundation for the study of the rapid detection method of Paenibacillus larvae.The PLMP gene was amplified by PCR and cloned into the prokaryotic expression vector pGEX-4 T-1,and then the recombinant vector was transformed into E.coliBL21(DE3).The PLMP recombinant protein was induced by different time and different IPTG concentrations,and purified by cutting SDS-PAGE gel extraction.After the four times immunization with purpose protein in male rabbits,sera were collected and polyclonal antibodies were prepared.The PLMP gene fragment of 1242 bp was successfully cloned,prokaryotic expression vector pGEX-4 T-1-PLMP was constructed,the recombinant protein about 70 ku was obtained after inducing the pGEX-4 T-1-PLMP with 0.8 mmol/L IPTG at 37℃for 5 hrs.The titer of the polyclonal antibodies against rabbit immunized with the recombinant protein was 1∶12 800 by ELISA and a band appeared on nitrocellulose by Western blot,this showed that the obtained antibodies could be applied to detect the PLMP antigen.The pGEX-4 T-1-PLMP was successfully constructed and PLMP recombinant protein was expressed.Simultaneously,the polyclonal antibodies were prepared,and it laid the foundation to establish the method for rapid detection of Paenibacillus larvae.
【Fund】: 新疆维吾尔自治区自然科学基金项目(2016D01B059)
【CateGory Index】: S895.131
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