Cloning,Prokaryotic Expression and Bioinformation Analysis of dhbC Gene of Brucella melitensis |
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| Authors: | LI Bao-bao NIE Xin YANG Xiao-jian CAO Rui-yong ZHU Shu HUANG Hai-feng ZHANG Zhen-xing PENG Dong-mei LI Guo-hua LI Ya-ying WANG Feng-yang DU Li |
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| Institution: | Key Laboratory of Animal Genetic Engineering of Haikou City, Key Laboratory of Tropical Animal Reproduction & Breeding and Epidemic Disease Research of Hainan Province, Institute of Tropical Agriculture and Forestry, Hainan University, Haikou 570228, China |
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| Abstract: | This study was aimed to clone and express dhbC gene of Brucella melitensis, and analyze the bioinformatics of its expressed protein. A pair of primers were designed by referring to dhbC gene sequence information of Brucella melitensis M5-90 strain in GenBank, and the dhbC gene fragment was amplified by PCR method. The obtained dhbC gene was ligated into pMD20-T vector to construct pMD20-T-dhbC recombinant plasmid and transformed into E.coli DH5α competent cells. The plasmid was identified by restriction enzyme digestion. The recombinant plasmid pET28a-dhbC was constructed and transformed into E.coli BL21 (DE3) competent cells. The expression was induced by IPTG. The expressed product was analyzed by SDS-PAGE and Western blotting. Bioinformatics analysis of the amino acid sequence encoded by dhbC gene was carried out using bioinformatics software DNAMAN and related online sites ProtParam, SOPMA and Protscale. The results showed that dhbC gene was cloned with the length of 1 093 bp, and protein expression was expressed. The expressed fusion protein was about 47 ku, and was mainly in the form of inclusion body. The molecular weight of the dhbC protein was C1866H2968N544O562S15, the molecular mass was 42 496.3 u, the theoretical isoelectric point (pI) was 5.81, the extinction coefficient was 33 835, the instability coefficient was 36.76, the hydrophobic index was 86.19, the total average hydrophobicity (GRAVY) was -0.215. The half-life of reticulocytes in mammals was predicted to be 30 h, and the secondary structure was dominated by α-helix (41.94%) and random coil (31.46%). |
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| Keywords: | Brucella melitensis dhbC gene cloning prokaryotic expression bioinformatics analysis |
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