牛支原体pdhc基因的克隆、表达及其亚细胞定位

doi:10.16431/j.cnki.1671-7236.2018.09.004

Abstract

Abstract:

To study the sequences characteristics of pdhc gene in M.bovis Wuwei strain and its location in M.bovis cells,the primers of pdhc gene were designed according to M.bovis HB0801 strain (accession No.:CP002058.1) in GenBank,and the pdhc gene of M.bovis Wuwei strain was amplified by PCR.Based on sequencing and gene analysis,the prokaryotic expression vector pET-pdhc was constructed,and then transformed into Escherichia coli Rosetta (DE3).After induced by IPTG,the M.bovis fusion protein PDHc-E2 was purified and New Zealand rabbits (Oryctolagus cuniculus) was immunized to prepare anti-serum against M.bovis rocombinant protein PDHc-E2.Subsequently,the distribution of PDHc-E2 in M.bovis cells was analyzed using iELISA and Western blotting.The results showed that the full-length sequence of pdhc gene CDS of M.bovis Wuwei strain was 735 bp and encoded 244 amino acids.It had 100.0% homology with the domestic M.bovis isolates,such as HB0801,Hubei-1,CQ-W70 and NM2012,and had 99.2% homology with the international standard strain PG45,the homology with Mycoplasma agalactiae (M.agalactiae) were 90.9% to 91.2%,the homology with the ST6 strain of Mycoplasma californicum (M.californicum) was only 78.4%,and the gene sequence was very conservative.By Overlap PCR,four TGA codons encoding tryptophan in this gene mutated to TGG;After point mutation the pdhc gene successfully expressed in Escherichia coli.The molecular weight of recombinant protein was approximately 29 ku,and it mainly existed in the form of soluble protein.iELISA results showed that recombinant protein PDHc-E2 had a high immunogenicity,and could stimulate New Zealand rabbit to produce high levels of antibodies,the iELISA titer of antiserum was approximately up to 1:100 000.The results of subcellular localization showed that the preparation of polyclonal antibody could bind specifically to recombinant protein PDHc-E2,total protein,membrane protein and cytoplasm of M.bovis,which indicated that the PDHc-E2 of M.bovis was distributed in both the membrane and the cytoplasm,and was a membrane-related protein,but the distribution in the cytoplasm was more than in the cell membrane.The results of this study laid a theoretical basis for further investigation on biological functions of M.bovis.

Key words: Mycoplasma bovis; dihydrolipoamide acetyltransferase; prokaryotic expression; subcellular localization

CLC Number:

S858.62

ZHANG Yi, XING Xiaoyong, WEN Fengqin, BAO Shijun. Cloning, Expression of pdhc Gene of Mycoplasma bovis and Its Subcellular Localization[J]. , 2018, 45(9): 2377-2385.

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Cloning, Expression of pdhc Gene of Mycoplasma bovis and Its Subcellular Localization

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