Prokaryotic Expression and Antigenic Analysis of Wbkc Gene from Brucella abortus

[ Objective ] This study aimed to clone and express formyltransferase （Wbkc） gene from Brucella abortus in E. coli, purify the expressed protein and analyze its immunogenicity. [Method] A gene encoding 27 -35 ku formyltransferase （Wbkc） was amplified from the genomic DNA of BruceUa abortus by PCR. The amplified fragments were digested with BamH I and Sal I, and inserted into pET28a vector. The constructed recombinant plasmid pET 28a-Wbkc was trans- formed into E. coli BL21 and was induced to express the fusion protein. Subsequently, the protein was purified by histidine-binding resin column chromatography, and the immunogenicity was detected by Western blot assay. The recombinant plasmid was identified by colony PCR, double digestion and sequencing analysis. [ Result] Wbkc was successfully cloned and expressed in E. coli. A specific protein band of 29 ku was detected by SDS-PAGE. Western blot showed specific im- munoreactivity of the purified fusion protein. [ Conclusion] This study provided a solid foundation for further investigating diagnostic proteins for brucellosis and developing Brucella gene-deletion vaccines.

Analysis of codon usage patterns in Bupleurum falcatum chloroplast genome

Objective In order to distinguish the traditional Chinese medicine Bupleurum falcatum and its adulterants effectively and develop a better understanding of the factors affecting synonymous codon usage,codon usage patterns of chloroplast genome,we determine the complete chloroplast(cp)genome of B.falcatum and clarify the main factors that influence codon usage patterns of 78 genes in B.falcatum chloroplast genome.Methods The total genomic DNA of fresh leaves from a single individual of B.falcatum was extracted with EASYspin plus Total DNA Isolation Kit and 2μg genome DNA was sequenced using Illumina Hiseq 2500 Sequencing Platform.The cp genome of B.falcatum was reconstructed with MITObim v1.8 and annotated in the program CPGAVAS2 with default parameters.Python script and Codon W were used to calculate the codon usage bias parameters.Results The full length of B.falcatum cp genome was 155851 bp,132 different genes were annotated in this cp genome containing 80 protein-coding genes,30 tRNA genes,and four rRNA genes.The codon usage models tended to use A/T-ending codons.The neutrality plot,ENC plot,PR2-Bias plot and correspondence analysis showed that both compositional constraint under selection and mutation could affect the codon usage models in B.falcatum cp genome.Furthermore,three optimal codons were identified and most of these three optimal codons ended with G/U.Conclusion The cp genome of B.falcatum has been characterized and the codon usage bias in B.falcatum cp genome is influenced by natural selection,mutation pressure and nucleotide composition.The results will provide much more barcode information for species discrimination and lay a foundation for future research on codon optimization of exogenous genes,genetic engineering and molecular evolution in B.falcatum.