Methionine and lysine are restrictive essential amino acids of livestock, they are also the most attentive indexes in the feed production to carry out the quality control and quality evaluation. Their contents in feed...Methionine and lysine are restrictive essential amino acids of livestock, they are also the most attentive indexes in the feed production to carry out the quality control and quality evaluation. Their contents in feed directly affect livestock protein synthesis. Bacillus natto has excellent probiotic properties. In this experiment, we used the genetic engineering method, fusion PCR technique, to connect methionine-rich gene （zein） from maize endosperm protein with lysine-rich gene （Cflr） from the pepper anther, then the fusion gene was inserted into the expression vector pHT43, and the recombinant plasmid pHT43/zein-Cflr was constructed. The recombinant plasmid was transferred into Bacillus natto, and induced by IPTG for the expression of the fusion gene. We found an apparent band at 40 ku site for the recombinant strain by SDS-PAGE. The contents of methionine and lysine were individually detected with HPLC, the quantities of methionine and lysine in the recombinant strain increased by 18.37% and 24.68% than the wild one, respectively. We also verified the stability of the recombinant bacterium during passaging, and found the stability was 100%. This study provided research-basis for the application of the recombined Bacillus natto as feed additive.展开更多
The outer membrane protein, ompA, ofAeromonas veronii has a role in the virulence of the organism and is a potential candidate for vaccine development. In this study, ompA I ofAeromonas veronii strain WA106 was cloned...The outer membrane protein, ompA, ofAeromonas veronii has a role in the virulence of the organism and is a potential candidate for vaccine development. In this study, ompA I ofAeromonas veronii strain WA106 was cloned and sequenced, then, it was expressed in Escherichia coli BL21. The nucleotide sequence of ompA I gene was 1 023 base pairs （GenBank Accession NO.KC748024）, which showed 100% homology with that of A. veronii （NO.AB290200.1）. This predicted protein was composed of 340 amino acid residues. Its molecular weight was 35.78 ku and isoelectric point was 5.18. The protein was a hydrophilic protein containing alpha helix and random coil with percentage of 35.0% and 49.7%, respectively. The tertiary structure, quaternary structure prediction showed that ompA I protein contained two peptide chains. SDS-PAGE showed that the actual value of the fusion protein was consistent with the expected result. It will facilitate further study of the role of ompA I protein.展开更多
基金Supported by the Funding of High Technology Project of Ministry of Science and Technology of China(863 Project,2013AA102504-03)
文摘Methionine and lysine are restrictive essential amino acids of livestock, they are also the most attentive indexes in the feed production to carry out the quality control and quality evaluation. Their contents in feed directly affect livestock protein synthesis. Bacillus natto has excellent probiotic properties. In this experiment, we used the genetic engineering method, fusion PCR technique, to connect methionine-rich gene （zein） from maize endosperm protein with lysine-rich gene （Cflr） from the pepper anther, then the fusion gene was inserted into the expression vector pHT43, and the recombinant plasmid pHT43/zein-Cflr was constructed. The recombinant plasmid was transferred into Bacillus natto, and induced by IPTG for the expression of the fusion gene. We found an apparent band at 40 ku site for the recombinant strain by SDS-PAGE. The contents of methionine and lysine were individually detected with HPLC, the quantities of methionine and lysine in the recombinant strain increased by 18.37% and 24.68% than the wild one, respectively. We also verified the stability of the recombinant bacterium during passaging, and found the stability was 100%. This study provided research-basis for the application of the recombined Bacillus natto as feed additive.
基金Supported by the Science&Technology Department of Sichuan Province(2013FZ0014)the Construction Project of Postgraduate Academic Degree in Southwest University for Nationalities(2015XWD-S071007)
文摘The outer membrane protein, ompA, ofAeromonas veronii has a role in the virulence of the organism and is a potential candidate for vaccine development. In this study, ompA I ofAeromonas veronii strain WA106 was cloned and sequenced, then, it was expressed in Escherichia coli BL21. The nucleotide sequence of ompA I gene was 1 023 base pairs （GenBank Accession NO.KC748024）, which showed 100% homology with that of A. veronii （NO.AB290200.1）. This predicted protein was composed of 340 amino acid residues. Its molecular weight was 35.78 ku and isoelectric point was 5.18. The protein was a hydrophilic protein containing alpha helix and random coil with percentage of 35.0% and 49.7%, respectively. The tertiary structure, quaternary structure prediction showed that ompA I protein contained two peptide chains. SDS-PAGE showed that the actual value of the fusion protein was consistent with the expected result. It will facilitate further study of the role of ompA I protein.
