Prokaryotic expression and preparation of polyclonal antibody of NbGAD1 protein from Nicotiana benthamiana
γ-aminobutyric acid is a non-protein amino acid,which is ubiquitous in plants,and GABA plays an impor-tant role in plant growth,development and stress resistance.Glutamate decarboxylase is a key protease for the synthesis of GABA.At present,the molecular mechanism of GAD-mediated GABA regulation against virus infection is unclear in plants.The NbGAD1 gene of Nicotiana benthamiana was cloned by RT-PCR,and connected with the prokaryotic ex-pression vector pET-28a to construct the recombinant vector pET-28a-NbGAD1.The recombinant protein was induced and purified by Ni-NTA column,and the antiserum was obtained by immunizing New Zealand White rabbits.The results showed that the prokaryotic expression vector pET-28a-NbGAD1 was successfully constructed,and the recombinant pro-tein NbGAD1 with molecular weight of about 57 ku was obtained after induction and purification from Escherichia coli.Western blotting results showed that the titer of the antiserum reached 1∶100 000,and the recombinant protein with the density of 0.005 mg·mL-1 could still be detected by the antiserum diluted 2 000 times.In addition,the endogenous NbGAD1 protein from N.benthamiana can be also detected using the antiserum.Overall,the antiserum has the advantages of strong specificity and high sensitivity,which provides a basis for further study on the mechanism of GAD in plant virus infection.
Nicotiana benthamianaγ-aminobutyric acidglutamate decarboxylaseprokaryotic expressionprepara-tion of antiserum
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