[Preparation of polyclonal antibody against insect antifreeze protein MpAFP149 and identification of MpAFP149 expression in transgenic tobacco].
OBJECTIVE
To prepare the polyclonal antibody against insect antifreeze protein MpAFP149 and use it to identify the expression of the heterologous antifreeze protein in transgenic tobacco.


METHODS
Eukaryotic expression vector pcDNA3-MpAFP149 was constructed as a DNA vaccine by inserting MpAFP149 gene into pcDNA3 vector. The gene fragment encoding MpAFP149 mature peptide was cloned into pGEX-4T-1 vector to yield recombinant pGEX-4T-1- MpsAFP149. The pGEX-4T-1MpsAFP149 was transformed into E.coli BL21(DE3). GST-sAFP149P fusion protein was obtained after IPTG induction, which was used as a protein vaccine. The polyclonal antibody was generated by the DNA prime-protein boost vaccination strategy and its speciality was analyzed by Western blot. Ultra-thin sections for leaves of transgenic tobacco were assayed for the expression and distribution of heterologous protein MpAFP149 by immunogold particle technique.


RESULTS
The prokaryotic and eukaryotic expression vectors carrying MpAFP149 were constructed. The recombinant protein GST-MpAFP149 was expressed with the expected molecular weight at 36 kDa. The results of Western blot and immunogold localization confirmed that mouse polyclonal antibody against MpAFP149 was obtained.


CONCLUSIONS
The recombinant expression vectors carrying MpAFP149 were successfully constructed and the polyclonal antibody against MpAFP149 was obtained. The immunogold localization by TEM (transmission electron microscope) showed that the heterologous MpAFP149 protein was mainly rocalized in the cell wall in apoplast of the transgenic tobacco plant.
Xinjiang Key Laboratory of Biological Resources and Genetic Engineering, College of Life Science and Technology, Xinjiang University, Urumqi 830046, China.
Yan
Y
Wang
Li-ming
LM
Qiu
Chun-ying
CY
Dai
Jing
J
Wang
Fu-chun
FC
Zhang
Ji
J
Ma
D000818:Animals; D021301:Antifreeze Proteins / Q000276:immunology*; D015153:Blotting, Western; D001517:Coleoptera; D004926:Escherichia coli / Q000235:genetics; D005260:Female; D019476:Insect Proteins / Q000276:immunology*; D051379:Mice; D030821:Plants, Genetically Modified / Q000235:genetics*; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000276:immunology; D014026:Nicotiana / Q000235:genetics*
D021301:Antifreeze Proteins; D019476:Insect Proteins; D011993:Recombinant Fusion Proteins