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Journal of Immunology Research
Volume 2016 (2016), Article ID 1781803, 13 pages
http://dx.doi.org/10.1155/2016/1781803
Research Article

Cloning, Expression, and Characterization of Prophenoloxidases from Asian Corn Borer, Ostrinia furnacalis (Gunée)

Department of Entomology, China Agricultural University, Beijing 100193, China

Received 27 July 2016; Revised 27 September 2016; Accepted 26 October 2016

Academic Editor: Daniela Cosentino-Gomes

Copyright © 2016 Shasha Zhang et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Supplementary Material

The following supplementary material is available: Table S1: Oligonucleotides used for cloning, RT-PCR, and plasmid construction and confirmation. Figure S1: Amino acid sequences of 4 recombinant O. furnacalis PPOs expressed in E. coli. The amino acid sequences were deduced from the nucleotide sequences of the insert in constructed plasmids via FastCloning. The amino acid residues from the expression vector pET28a were underlined. Figure S2: Phylogenetic analysis of O. furnacalis PPOs with other insect tyrosinase- and laccase-type PPOs. The analysis was performed using the MEGA 5 program. The consensus tree was constructed using the neighbor-joining method. Gaps were treated as characters and statistical analysis was performed by the bootstrap method with 1000 repetitions. Only bootstrap values greater than 90 were shown. The names of used genes were shown as scientific name of species followed by NCBI accession number of this specific gene. The branches specific for tyrosinase- and laccase-type PPOs were shaded in yellow and blue, respectively. The circled bootstrap values indicated that O. furnacalis PPOs belong to tyrosinase-type PPOs. Figure S3: Co-expression of O. furnacalis PPOs in E. coli. (A) Confirmation of dual plasmid transformation by PCR. Plasmids encoding OfPPO1a or OfPPO1b were respectively transformed to E. coli BL21 (DE3) cells which already contained plasmids encoding OfPPO2 or OfPPO3. After selection by kanamycin, a single clone was used as template for PCR using two pairs of primers listed in Table S1. The PCR products were separated by electrophoresis on a 1.0% agarose gel. (B) SDS-PAGE analysis of recombinant PPOs. O. furnacalis PPOs were expressed in E. coli at 18°C for 12 h. The cultured cells were harvested and treated as described in “Materials and Methods”. The obtained protein samples were then subjected to 10% SDS-PAGE and visualized by Coomassie brilliant blue staining. Lane 1 : 10 μL of cell lysate collected before the addition of IPTG. Lane 2–5 : 5 μL of cell lysate collected 12 h after the addition of IPTG, which contained recombinant OfPPO1a, OfPPO1b, OfPPO2, and OfPPO3, respectively. Lane 6 and 7 : 10 μL of cell lysate simultaneously expressing recombinant OfPPO1a and OfPPO2 (OfPPO1a/2), collected before or after the addition of IPTG, respectively. Lane 8 and 9 : 10 μL of cell lysate simultaneously expressing recombinant OfPPO1a and OfPPO3 (OfPPO1a/3), collected before or after the addition of IPTG, respectively. Lane 10 and 11 : 10 μL of cell lysate simultaneously expressing recombinant OfPPO1b and OfPPO2 (OfPPO1b/2), collected before or after the addition of IPTG, respectively. Lane 12 and 13 : 10 μL of cell lysate simultaneously expressing recombinant OfPPO1b and OfPPO3 (OfPPO1b/3), collected before or after the addition of IPTG, respectively. (C) Western blot analysis of recombinant PPOs. The obtained samples in (B) were subjected to immunoblotting using mouse anti-His as primary antibodies. Lane 1 : 20 μL of cell lysate collected before the addition of IPTG. Lane 2 : 10 μL of cell lysate collected 12 h after the addition of IPTG. Lane 3 : 20 of supernatant of cell lysate treated by sonication (soluble faction). Lane 4 : 20 μL of precipitate of cell lysate treated by sonication (insoluble fraction).

  1. Supplementary Material