الفهرس 
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Abstract
The present study aimed to elucidate the contributions of phenoloxidase activity as an antimicrobial activity of the reactive compounds in vitro against infected tissues and established that the reactive intermediates yielded by PO had a broad-spectrum bactericidal activity. It evaluates the antimicrobial activity of PO as consequence of exposure to Bt. induction sublethal dose. Also, the study characterizes the molecular structure of PO extracted from Sp. littoralis hemolymph.
1- Immune induction study
This study shows that the estimated LC20 is 2X1060 cells/ml, and it was found that this concentration did not cause high mortality while inducing the Spodoptera’s immune response. Therefore, this concentration of 2X1060 cells/ml was used as the sublethal concentration to study subsequent experiments.
2. Molecular characterization of PO in Sp. littoralis
2.1. cDNA synthesis of PO gene
For determination and identification of PO gene molecular characterization in Sp. Littoralis, Whole larvae and hemolymph was collected and kept at –80C for RNA extraction, profiling of gene and protein expression of PO, respectively.
cDNA had been synthesized using Applied Biosystems™ High-Capacity cDNA Reverse Transcription with inhibitor kit.
2.2. Amplification of PO gene:
The synthesized cDNA was amplified to obtain the full length nucleotide sequences of PO using PO primers. The amplification of a fragment of PO produced specific band of 600bp. The PCR products of PO were identifying using freshly prepared 2% agarose gel as a preparation for gene sequencing.
2.3. PO sequencing and searching for sequence homologies in protein databases:
After purifying the PO transcripts from hemolymph of Sp. littoralis larvae ,the produced sequences were represented for forward and reverse directions. PO sequence analysis via Basic Local Alignment Search Tool (BLAST) showed that regions of local similarity between PO sequence from haemolymph of S. littoralis, and compared the PO sequence to sequence databases. The comparison revealed that there are similarities ranges between 57 and 59 sequences of PPO and PO. There was high percentage of identity 97%.
3. Protein electrophoretic analysis of the hemolymph collected from Sp. littoralis larvae
After 48 hours from Bt injection, haemolymph of cotton leaf worm had been withdrawed (test group), and haemolymph from uninjected Spodoptera had been collected as a (control group).
The HiTrapTM CM FF 1ml column used for purification of PO from the haemolymph, A confirmatory step to verify the purification of activated PO in the haemolymph had been done by analyzed the purified haemolymph using SDS-polyacrylamide gel .
The protein profile of the PO of Spodoptera hemolymph is separated by electrophoresis according to the two groups used. Comparison of the electrophoresis patterns of Sp. littoralis PO showed that the protein composition of the two groups was almost similar. PO protein bands in induced and intact larvae were observed to be 70 kDa for the control uninjected group and 65 kDa for the injected group. This may indicate that the protein forms of active PO and PPO are almost similar
4. Bacterial susceptibility against the PO:
Two gram positive bacteria ( Staph aureus and Enterococci) and four gram negative bacteria (Pseudomonas , E.coli, Acinetobacter and Klebsiella) had been prepared in a standard 0.5 McFarland bacterial suspension.
Four dilutions had been prepared from the purified PO, Plating for bacterial suspension had been done, then adding 10µ/DROP from each tube to the bacterial plate, with three replicates each, incubation for 48hr had been done in 40ºC incubator.
The concentrated purified PO was the only concentration made inhibition zone in the bacterial culture, it affect against gram +ve bacteria with average 1 cm inhibition zone in Enterococci and average 1.5 cm inhibition zone in Staph. aureus.
For the gram –ve bacteria, the PO affect only E.coli with average inhibition zone 1.5 cm
No change happened in the remaining tested gram negative bacterial culture which revealed that the PO has wide antimicrobial activity against gram +ve bacteria more than gram –ve.
Finally, it could be concluded from the present work the following:
PO has an antimicrobial activity against pathogenic gram positive bacteria more than pathogenic gram negative bacteria; this might be because of differentiation of cell wall structure of the two groups of bacteria.
The immediate purified concentrated PO is more effective than the diluted form, also time weaken the antimicrobial effect of PO.