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Journal of Mosquito Research, 2013, Vol.3, No.4, 21
-
32
ISSN 1927-646X
http://jmr.sophiapublisher.com
21
Research Report Open Access
Cloning and Expression of the Recombinant Transferrin Protein from
Culex
quinquefasciatus
Say (Diptera: Culicidae) and Study of Its Antimicrobial
Activity
Emtithal M. Abd El-Samiee
1
, Heba M. Hamama
1
, Nawal M. Zohdy
1
, Mohamed M. Shamseldean
2
1. Entomology Department, Faculty of Science, Cairo University, Egypt
2. Nematology Department, Faculty of Agriculture, Cairo University, Egypt
Corresponding author email:
hebahamama@gmail.com;
Authors
Journal of Mosquito Research, 2013, Vol.3, No.4 doi: 10.5376/jmr.2013.03.0004
Received: 12 Jan., 2013
Accepted: 23 Jan., 2013
Published: 27 Feb., 2013
This is an open access article published under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction
in any medium, provided the original work is properly cited.
Preferred citation for this article:
Abd El-Samiee et al., 2013, Cloning and Expression of the Recombinant Transferrin Protein from
Culex quinquefasciatus
Say (Diptera: Culicidae) and Study of
Its Antimicrobial Activity, Journal of Mosquito Research, Vol.3, No.4 21-32 (doi: 10.5376/jmr.2013.03.0004)
Abstract
Mosquitoes as in other examined insects so far have an abundant haemolymph transferrin (Tsf). The Tsf gene of
Culex
quinquefasciatus
has been cloned and sequenced. Total RNA was successfully isolated from the whole-body of the 4
th
instar larvae of
the control and treated insects which were subjected to sub-lethal concentrations of nematode infective juveniles of
Heterorhabditis
bacteriophora
(HB) and
Steinernema carpocapsae
(SC). Transferrin transcripts were detected in control, HB- and SC-treated insects
after 3, 6, 9, and 12 hr post infection. cDNA was prepared and the gene coding for Tsf protein was amplified using conventional
polymerase chain reaction (PCR). The gene was then cloned into Mach1™- T1
®
Escherichia coli
cells, then subcloned into the
over-expression vector pET-28a (+) using BL21 expression bacteria. The recombinant Tsf protein was purified using Ni-NTA resin.
The putative Trf was 248 amino acids with a molecular mass of approximately 63 kDa. The deduced amino acid sequence showed
significant homology with known insect transferrins. Tsf protein showed antibacterial activity against
Escherichia coli
. The results
demonstrated that the mosquito,
Cx. quinquefasciatus
has the capacity to respond to entomopathogenic nematode-bacteria
inoculation with a rapid up-regulation of transcripts of immune peptides Trf when using Steinernematid nematodes only.
Keywords
Entomopathogenic nematode; Cloning; Antimicrobial activity; Transferrin (Tsf) gene;
Culex quinquefasciatus
Introduction
Mosquitoes are medically important insects as vectors
of several tropical diseases including malaria,
filariasis and numerous viral diseases. Control
measures are generally directed against only a few of
the most important species. The mosquito
Culex
quinquefasciatus
Say (Diptera: Culicidae) is the
vector of the filarial parasite
Wuchereria bancrofti
(Cobbold) (Spirurida: Onchocercidae), causing human
bancroftian filariasis. Numerous studies have been
undertaken for unconventional control agents owing
to the hazards of conventional pesticides. Among such
agents are the entomopathogenic nematodes (EPN)
belonging to the families, Heterorhabditidae and
Steinernematidae (Kaya and Gaugler, 1993).
The immunological responses of mosquitoes to
entomopathogenic Steinernematids and Heterorhabditids
are poorly studied especially in
Culex
spp. Ample
evidence from mosquitoes and other medically
important insect vectors suggests that endogenous
innate immune molecules can hinder the development
of parasites (Smart, 1995; Bartholomay et al., 2003).
Vector insects are equipped with both cellular and
humoral defense systems (Hultmark, 1993; Hoffmann,
1995; Richman and Kafatos, 1996). Many, if not all
species of mosquitoes are capable of encapsulating
S.
carpocapsae
at low densities, whereas, at higher
densities the parasite can overcome the host’s immune
response (Paily and Jayachandran, 1987). In contrast,
only a few species have been found to encapsulate
themermithid
Reesimermis Nielseni
(Paily and
Jayachandran, 1987).
Transferrin (Tsf) is a defense protein found in both
vertebrates and invertebrates. It is classified as an
acute-phase protein because, under conditions of