Biology Reference
In-Depth Information
EcoRI
A
Amp r
R. rhodnii ori
EcoRI
BamHI
pRrMDWK6
7.4 KB
ColE1 ori
Kan r
Xbal
MK
α
DB3V H /K
SacII
BamHI
BamHI
Xbal
MK
DB3V H /K
α
B
Xbal SacII Xhol Sacl Xbal
MKa Regulatory
Sequence
kappa
V H
FIGURE 6.4 (A) The shuttle plasmid pRrMDWK6. (B) MKbDB3WV H K: rDB3 expression/secretion cassette.
[From Durvasula, R.V., Gumbs, A., Panackal, A., Kruglov, O., Taneja, J., Kang, A.S., Cordon-Rosales, C.,
Richards, F.F., Whitham, R.G., and Beard, C.B. (1999a). Med. Vet. Entomol. 13: 115Ï119. With permission.]
systems. These antibody fragments are composed of variable regions of heavy chains linked to
kappa light chains (VH-Kappa) and are selected from combinatorial libraries of heavy- and light-
chain genes. Selective panning of VH-Kappa fragments in a phage-display system yields mole-
cules with target speciÝcity and afÝnity that approximate parent IgG.
Insects lack immunoglobulin-mediated defenses and secondary cascades such as complements.
Hence, a strategy to express recombinant single-chain antibody fragments that directly disrupt
transmission of a pathogen by an arthropod is highly desirable.
In 1999, we Ýrst described the expression of a functional single-chain antibody in an insect
(Durvasula et al., 1999a). Rhodoccus rhodnii was transformed using the expression plasmid
pRrMDWK6 to secrete the single-chain antibody rDB3 (Figure 6.4), a murine VH-Kappa fragment
that binds progesterone and served as a marker antibody in our system.
Expression and secretion of rDB3 was under the control of the heterologous element Mkb
derived from Mycobacterium kansasii . R. rhodnii transformed with pRrMDWK6 were introduced
via blood meal to aposymbiotic Ýrst-instar nymphs of Rhodnius prolixus . The recombinant DB3
antibody fragment was synthesized and secreted into the insect gut lumen by the engineered
symbiont in a stable fashion for the 6-month duration of the study. Gut contents of the experimental-
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