Molecular cloning, purification and characterisation of myosin of human lymphatic filarial parasite Brugia malayi

Abstract

Global efforts have been made towards development of vaccine for prevention of lymphatic filariasis. However, lack of thorough knowledge about developmental biology and pathogenesis of filarial parasite restricts us from developing an effective vaccine. A limited number of immunodominant antigens of human lymphatic filariid Brugia malayi have been characterised; however, none of these recombinant antigens so far induced significant degree of protective immunity to challenge infection. In the present study, we identified a approximately 2.0 Kb cDNA clone by immunoscreening of cDNA library of adult female Brugia malayi. The nucleotide sequence of the identified clone showed 94.3% homology with C-terminal part of myosin heavy chain gene of Brugia malayi. This cDNA insert was sub-cloned into pET28b vector and expressed in BL21(DE3). The recombinant protein was purified to near homogeneity by immobilised metal affinity chromatography (IMAC) with yield of approximately 25 mg/l. The purified protein was recognised in western blot with anti-His tag antibody as also with the antibodies present in the sera of human W. bancrofti patients of all categories and infected/immunized rodent serum demonstrating its functional role. Recombinant myosin induced marked cellular immune response as observed by lymphoproliferation assay. The present findings demonstrate the usefulness of B. malayi recombinant myosin as vaccine candidate against human lymphatic filariasis.

Cloning, expression, and characterization of salivary apyrase from Aedes albopictus

“…Numbers of significant proteins have been expressed with the prokaryotic expression system during the past few years (Verma et al 2008;Liu et al 2010;Yang et al 2010). Purification was done by Ni-NTA under denaturing condition.…”

Molecular cloning and characterization of a novel immunoreactive ATPase/RNA helicase in human filarial parasite Brugia malayi

“…Larvae were exposed to Cobalt 60 irradiation at dose of 25 krad and inoculated subcutaneously to mastomys (∼100 larvae) three times at 4-week intervals (Weil et al 1992) until the animals developed high antibody titer to L 3 . Serum antibody titer was assessed by ELISA using somatic soluble antigen of L 3 as mentioned earlier (Verma et al 2007). A B. malayi L 3 cDNA library (SAW94WL-BmL3 library) constructed in the lambda UniZap XR vector (Stratagene) kindly provided by Prof. S.A. Williams, Smith College, Northampton, Massachusetts, USA was used for immunoscreening with high titer pooled L 3 resistant serum (Verma et al 2007).…”

“…Serum antibody titer was assessed by ELISA using somatic soluble antigen of L 3 as mentioned earlier (Verma et al 2007). A B. malayi L 3 cDNA library (SAW94WL-BmL3 library) constructed in the lambda UniZap XR vector (Stratagene) kindly provided by Prof. S.A. Williams, Smith College, Northampton, Massachusetts, USA was used for immunoscreening with high titer pooled L 3 resistant serum (Verma et al 2007). Nucleotide sequencing of the 5′ and 3′ ends of the inserts using universal T3 and T7 primers was performed at the UDSC Department of Biochemistry, University of Delhi, South Campus, New Delhi, India, using automatic sequencer ABI Prism (Version 3.0.…”

Immunization with a multisubunit vaccine considerably reduces establishment of infective larvae in a rodent model of Brugia malayi

“…cDNA coding for B. malayi BmAF-Myo was picked up earlier [15] by immunoscreening of adult female B. malayi cDNA expression library with human bancroftian sera and subsequently subcloned in pET28b expression vector followed by transformation into DH5␣ Escherichia coli cells (Qiagen). The recombinant plasmid was further transformed into BL21 (DE3) E. coli cells (Qiagen) and grown in 5 ml Luria-Bertani (LB) medium overnight (O/N) in the presence of 50 g/ml of kanamycin at 37 • C with 220 rpm shaking.…”

“…malayi myosin was selected on the basis of its high reactivity with endemic normal human sera from bancroftian endemic area [15]. It is a body wall muscle protein and recently its presence has been shown in excretory secretary product of adult worms [16].…”