Molecular cloning, purification and characterisation of myosin of human lymphatic filarial parasite Brugia malayi.
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.
Verma
S K
SK
Division of Parasitology, Central Drug Research Institute, Post Box 173, Chattar Manzil Palace, Lucknow 226001, Uttar Pradesh, India.
Bansal
I
I
Vedi
S
S
Saxena
J K
JK
Katoch
V M
VM
Bhattacharya
S M
SM
D000818:Animals; D000909:Antibodies, Helminth / Q000097:blood; D000947:Antigens, Helminth / Q000097:blood; D017178:Brugia malayi / Q000235:genetics*; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics; D018509:DNA, Helminth / Q000235:genetics; D004605:Elephantiasis, Filarial / Q000276:immunology*; D004797:Enzyme-Linked Immunosorbent Assay; D005368:Filariasis / Q000276:immunology*; D006801:Humans; D008208:Lymphatic System / Q000469:parasitology; D009218:Myosins / Q000235:genetics* / Q000302:isolation & purification; D015183:Restriction Mapping; D014612:Vaccines
D000909:Antibodies, Helminth; D000947:Antigens, Helminth; D018076:DNA, Complementary; D018509:DNA, Helminth; D014612:Vaccines; D009218:Myosins