ABSTRACT
Specific primers were designed and synthesized based on the reported glyceraldehyde-3-phosphate dehydrogenase (BmG3PD) gene of Brugia malayi (GenBank Accession No. U18137). Total RNA was extracted from Brugia malayi and its BmG3PD gene was amplified by reverse transcription-polymerase chain reaction (RT-PCR). The PCR product was purified and cloned into plasmid pGEM-T, then transformed into Escherichia coli DH5alpha. The recombinant plasmids were screened and identified by digestion with restriction enzyme and PCR amplification. The positive recombinant plasmid pGEM-T-BmG3PD was confirmed by sequencing and homology comparison. Five parameters and methods were used to predict B-cell epitopes in amino acid sequence of BmG3PD. The amplified DNA fragment (1,020 bp) had a high identity of 99% with the BmG3PD gene sequence of Brugia malayi. B-cell epitopes of BmG3PD were probably at or adjacent to 22-36, 242-255, 303-318 and 326-336 in its amino acid sequence.
Subject(s)
Antigens, Helminth/genetics , Brugia malayi/genetics , Epitopes, B-Lymphocyte/genetics , Glyceraldehyde-3-Phosphate Dehydrogenases/genetics , Animals , Antigens, Helminth/immunology , Brugia malayi/enzymology , Cloning, Molecular , Glyceraldehyde-3-Phosphate Dehydrogenases/immunology , Sequence Analysis, DNAABSTRACT
Total RNA was extracted from periodic microfilariae of Brugia malayi and its myosin partial gene (Bm-M55) was amplified by RT-PCR. The PCR product was cloned and then subcloned into pcDNA3.1 (+)vector. The recombinant eukaryotic plasmids were screened and identified by digestion with restriction enzyme and PCR amplification, and was transfected into COS-7 cells subsequently. The expressed protein was identified by SDS-PAGE. Bm-M55 mRNA was highly expressed in transfected COS-7 cells. The deduced amino acid sequence showed to be identical with that of Bm-M55, and the recombinant protein was about Mr 55000.
