Cloning, sequencing of myosin gene from periodic Brugia malayi and prediction of B cell epitopes in its amino acid sequence

ObjectiveTo clone and sequence the myosin gene of periodic Brugia malayi(BmM yosin)and to predict the Bcell epitopes of encoded peptide sequence of the gene.MethodsTotal RNA was extracted from periodic Brugia malayi.Acouple of specific primers were designed on the basis of known sequences of myosin gene from Brugia malayi.The desired gene was amplified by PCR technique from cDNA.The PCR products were purified and cloned in to plasmid pGEM-T by T-A cloning method,and transformed in to Escherichia coli(E.coli)strain DH 5α.The recom binant plasmids were screened and identified by digestion with restriction enzyme and PCR amplification.The positive recombinant plasmid of pGEM-T BmM yosin was confirmed by sequencing and homology comparison.Five parameters and methods were used to predicate B-cell epitopes in aminoacide sequence of BmM yosin.ResultsA specific band around 1 292 bp was amplified by RTPCR.The same product was obtained by double restriction enzyme digestion of recombinant plasmid and PCR.The result of DNA sequencing shows that BmM yosin shares 98.45% nucleotide sequence with that of known sequences in the gene bank.B cell epitopes of BmM yosin are probably at or adjacent to 287-300,339-350 and 416-422 in its am ino ac id sequence.ConclusionpGEM-T BmM yosin of periodic Brugia malayi was successfully constructed,which provides the basis for further study of BmM yosin expression and functions.