马来丝虫3-磷酸甘油醛脱氢酶基因的克隆、序列分析及编码产物B细胞表位预测

Cloning, Sequencing of G3PD Gene from Brugia malayi and Prediction of B cell Epitopes in its Amino Acid Sequence

根据GenBank中马来丝虫3-磷酸甘油醛脱氢酶基因(BmG3PD基因)序列设计引物,以马来丝虫mRNA为模板,RT-PCR扩增BmG3PD基因,将其克隆入pGEM-T载体,转化大肠埃希菌(E.coli)DH5α,筛选阳性克隆。经EcoRⅠ和XhoⅠ双酶切及PCR鉴定,获得阳性重组质粒pGEM-BmG3PD,经序列分析及同源性比较,以及对其编码产物进行B细胞表位预测,结果表明PCR扩增的特异性条带为1020bp,与预期相符,与GenBank已知基因序列同源性为99%。编码产物B细胞表位预测,氨基酸区域可能在22～36、242～255、303～318和326～336位。

Specific primers were designed and synthesized based on the reported glyeeraldehydes-3-phosphate dehydrogenase （BmG3PD） gene of Bragia malayi （GenBank Accession No. U18137）. Total RNA was extracted from Brugia malayi and its BmG3PD gene was amplified by reverse transcription-polymerase chain faction （RT-PCR）. The PCR product was purified and cloned into plasmid pGEM-T, then transformed into Escherichia colt DHSα. The recombinant plasmids were screened and identified by digestion with restriction enzyme and PCR amplification. The positive recombinant plasmid pGEM-T-BmG3PD was conffirmed 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 BrnG3PD were probably at or adjacent to 22-36, 242-255, 303-318 and 326-336 in its amino acid sequence.