Xie Dong-Fang, Fang Zheng, Tong Hai-Yan, Xu Bang-Sheng, Huang Wei-Qun, Fang Hao, Shen Qin
Department of Parasitology, School of Basic Medical Sciences, Nantong University, Nantong 226001, China.
Zhongguo Ji Sheng Chong Xue Yu Ji Sheng Chong Bing Za Zhi. 2009 Jun;27(3):226-8.
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.
根据已报道的马来布鲁线虫甘油醛-3-磷酸脱氢酶（BmG3PD）基因（GenBank登录号：U18137）设计并合成特异性引物。从马来布鲁线虫中提取总RNA，通过逆转录聚合酶链反应（RT-PCR）扩增其BmG3PD基因。将PCR产物纯化并克隆到质粒pGEM-T中，然后转化到大肠杆菌DH5α中。通过限制性内切酶消化和PCR扩增对重组质粒进行筛选和鉴定。通过测序和同源性比较确认阳性重组质粒pGEM-T-BmG3PD。使用五个参数和方法预测BmG3PD氨基酸序列中的B细胞表位。扩增的DNA片段（1020 bp）与马来布鲁线虫的BmG3PD基因序列具有99%的高度同一性。BmG3PD的B细胞表位可能位于其氨基酸序列的22-36、242-255、303-318和326-336处或其附近。
