马来丝虫肌球蛋白基因序列及编码产物预测

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

目的克隆周期型马来丝虫肌球蛋白（BmMyosin）基因,进行序列测定、分析及编码产物的B细胞表位预测。方法依据公布的BmMyosin基因序列设计引物,以周期型马来丝虫总RNA为模板,反转录PCR（RT-PCR）扩增目的编码基因。扩增产物经初步鉴定后将其克隆入pGEM-T载体,转化大肠埃希菌（E.coli）DH5α,筛选阳性克隆,进行双酶切及PCR扩增鉴定,获得阳性重组质粒pGEM-BmMyosin,经测序验证,并进行同源性比较。应用5种参数和方法对其编码产物进行B细胞表位预测。结果RT-PCR扩增出一条约1 292 bp大小的特异性条带,重组质粒双酶切的PCR结果与预期相符,DNA序列分析与GeneBank已知的基因序列同源性为98.45%。经表位预测分析,BmMyosin的B细胞表位可能在287-300位、339-350位和416-422位氨基酸区域。结论成功构建了周期型马来丝虫肌球蛋白重组质粒pGEM-T克隆载体,对其编码产物进行B细胞表位预测。为蛋白质特性分析及免疫学研究提供基础依据。

Objective To clone and sequence the myosin gene of periodic Brugia malayi（BmMyosin） and to predict the B cell epitopes of encoded peptide sequence of the gene. Methods Total RNA was extracted from periodic Brugia rnalayi. A couple 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 eDNA. The PCR products were purified and cloned into plasmid pGEM-T by T-A cloning method,and transformed into Escherichia coli （E. coli） strain DH5α. The recombinant plasmids were screened and identified by digestion with restriction enzyme and PCR amplification. The positive recombinant plasmid of pGEM-T-BmMyosin was confirmed by sequencing and homology comparison. Five parameters and methods were used to predicate B-cell epitopes in amino acide sequence of BmMyosin. Results A specific band around 1 292 bp was amplified by RT-PCR. The same product was obtained by double restriction enzyme digestion of recombinant plasmid and PCR. The result of DNA sequencing shows that BmMyosin shares 98.45% nucleotide sequence with that of known sequences in the gene bank. B-cell epitopes of BmMyosin are probably at or adjacent to 287 -300,339 -350 and 416 -422 in its amino acid sequence. Conclusion pGEM-T-BmMyosin of periodic Brugia malayi was successfully constructed, which provides the basis for further study of BmMyosin expression and functions.