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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 BmMyosin gene of the periodic Malay worm, and analyze the sequence of BmMyosin. Methods Primers were designed according to published sequence of BmMyosin gene, and the target gene was amplified by reverse transcription polymerase chain reaction (RT - PCR). After preliminary identification, the amplified product was cloned into pGEM-T vector and transformed into E. coli DH5α. The positive clones were screened for double enzyme digestion and PCR amplification, and the positive recombinant plasmid pGEM-BmMyosin was obtained. After sequencing verification, and homology comparison. Five kinds of parameters and methods were used to predict the B cell epitopes of the encoded products. Results A specific band of about 1 292 bp was amplified by RT-PCR. The result of double-digested PCR was consistent with the expectation. The sequence homology between DNA sequence analysis and GeneBank was 98.45%. According to the epitope prediction analysis, the B cell epitopes of BmMyosin may be in the amino acid regions of 287 to 300, 339 to 350 and 416 to 422. Conclusion The recombinant plasmid pGEM-T of Malassezia was successfully constructed, and its coding product was predicted by B cell epitope. Provide a basis for protein characterization and immunological studies.