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目的克隆周期型马来丝虫副肌球蛋白(BmPmy)基因,进行序列测定、分析及编码产物的B细胞表位预测。方法从周期型马来丝虫虫体中抽提总RNA,以mRNA为模板,RT-PCR法体外扩增BmPmy基因,扩增产物经初步鉴定后将其克隆入pGEM-T载体,转化E.coli DH5a,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-T-BmPmy,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2640bp的特异性条带,重组质粒双酶切的PCR结果与预期相符,DNA序列分析与基因库已知的基因序列同源性为99%。经表位预测分析,BmPmy的B细胞表位可能在54～66位、144～152位、770～780位和834～845位氨基酸区域。结论成功构建了BmPmy重组质粒pGEM-T克隆载体,为进一步研究该基因的功能提供了条件。
Objective To clone the BmPmy gene of Cyclosporidium malariae and make sequencing and analysis of the BmPmy gene. Methods The total RNA was extracted from the periodontal worms and the mRNA was used as template. The BmPmy gene was amplified by RT-PCR in vitro. The amplified product was cloned into pGEM-T vector and transformed into E. coli. The recombinant plasmid pGEM-T-BmPmy was screened by double enzyme digestion and PCR amplification. The positive clones were verified by sequencing 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 2640bp was amplified by RT-PCR. The PCR result of double-digested recombinant plasmids was in accordance with the expectation. The homology of DNA sequence analysis and gene library was 99%. According to the epitope prediction analysis, the B cell epitopes of BmPmy may be located in amino acid regions 54 to 66, 144 to 152, 770 to 780 and 834 to 845. Conclusion The recombinant plasmid pGEM-T of BmPmy was successfully constructed, which provided conditions for further study on the function of this gene.