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[目的]构建我国流行的周期型马来丝虫3-磷酸甘油醛脱氢酶(BmGAPDH)部分编码基因原核和真核表达质粒以及基因序列分析,为进一步的研究奠定基础。[方法]根据GeneBank中马来丝虫GAPDH基因的已知序列设计引物,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,转化大肠杆菌(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-BmGAPDH,经测序验证,并进行同源性比较。亚克隆至真核表达质粒pcDNA3.1(+),构建真核表达载体pcD-NA3.1(+)-BmGAPDH,转染COS-7细胞后进行RT-PCR验证。[结果]RT-PCR扩增出一条约877 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99%。转染的COS-7细胞高水平表达BmGAPDH的mRNA,根据克隆的目的基因序列推导的氨基酸序列与GenBank登录的一致。[结论]成功构建了周期型马来丝虫3-磷酸甘油醛脱氢酶部分编码基因原核及真核表达载体,为进一步功能研究提供了条件。
[Objective] To construct the prokaryotic and eukaryotic expression plasmids and gene sequence analysis of the partially encoded BmGAPDH gene of the periodic malayian worm in China, which laid the foundation for further research. [Method] Primers were designed according to the known sequence of GAPDH gene in GeneBank, and the target gene was amplified by reverse transcription polymerase chain reaction (RT-PCR) using the total RNA of the periodic Malayan worm as a template. 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. The positive recombinant plasmid pGEM-BmGAPDH was obtained and sequenced Validation, and homology comparison. The recombinant plasmid pcDNA3.1 (+) was subcloned into the eukaryotic expression vector pcDNA3.1 (+). The eukaryotic expression vector pcD-NA3.1 (+) - BmGAPDH was constructed and transfected into COS-7 cells. [Result] A specific band about 877 bp was amplified by RT-PCR. The double-digested PCR result of the recombinant plasmids was consistent with the expectation. The sequence homology between DNA sequence and GeneBank was 99%. The transfected COS-7 cells expressed BmGAPDH mRNA at high level. The deduced amino acid sequence of the cloned gene was identical to that of GenBank. [Conclusion] The prokaryotic and eukaryotic expression vectors of the partial coding gene of glyceraldehyde-3-phosphate dehydrogenase of Cyclosporidium malayi were successfully constructed, which provided the conditions for further functional studies.