大鼠源卡氏肺孢子菌p55抗原基因克隆和序列分析

Cloning and sequence analysis of the p55 gene in Pneumocystis carinii from rats

目的克隆大鼠源卡氏肺孢子菌55kDa抗原(p55)基因。方法取肺孢子菌病大鼠肺组织制备肺孢子菌DNA,并以此模板扩增p55基因,将PCR产物与pGEM-T载体连接,转化E.coli DH5a,在含氨苄青霉素(Amp)的LB培养基上筛选出重组子,提取质粒进行PCR扩增、酶切鉴定、序列测定及序列比较分析。结果p55基因体外扩增产物长度为1286bp,重组质粒经PCR及酶切鉴定结果表明获得正确重组子,测序结果与文献报道同源性为99.8%。结论成功扩增了p55基因,并插入克隆载体pGEM-T中。p55基因的克隆为进一步建立基因分型、疫苗等研究打下基础。

The Pneumocystis carinii DNA was extracted from lung tissues of rats infected with P. carinii, and it was used as template to amplify the p55 gene by PCR. Meanwhile, the PCR products were purified and inserted to vector pGEM-T, Easy. E. coli DH5a was then transformed in LB culture medium containing ampicillin and the recombinant clones were selected. The positive clones of recombinants were extracted and identified by digestion with enzymes. Then they were sequenced and analyzed. It was demonstrated that a gene fragment with 1 286 bp was obtained by PCR amplification and the correct recombinant plasmid was isolated and confirmed by PCR and restriction enzyme analysis. The sequence identity between nucleotides of the cloned gene and that of the published sequence was 99.8%. It is concluded that the p55 gene in P. carinii from rats was successfully amplified and cloned, thus it would provide basis for the further studies on the genotypes of p55 gene and for the development of the corresponding DNA vaccine.