羊种布鲁氏菌侵染HPT-8细胞cDNA文库的构建及EST序列分析

Construction of cDNA Library of HPT-8 Cells Infected with Brucella menlitensis and Analysis of EST Sequence

拟建立羊种布鲁氏菌侵染HPT-8细胞的cDNA文库。分别提取布鲁氏菌侵染20min、1h、2h、3h、4h后HPT-8细胞总RNA,逆转录合成cDNA,同源重组法构建布鲁氏菌侵染HPT-8细胞的cDNA文库,测定其库容量和重组率,对文库63个克隆进行测序分析,采用BLASTx和BLASTn进行序列同源性比对,并将63个基因进行了功能分类。结果得到的羊种布鲁氏菌侵染人胚胎滋养层细胞HPT-8cDNA文库的库容为1.43×106,重组率是96.92%,插入片段大小为0.2-5.0kb。在63个基因中,与转录、能量代谢、运输及细胞因子相关的基因所占比例较高。构建的羊种布鲁氏菌侵染HPT-8细胞的cDNA文库,为研究宿主细胞受体和布鲁氏菌入侵途径,进一步了解布鲁氏菌的致病机理奠定了基础。

The cDNA library of HPT-8 ceils infected with Brucella melitensis 027 strain was constructed in this study. Total RNA were extracted from HPT-8 cells at 20 rain, 1 h, 2 h, 3 h and 4 h post infection respectively. With eDNA synthesized by reverse transcriptase, homologous recombination construct infected HPT-8 cell eDNA library. The partial genes were sequenced and classifid by function. The capacity of eDNA library was 1.43 - 106, the recombinant rate was 96.92 percent. The size of the inserted fragments is between 0.2-5.0 kb. 63 clones of eDNA library were random selected and sequenced. The rates of transcription, energy metabolism, transporter, cytokine are relatively high. The results showed that cDNA library was successfully constructed. The cDNA library of HPT-8 cells infected with Brucella melitensis 027 strain may facilitate to identify the receptors associated with the resistance against Brucella in host cells and to cast new light on the mechanism of cellular tropism.