经猪血管内皮细胞多次传代的CSFV E2基因的遗传变异研究

Research of genetic diversity of E2 gene of classical swine fever virus that poly-passed in the swine umbilical vein endothelial cells

【目的】研究猪瘟病毒（CSFV）E2基因在猪脐静脉血管内皮细胞中多次传代后的遗传变异情况,为CSFV的致病机理研究提供理论依据。【方法】分离并培养猪脐静脉血管内皮细胞,接种猪瘟病毒石门株脾毒后继续培养,染毒细胞经3次连续传代后全部死亡、脱落,收集每代细胞并提取总RNA,采用RT-PCR方法扩增CSFVE2基因。将获得的目的基因克隆入T载体并转化DH5α感受态细胞,提取重组质粒,进行PCR和BamHⅠ、HindⅢ酶切鉴定,将阳性的重组质粒进行测序,并用DNAStar软件进行序列分析。【结果】扩增出了E2基因,重组质粒PMD18-T-E2的PCR和双酶切鉴定结果表明,E2基因与pMD18-T载体连接成功。各代猪脐静脉血管内皮细胞中CSFVE2基因之间的核苷酸序列同源性为99.4%-99.9%,氨基酸序列同源性为98.9%-99.8%;各代猪脐静脉血管内皮细胞中的CSFVE2基因与标准病毒石门株之间的核苷酸序列同源性为98.9%-99.3%,氨基酸序列同源性为98.9%-99.2%。【结论】猪瘟病毒石门株在猪血管内皮细胞上传代的过程中E2基因无明显的变异,能保持遗传的稳定性。

[Objective] The study researched the genetic diversity of E2 gene of classical swine fever virus that poly-passed in the swine umbilical vein endothelial cells to reveal the virus＇s pathopoiesis mechanism. [Method] Swine umbilical vein endothelial cells were isolated and cultivated continuously after CSFV were incoulated. Passaged for three times,the cells were all exfoliated and dead, the RNA was extracted from the collected cells. E2 gene was amplified by reverse transcription-polymerase chain reaction （RTPCR） using the special primers. The PCR product was cloned into PMD-18T vector,then transformed into E. coli,the recombinant plasmid was extracted. The positive plasmid was sequenced after identified by PCR and restriction enzyme,the sequences were analyzed by DNAstar software. [Result] The results showed that E2 gene was amplified,and was inserted the recombinant plasmids PMD18-T-E2 exactly by PCR and restriction digestion. The nucleotide homology of E2 gene among each passage virus was 99.4%-99.9%,the amino acid homogeneity 98.8%-99.8% ;the nucleotide homology of E2 gene between each passage virus and shimen virus was 98.9%-99.3%,the amino acid homogeneity 98.9%-99.2%. [Conclusion] After poly-passaging in the swine umbilical vein endothelial cells,E2 genes of CSFV were genetically stable.