感染强、弱PRRSV毒株的Marc-145细胞差异基因文库的构建

Construction of a library for genes differentially expressed in Marc-145 cells infected with high-virulence and low-virulence strains of PRRSV GD strain

【目的】用强、弱PRRSV GD株传代毒株感染Marc-145细胞,构建Marc-145细胞的差异基因cDNA文库。【方法】以感染PRRSV GD株第100代弱毒株的Marc-145 cDNA为测试组(Tester),以感染PRRSV GD株第5代强毒株的Marc-145 cDNA为对照组(Driver),采用抑制性消减杂交方法,构建强、弱PRRSV GD株感染的Marc-145细胞差异基因cDNA文库,并使用LAMP(Loop-mediated isothermal amplification)技术对试验结果进行验证。【结果】经4次抑制性消减杂交重复试验,得到S8、S29、L6、L26及L30等5个稳定差异表达的基因。LAMP结果显示,S8、S29、L6、L26及L30均为差异表达基因,这与抑制性消减杂交结果吻合。【结论】成功构建了感染强、弱PRRSV毒株的Marc-145细胞差异基因文库,为研究S8、S29、L6、L26和L30基因与PRRSV感染体外培养细胞Marc-145的关系奠定了基础。

【Objective】 The goal of this research is to construct a cDNA library of genes differentially expressed in host cells following infection of Marc-145 cells with low-virulent strain of PRRSV GD100 and strong-virulent strain of PRRSV GD5.【Method】 With GD100-infected Marc-145 cells as the test group（Tester） and GD5-infected Marc-145 cells as the control group（Driver）,suppression subtractive hybridization（SSH） was applied to derive the differences in gene expression of host cells between the test group and the control group.The reverse transcription-loop-mediated isothermal amplification（LAMP） method was used to validate the differential genes screened out.【Result】 After four repeated trials of SSH,five stably differentially expressed genes were obtained,including S8,S29,L6,L26 and L30,which were validated to be differentially expressed genes by the LAMP method,tallying with the results of SSH.【Conclusion】 A cDNA library of genes differentially expressed in host cells following infection of Marc-145 cells with low-virulent strain of porcine reproductive and PRRSV GD100 and strong-virulent strain of PRRSV GD5 was constructed successfully,which laid the foundation for studies revealing the association between S8,S29,L6,L26 and L30 genes and PRRSV infection.