利用Rde/ET技术构建表达H5亚型禽流感病毒HA基因的重组火鸡疱疹病毒

Generation of a recombinant HVT virus expressing the HA gene of avian influenza virus H5N1 by Rde/ET

摘要:

【目的】本研究以火鸡疱疹病毒（HVT） BAC分子克隆为平台，构建表达禽流感HA基因的重组火鸡疱疹病毒，以开发新型病毒活载体疫苗。【方法】利用Red/ET重组技术，经过两步法重组：第一步，用两端带有50 bp大小左、右同源臂a和b的选择标记基因rpsL-neo表达盒替换HVT基因组US2区；第二步，用两端带有同样的50 bp左、右同源臂a和b的HA基因表达盒替换选择标记基因rpsL-neo表达盒。在含有氯霉素和链霉素双抗性的平板上筛选阳性克隆，经卡那霉素抗性反向筛选和PCR进一步鉴定，鉴定正确的克隆命名为pHVT-HA。提取并纯化pHVT-HA DNA，转染原代鸡胚成纤维细胞（CEF），以完成重组病毒的拯救。【结果】命名为pHVT-HA3的BAC克隆转染CEF后第4天，出现病毒噬斑，噬斑形态与野生型HVT相似，获得拯救的重组病毒，命名为rHVT-HA3，将重组病毒rHVT-HA3在CEF上连续传代培养，经PCR和间接免疫荧光检测表明，重组病毒在连续传代过程中仍能稳定表达HA蛋白。【结论】本研究以HVT BAC为平台，利用Red/ET重组技术，构建了表达禽流感A/Goose/Guangdong/3/96(H5N1) 毒株的血凝素（HA）基因的重组火鸡疱疹病毒，为新型禽流感重组活载体疫苗开发奠定基础。

Abstract:

[Objective] In recent years, manipulation of large herpesvirus genomes has been facilitated by using bacterial artificial chromosome (BAC) vectors. We have previously reported the construction of the BAC clones (HVT BACs) of herpesvirus of turkey (HVT). [Methods] With these BAC clones in hand, we manipulated the genome of HVT by utilizing Red/ET recombination system, and we were trying to develop a biologically safe live vaccine based on the HVT BACs. In this two-step approach, we first transformed the plasmid pRedET into the DH10B competent cells that carried the HVT BACs, and then added inducer L-arabinose into the cells and shifted the temperature from 30℃ to 37℃, after 45 minutes induction, we prepared the cells into competent cells,then we electroporated the linear rpsL-neo counter-selection/selection cassette flanked by the 50 bp long homology arms into the cells, RecE and RecT catalyze the homologous recombination reaction, the functional cassette was inserted into the US2 locus to be modified of HVT. Only colonies carrying the modified BAC would survive Kanamycin selection on the agar plates. The successful integration of the rpsL-neo cassette was monitored by PCR and Streptomycin selection, for the insertion of rpsL-neo cassette cells will become Streptomycin sensitive. Secondly, in the same way, we replaced the rpsL-neo cassette with the hemagglutinin(HA )gene of (HPAIV)A/Goose/Guangdong/1/96(H5N1)flanked by the same homology arms. Only colonies which lost the rpsL-neo cassette will grow on Streptomycin containing plates. [Results]Finally, we obtained many colonies of which the HA gene of the AIV was inserted into the US2 locus to be modified of HVT. And we reconstituted one recombinant virus from transfecting one of these BAC clones DNA into chick embryo fibroblasts(CEFs). [Conclusion]We achieved one rescued recombinant virus which designated as rHVT-HA3.The H5 subtype HA gene expression in this recombinant virus rHVT-HA3 was confirmed by immunofluorescence assay.