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Cloning of equine herpesvirus type 1 438/77 strain genome as a bacterial artificial chromosome

文献类型： 外文期刊

作者： Sun, Xuegiang 1 ; Yao, Huochun 1 ; Zhang, Cun 2 ; Lu, Chengping 1 ;

作者机构： 1.Nanjing Agr Univ, Coll Vet Med, Nanjing 210095, Peoples R China

2.Zhejiang Acad Agr Sci, Inst Anim Husb & Vet Sci, Hangzhou, Zhejiang, Peoples R China

关键词： EHV-1;herpesvirus 438/77;recombinant virus;bacterial artificial chromosome;BAC;EHV-1;Herpesvirus 438/77;Rekombinantes Virus;bakterielles artifizielles Chromosome;BAC

期刊名称：BERLINER UND MUNCHENER TIERARZTLICHE WOCHENSCHRIFT （ 影响因子：0.328； 五年影响因子：0.488 ）

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收录情况： SCI

摘要： Equine herpesvirus type 1 (EHV-1) is a major cause of respiratory and reproductive diseases in horses worldwide. The genome of EHV-1 strain 438/77 (isolated from an aborted equine fetus) was cloned as a bacterial artificial chromosome (BAC) in ￡ coliwithout any gene deletions. The mini-F plasmid sequence was inserted in the middle of ORF19 and 20 via homologous recombination following co-transfection of viral DNA and plasmid pE19_20/HA into RK13 cells. Circular viral DNA was extracted from RK13 cells infected with purified recombinant virus expressing green fluorescent protein (GFP) and electrophorated into E coli DH10B cells. The clone harboring the BAC was screened and analyzed by PCR and RFLR Reconstitution of the recombinant virus was achievedsuccessfully by transfec-tion of the BAC DNA into RK13 cells. The mini-F sequence in the reconstituted virus was subsequently removed by homologous recombination between virus DNA and plasmid pE1920XM, inducing a point mutation in the Xbal site in ORF19. Comparison of RFLP profiles of the rescued, recovered and the wild-type viral genome demonstrated that no unexpected changes occurred during mutagenesis. In vitro replication assays showed that BAC-reconstituted virus mutant growth kinetics and plaqueformation morphology/size were indistinguishable to those measured for wild-type virus.

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