| Complete Genomic Sequence of Porcine Reproductiveand Respiratory Syndrome Virus Strain CH-la &Structural and Functional Analyses of Its Envelope ProteinPorcine reproductive and respiratory syndrome virus (PRRSV) is the causative agent of porcine reproductive and respiratory syndrome (PRRS), a newly recognized pig disease of economic importance, characterized by reproductive failures (abortions, stillbirths, mummied fetuses) and respiratory problems in any age. PRRSV strain CH- 1 a, a Chinese reference strain, was isolated in the suburb of Beijing in China during a PRRS outbreak in the middle of 1990s.The cDNAs of strain CH-1 a genomic RNA were amplified using a set of primers based on the published sequence of strain VR-2332 by reverse transcription and polymerase chain reaction (RT-PCR). The complete genomic sequence of the strain was then determined and analyzed using a package of softwares. The genome of CH- 1 a is 15 432 bases (b) in length, comprising 8 open reading frames (ORFs). The results showed that the Chinese isolate CH- I a is more closely related to North American genotype than European genotype, though probably representing a subtype different from VR-2332 within North American genotype.Pseudorabies virus (PRV) strain Bartha-K6 I, a widely used attenuated live vaccine in the world, has been shown previously to be defective for glycoprotein E (gE) gene. PRV genomic DNA was extracted from PRV-infected PK-1 5 cells and over-digested by the restriction endonuclease (RE) KpnI. The KpnI-J fragment of 5.9 kilo-base pairs (kb) or so was shown to contain thymidine kinase (TK) gene by polymerase chain reaction (PCR), and it was cloned into pUC 119, creating a recombinant pBTK5 .9. The RE and PCR analyses confirmed that the TK gene was located within the 2.6 kb PstI-KpnI fragment within the former fragment. This minor fragment was then subcloned to produce recombinant pBTK2.6. A fragment containing the immediate early promoter of cytomegalovirus (CMV) and bovine growth hormone (BGH) polyadenylation signal derived from pCR3-Uni, a eukaryotic expression plasmid, was amplified and used to replace the 378bp AccI fragment of pBTK2.6, creating pBdTK-Uni. The KpnI fragment of 1.7 kb from pSTK containing BamHI- 11 fragment was cloned into the KpnI site ofpBdTK-Uni. The resulting transfer vector pBdTK-Uni2 can be used as a universal PRV transfer vector for developing 1K- and gE-deleted recombinant PRV expressing foreign gene(s). The GP5 gene of PRRSV was cloned into the transfer vector, and the resultant recombinant plasmid was used to co-transfect PK-15 cells with Bartha-K61 genomic DNA using liposome-mediated transfection. A recombinant PRV was obtained after several times of screening with 5-bromo-2?deoxy-uridine. The recombinant virus was shown to express recombinant GP5 as confirmed by immunofluorescent antibody and Western blotting analyses using PRRS V-specific antisera. The double-gene-deleted recombinant PRV expressing PRRSV GP5 could be a competitive genetically engineered vaccine candidate against reproductive failures caused by pseudorabies and PRRS when tested in animals.The GP5 gene was then inserted into plasmid pBlueBacHis B, a baculovirus transfer vector. The resulting transfer plasmid harboring the GP5 gene was used to co-transfect sf9 cells with linearized wild-type baculovirus DNA mediated by liposome. A recombinant baculovirus containing the GP5 gene was obtained after blue-plaque screening and three cycles of plaque purification. The GP5 gene was expressed by the recombinant virus in sf9 cells as indicated by immunofluorescent antibody and Western blotting assays. The expressed recombinant protein fused with His-tag had a molecular weight (MW) of 22, 25, 30 ku, respectively, accounting for 26.4% or so in total of the whole cellular proteins. The recombinant GP5 protein is a potential subunit vaccine against PRRS.A plasmid expressing PRRSV ORES under the control of immediate early promoter of CMV was also constructed. MARC-145 cell monolayer was tran...
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