Construction of recombinant PRRSV ORF5 gene by DNA shuffling technique / 生物工程学报

Recombinant PRRSV △2ORF5 gene was constructed using DNA shuffling from four genetically different strains of PRRSV to study its heterologous cross-neutralizing ability. The △2ORF5 mutant gene was cloned into the vector pET-32a and transferred into E. coli BL21. SDS-PAGE confirmed that the molecular weight of the recombinant △2ORF5 was about 42 kDa, consistent with the predicted result. Then the purified recombinant protein was injected into BALB/c mouse to obtain polyclonal antibody. Western blotting analysis with mouse-anti-△2ORF5 polyclonal serum indicated that the parental virus recombinant GP5 protein reacted with the specific antibodies. Four parental viruses could be inhibited by the anti-△2ORF5 polyclonal antibody and the inhibition rates were higher than 53%. This work has laid a foundation for further development vaccine for PRRSV.

Prediction of the secondary structure and B-cell epitopes of Mycoplasma suis ORF5 protein / 中国人兽共患病学报

The objective of the study is to predict the spatial structure and B‐cell epitopes of Mycoplasma suis ORF5 pro‐tein .The secondary structure ,hydrophilicity ,flexible region ,antigenic index and surface probability were analyzed and predic‐ted by the Protean module in DNAStar software and B Cell Epitope Prediction Tools of IDEB ,then B‐cell epitopes were predic‐ted by aggregate analysis .Results showed that the secondary structure of Mycoplasma suis ORF5 protein was relatively regu‐lar ,in which the potential B cell antigenic epitopes were located at GGVDGGRD ,GMRLPEDSR ,and EGHPDLESAR .The methods of prediction of the secondary structure and B‐cell epitopes of Mycoplasma suis ORF5 protein may provide a new method for the study of M .suis immunogenicity ,and provides a new idea for the study on immunogenicity of pathogenic micro‐organisms .

Positive effects of porcine IL-2 and IL-4 on virus-specific immune responses induced by the porcine reproductive and respiratory syndrome virus (PRRSV) ORF5 DNA vaccine in swine

The purpose of this study was to investigate the effects of porcine interleukin (IL)-2 and IL-4 genes on enhancing the immunogenicity of a porcine reproductive and respiratory syndrome virus ORF5 DNA vaccine in piglets. Eukaryotic expression plasmids pcDNA-ORF5, pcDNA-IL-2, and pcDNA-IL-4 were constructed and then expressed in Marc-145 cells. The effects of these genes were detected using an indirect immunofluorescent assay and reverse transcription polymerase chain reaction (RT-PCR). Characteristic fluorescence was observed at different times after pcDNA-ORF5 was expressed in the Marc-145 cells, and PCR products corresponding to ORF5, IL-2, and IL-4 genes were detected at 48 h. Based on these data, healthy piglets were injected intramuscularly with different combinations of the purified plasmids: pcDNA-ORF5 alone, pcDNA-ORF5 + pcDNA-IL-2, pcDNA-ORF5 + pcDNA-IL-4, and pcDNA-ORF5 + pcDNAIL-4 + pcDNA-IL-2. The ensuing humoral immune responses, percentages of CD4+ and CD8+ T lymphocytes, proliferation indices, and interferon-gamma expression were analyzed. Results revealed that the piglets co-immunized with pcDNA-ORF5 + pcDNA-IL-4 + pcDNA-IL-2 plasmids developed significantly higher antibody titers and neutralizing antibody levels, had significantly increased levels of specific T lymphocyte proliferation, elevated percentages of CD4+ and CD8+ T lymphocytes, and significantly higher IFN-gamma production than the other inoculated pigs (p < 0.05).

Sequence Analysis and Expression of ORF5 in Korean Isolate of Porcine Reproductive and Respiratory Syndrome Virus

Porcine reproductive and respiratory syndrome virus (PRRSV) is the causative agent of reproductive failure and respiratory disorders in pigs. The viral genome consists of eight overlapping open reading frames (ORFs). ORF5 encodes one of the major glycoproteins and is known as an immunologically important structural protein associated with virus neutralization. The ORF5 gene of the Korean PRRSV isolate, CNV-1, was amplified by reverse transcription-polymerase chain reaction (RT-PCR), cloned and sequenced. The nucleotide and amino acid sequences of CNV-1 ORF5 shared 91% and 83% identity, respectively, with the American isolate (VR2332 strain) and 57% and 49% identity with the European isolate. For the expression and easy purification of ORF5, the cDNA containing the complete ORF5 sequence fused in-frame with sequence encoding glutathione S-transferase (GST) was cloned into a baculovirus transfer vector and transfected into Sf9 cells. The GST-ORF5 fusion protein produced in Sf9 cells was detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting. Sequencing results confirmed that the recombinant baculovirus from Sf9 cells contains the complete ORF5 gene. Further studies in this direction will address whether ORF5 can be a good candidate for a subunit vaccine against PRRSV in Korea.

Genetic analysis of ORF5 of recent Korean porcine reproductive and respiratory syndrome viruses (PRRSVs) in viremic sera collected from MLV-vaccinating or non-vaccinating farms

The 23 open reading frame (ORF) 5 sequences of Korean type II porcine reproductive and respiratory syndrome virus (PRRSV) were collected from viremic sera from the (modified live vaccine) MLV-vaccinating and non-vaccinating farms from 2007 to 2008. The samples were phylogenetically analyzed with previous ORF5 sequences, including type I Korean PRRSV, and previously reported or collected sequences from 1997 to 2008. A MN184-like subgroup of type II Korean PRRSV was newly identified in the viremic sera collected from 2007 to 2008. And of the type I PRRSVs, one subgroup had 87.2~88.9% similarity with the Lelystad virus, showing a close relationship with the 27~2003 strain of Spain. The maximum parsimony tree of type II PRRSV from 1997 to 2008 showed that they had evolved to four lineages, subgroups 1, 2, 3 and 4. Most of the recently collected type II PRRSVs belonged to subgroup 4 (48%). The region of three B-cell epitopes and two T-cell epitopes of ORF5 amino acids sequences was considerably different from the MLV in subgroups 3 and 4. In conclusion, the existence of type I PRRSV, which was genetically different from Lelystad virus (Prototype of type I PRRSV), and heterologous type II PRRSVs of viremic pigs detected even in the MLV-vaccinating farms indicated the need for new vaccine approaches for the control of PRRSV in Korea.

Expression of Open Reading Frame 5 Protein of Porcine Reproductive and Respiratory Syndrome Virus Using Semliki Forest Virus Expression System

The ORF5 gene encodes a major envelope glycoprotein (GP5), which is one of the three major proteins of porcine reproductive and respiratory syndrome virus (PRRSV). The GP5 protein has been known to be a 24.5-26kDa N-glycosylated envelope protein. The GP5 is involved in inducing neutralizing antibodies. For this reason, the GP5 is primary candidate for the PRRSV subunit vaccine. To produce the native form of GP5 in mammalian cells, we have cloned the ORF5 gene from PRRSV CNV-1 into the Semliki Forest virus (SFV)-based expression vector, resulting in recombinant pSFV-ORF5. By the infection with recombinant pSFV-ORF5 to BHK-21 cells, the GP5 expression was confirmed by immunocytochemistry and immunoblotting assay. The recombinant virus particle harboring ORF5 gene was infectious to BHK-21 and MARC-145. The RNA synthesis and expression of GP5 in the infected cell was also confirmed by RT-PCR.

Study on immunological responses to eukaryotic expression vector containing PRRSV ORF5 and CpG motifs / 中国免疫学杂志

Objective:To enhance the immune efficiency of porcine reproductive and respiratory syndrome virus(PRRSV)DNA vaccine,the ORF5 genes were used as candidate genes to construct the recombinant plasmids CpG-pVAX1-ORF5 by pVAX1 as eukaryotic expression vector.The aim is to analyze the immune responses and the effects of CpG-ODN induced by GP5 recombinant plasmids of PRRSV.Methods:Piglets were immunized with recombinant DNA plasmids which expressed PRRSV GP5 for three inoculations.The PRRSV antibody in serum,the concentration of IL-2 and the lymphocyte proliferation test(MTT) in peripheral blood of vaccinated piglets were detected.The vaccinated pigs were challenged intranasally with PRRSV SD2.Results:GP5 DNA immunization with CpG resulted in the production of both PRRSV antibodies and cellular immune(a significant enhancement of a lymphoproliferative response ).The CpG-pVAX1-ORF5 was showed significantly better protection from the PRRSV challenge compared with the control plasmid.This immune response was characterized by a significantly decreased frequency of viraemia with decrease of 80% compared with the control group.There were little clinical symptoms and protection in some extent from lung damage.Conclusion:The results indicate that CpG-ODN could be used as immune adjuvant of PRRSV DNA vaccine.

Studies of the pig interleukin 2(IL-2) eukaryon expression plasmid on cellular immune responses of BALB/c mice immuned with pcDNA-PRRSV-ORF5 DNA vaccine / 中国免疫学杂志

Objective:To study the effect of pig interleukin 2(IL 2) eukaryon expression plasmid on cellular immune responses of BALB/c mice immuned with pcDNA PRRSV ORF5 DNA vaccine.Methods:BALB/c mice were immunized with pcDNA PRRSV ORF5 DNA vaccine and pig interleukin 2(IL 2) eukaryon expression plasmid by the routes of co injection and DNA vaccine injection alone respectively, with PBS and pcDNA3 1(+) as controls. Fluoresecence Activated Cell Sorter(FACS),T lymphocyte proliferation test(MTT) were used to detect the number of CD4 +、CD8 + and the T lymphocyte proliferation in peripheral blood of mice vaccinated.Results:ConA response of T lymphocytes in blood was higher in experiment group than the control group ( P