1.A method for immortalizing swine monoclonal B cells secreting anti-PRRSV antibodies.
Jian WANG ; Jing ZHANG ; Kun LI ; Pu SUN ; Guoxiu LI ; Jiaoyang LI ; Yimei CAO ; Zhixun ZHAO ; Hong YUAN ; Yuanfang FU ; Pinghua LI ; Dong LI ; Zaixin LIU ; Zengjun LU
Chinese Journal of Biotechnology 2022;38(8):2872-2882
Porcine reproductive and respiratory syndrome (PRRS) is a highly contagious disease caused by porcine reproductive and respiratory syndrome virus (PRRSV), which causes great economic losses. At the moment, no effective neutralizing antibody is available for scientific research and treatment. Therefore, developing a method for screening the neutralizing monoclonal antibodies is of great significance for the prevention and treatment of PRRSV and the screening of antigen sites. Monoclonal antibodies have been widely used in the treatment and diagnosis of many human and animal diseases. Therefore, screening effective neutralizing antibodies for different pathogens is an urgent task. Among the methods for monoclonal antibody screening, B cell immortalization is an effective method to obtain neutralizing monoclonal antibody. Specifically, in this study, the bcl-6 and bcl-xl genes were connected by f2a and then the yielded product was ligated to a vector for retrovirus packaging. The swine lymphocytes immunized with PRRSV were infected the yielded mature viruses and cultured in the complete medium containing CD40L and IL21 cytokines. Then, CD21 was used as the marker to screen B cells with the magnetic bead method. Finally, monoclonal B cells were obtained and the secretion of antibodies was tested. The results showed that the plasmid, either being transfected alone or with the packaged plasmids, could be expressed, and that the packaged retrovirus could infect the cells. Moreover, the infected lymphocytes secreted antibodies, so did the screened B cells. Therefore, the method for screening monoclonal antibody against PRRSV was successfully established.
Animals
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Antibodies, Monoclonal
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Antibodies, Neutralizing
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Antibodies, Viral
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Humans
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Porcine Reproductive and Respiratory Syndrome/prevention & control*
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Porcine respiratory and reproductive syndrome virus/genetics*
;
Swine
2.Eukaryotic expression of GP5 and M protein of porcine reproductive and respiratory syndrome virus and immunogenicity evaluation.
Huicong LOU ; Runshan LIN ; Yabo LI ; Yuna ZHAO ; Pengtao JIAO ; Tingrong LUO ; Wenjun LIU
Chinese Journal of Biotechnology 2023;39(12):4809-4823
In order to understand the prevalence and evolution of porcine reproductive and respiratory syndrome virus (PRRSV) in China and to develop subunit vaccine against the epidemic lineage, the genetic evolution analysis of PRRSV strains isolated in China from 2001 to 2021 was performed. The representative strains of the dominant epidemic lineage were selected to optimize the membrane protein GP5 and M nucleotide sequences, which were used, with the interferon and the Fc region of immunoglobulin, to construct the eukaryotic expression plasmids pCDNA3.4-IFNα-GP5-Fc and pCDNA3.4-IFNα-M-Fc. Subsequently, the recombinant proteins IFNα-GP5-Fc and IFNα-M-Fc were expressed by HEK293T eukaryotic expression system. The two recombinant proteins were mixed with ISA206VG adjuvant to immunize weaned piglets. The humoral immunity level was evaluated by ELISA and neutralization test, and the cellular immunity level was detected by ELISPOT test. The results showed that the NADC30-like lineage was the main epidemic lineage in China in recent years, and the combination of IFNα-GP5-Fc and IFNα-M-Fc could induce high levels of antibody and cellular immunity in piglets. This study may facilitate the preparation of a safer and more effective new PRRSV subunit vaccine.
Humans
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Animals
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Swine
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Porcine respiratory and reproductive syndrome virus/genetics*
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Porcine Reproductive and Respiratory Syndrome/prevention & control*
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HEK293 Cells
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Viral Envelope Proteins/genetics*
;
Antibodies, Viral
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Viral Vaccines/genetics*
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Recombinant Proteins
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Vaccines, Subunit
3.Construction and immunogenicity of recombinant adenoviruses expressing Cap protein of PCV2 and GP5 protein of PRRSV in mice.
Xianwei WANG ; Yufeng LI ; Ping JIANG
Chinese Journal of Biotechnology 2009;25(11):1639-1645
Porcine circovirus 2 (PCV2) has been implicated as the etiological agent of postweaning multisystemic wasting syndrome (PMWS). Co-infection of PCV2 and porcine reproductive and respiratory syndrome virus (PRRSV) can result in severe economic losses to the swine industry. In this study, we constructed the recombinant adenovirus rAd-Cap-GP5 expressing Cap of PCV2 and GP5 of PRRSV. And the expression of Cap and GP5 protein in the HEK-293 cells inoculated with rAd-Cap-GP5 were confirmed by immunoperoxidase monolayer assay (IPMA), indirect immunofluorescence assay (IFA) and Western blotting, respectively. The immunogenicity of recombinant adenoviruses rAd-Cap-GP5 was examined in mice by vaccination with the recombinant adenovirus. The results showed that the mice could produce anti-PCV2 and PRRSV antibodies detected by indirect ELISA and virus neutralization assay. It indicated that rAd-Cap-GP5 could provide humoral immunity responses in mice. The recombinant adenovirus rAd-Cap-GP5 might be an attractive candidate vaccine for preventing the disease associated with PCV2 and PRRSV infection.
Adenoviridae
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genetics
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metabolism
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Animals
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Capsid Proteins
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biosynthesis
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genetics
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immunology
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Cell Line
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Circoviridae Infections
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prevention & control
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virology
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Circovirus
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genetics
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metabolism
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Immunization
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Mice
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Porcine Reproductive and Respiratory Syndrome
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prevention & control
;
virology
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Porcine respiratory and reproductive syndrome virus
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genetics
;
metabolism
;
Recombinant Fusion Proteins
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biosynthesis
;
genetics
;
immunology
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Swine
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Transfection
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Viral Envelope Proteins
;
biosynthesis
;
genetics
;
immunology
4.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.
Deyuan TANG ; Jian LIU ; Chunyan LI ; Hua ZHANG ; Ping MA ; Xianfeng LUO ; Zhiyong ZENG ; Nining HONG ; Xia LIU ; Bin WANG ; Feng WANG ; Zhenlei GAN ; Fei HAO
Journal of Veterinary Science 2014;15(1):99-109
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).
Animals
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Cell Line
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Escherichia coli/genetics
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Haplorhini
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Immunity, Cellular
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Interleukin-2/genetics/*metabolism
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Interleukin-4/genetics/*metabolism
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Neutralization Tests/veterinary
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Plasmids
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Porcine Reproductive and Respiratory Syndrome/*prevention & control
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Porcine respiratory and reproductive syndrome virus/*immunology
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Recombinant Proteins/genetics/metabolism
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Swine
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Vaccines, DNA/immunology
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Viral Envelope Proteins/*genetics/metabolism
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Viral Vaccines/*immunology
5.Construction and biological characteristic for the recombinant modified vaccinia virus ankara co-expressing modified GP5 and M protein of porcine reproductive and respiratory syndrome virus.
Qisheng ZHENG ; Peng LI ; Ruibing CAO ; Jibo HOU ; Puyan CHEN
Chinese Journal of Biotechnology 2008;24(5):766-773
Modified ORF5 (MORF5) and ORF6 gene of PRRSV were cloned into two multiple cloning sites of MVA transfer vector pLR-gpt to construct the recombinant plasmid pLR-MORF5/ORF6. Homologous recombination between pLR-MORF5/ORF6 and the wtMVA on BHK-21 cell line was mediated with liposome by infecting the cell with 0.01 MOI wtMVA two hours before transfecting the recombinant plasmid into the cell. When the cytopathic effect (CPE) was obvious, virus was collected from the cell plate and the recombinant virus was selected with drug selecting medium (2% MXHAT). After 12 cycles of selection, rMVA with a selection marker Eco gpt was obtained and named as rMVAgpt-MGP5/M. By infecting BHK-Cre expressing Cre recombinant enzyme, the Eco gpt marker in rMVAgpt-MGP5/M was deleted and this rMVA was named as rMVA-MGP5/M. The insertion of MORF5 and ORF6 into the MVA genome was confirmed with PCR analysis and the expression of MGP5 and M protein was identified with Western blot and IFA. Through biological study on the recombinant MVA, no obvious difference was observed between rMVA-MGP5/M and the wtMVA regarding to the CPE and growth curve. The recombinant MVA constructed in this study could coexpress the modified GP5 and M protein and the expressed product had good immunocompetence. Furthermore, the insertion of the MORF5 and ORF6 into MVA genome had no obvious effect on the replication and biological characteristics of this virus.
Animals
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Cell Line
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Genetic Vectors
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genetics
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Porcine Reproductive and Respiratory Syndrome
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prevention & control
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Porcine respiratory and reproductive syndrome virus
;
genetics
;
Recombinant Fusion Proteins
;
biosynthesis
;
genetics
;
immunology
;
Recombination, Genetic
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Swine
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Transfection
;
Vaccines, DNA
;
genetics
;
immunology
;
Vaccinia virus
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classification
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genetics
;
metabolism
;
Viral Envelope Proteins
;
biosynthesis
;
genetics
;
Viral Matrix Proteins
;
biosynthesis
;
genetics
6.Immunogenicity of DNA vaccine expressing GP5 of porcine reproductive and respiratory syndrome virus fused with VP22 of bovine herpesvirus 1.
Wu ZHAO ; Shao-Bo XIAO ; Liu-Rong FANG ; Yun-Bo JIANG ; Yun-Feng SONG ; Lin YAN ; Xiao-Lan YU ; Huan-Chun CHEN
Chinese Journal of Biotechnology 2005;21(5):725-730
To enhance the immuogenicity of DNA vaccines expressing the GP5 protein of Porcine Reproductive and Respiratory Syndrome Virus (PRRSV), the tegument protein VP22 (encoded by VP22 gene) of Bovine Herpesvirus 1 (BHV-1), which has been demonstrated to exhibit the unusual protein transduction property, was fused to N-terminus of GP5 of DNA vaccine construct pCI-ORF5M, resulting in pCI-VP22-ORF5M expressing VP22-GP5 fusion protein. The expression of VP22-GP5 fusion protein was confirmed by both indirect immunofluorescence assay (IFA) and Western blot. To investigate its immunogenicity, BALB/c mice were immunized with the fusion expression plasmid pCI-VP22-ORF5M and non-fusion expression plasmid pCI-ORF5M, respectively. The GP5-specific ELISA antibodies, neutralizing antibodies and lymphocyte proliferative responses were evaluated at various time points after primary immunization. The results showed that GP5-specific ELISA antibodies, neutralizing antibodies, and lymphocyte proliferative responses induced by DNA vaccine pCI-VP22-ORF5M were higher significantly than those of DNA vaccine pCI-ORF5M, indicating that fusion expression with BHV-1 VP22 significantly enhances the immuogenicity of DNA vaccine expressing the PRRSV GP5 protein, and that this strategy may also be useful to develop more efficient DNA vaccines against other pathogens.
Animals
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Antigens, Viral
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genetics
;
immunology
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Artificial Gene Fusion
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Female
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Mice
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Mice, Inbred BALB C
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Porcine Reproductive and Respiratory Syndrome
;
prevention & control
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Random Allocation
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Vaccines, DNA
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genetics
;
immunology
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Viral Envelope Proteins
;
genetics
;
immunology
;
Viral Structural Proteins
;
genetics
;
Viral Vaccines
;
genetics
;
immunology
7.Influence of epitope A modification and N-linked glycosylated site mutation of PRRSV NJ-a strain ORF5 gene on the ability to induce neutralizing antibodies and T cell proliferation response.
Qi-Sheng ZHENG ; Peng LI ; Zhi-Xiang BI ; Ming-Fu NIU ; Rui-Bing CAO ; Bin ZHOU ; De-Sheng CHEN ; Pu-Yan CHEN
Chinese Journal of Biotechnology 2007;23(1):33-39
To enhance the DNA immunogencity of PRRSV ORF5 gene, CpG sequence and the universal helper T cell antigen epitope (PADRE) sequence were inserted between the decoy epitope and the neutralizing epitope. At the same time, site-mutations were introduced at N33 and N51 to diminish the coverage effect to epitope B from the polysaccharides. Subsequently, the modified ORF5 gene (MORF5) and PRRSV ORF6 gene were cloned into the eukaryotic expression vector pcDNA3.0 under the control of two CMV promoters, respectively. With indirect immunofluorescence assay and Western-blot the expression in vitro of the two genes was confirmed, then six-week-old Balb/C mouse were immunized with the modified expression plasmid pcDNA-M5A-6A. The non-modified expression plasmid pcDNA-5A-6A, the blank eukaryotic expression plasmid pcDNA3.0, living attenuated vaccine and inactivated vaccine were used as controls. The PRRSV specific neutralizing antibodies and the T cell proliferation response were elevated with virus neutralization assay and MTf method. Results indicate that the modified plasmid pcDNA-M5A-6A can elicit not only higher titer of neutralizing antibodies in a rapid time, but also more vigorous T cell proliferation response compared with the non-modified plasmid pcDNA-5A-6A and commercial vaccines, indicating that DNA vaccine pcDNA-M5A-6A maybe a promising candidate for PRRS prevention.
Animals
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Antibodies, Neutralizing
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immunology
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Antibodies, Viral
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blood
;
immunology
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Binding Sites
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genetics
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Blotting, Western
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CHO Cells
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Cell Proliferation
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Cricetinae
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Cricetulus
;
Female
;
Glycosylation
;
Mice
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Mice, Inbred BALB C
;
Mutation
;
Open Reading Frames
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genetics
;
Porcine Reproductive and Respiratory Syndrome
;
immunology
;
prevention & control
;
Porcine respiratory and reproductive syndrome virus
;
genetics
;
immunology
;
metabolism
;
Swine
;
virology
;
T-Lymphocytes
;
cytology
;
immunology
;
metabolism
;
Vaccines, DNA
;
administration & dosage
;
immunology
;
Viral Proteins
;
genetics
;
immunology
;
metabolism
;
Viral Vaccines
;
administration & dosage
;
immunology
8.Construction and identification of a recombinant PRRSV expressing protective antigens of type O foot-and-mouth disease virus.
Wu TONG ; Yanzhao XU ; Yanjun ZHOU ; Yifeng JIANG ; Shanrui ZHANG ; Yaxin WANG ; Jianping ZHU ; Lingxue YU ; Jing SUN ; Huanchun CHEN ; Guangzhi TONG
Chinese Journal of Biotechnology 2012;28(12):1431-1440
Using mutation PCR, we cloned the target gene containing 421-480nt (141-160aa) and 598-639nt (200-213aa) of VP1 gene of foot and mouth disease virus (FMDV) into the deleted region (508-532aa) of Nsp2 gene of a highly pathogenic porcine reproductive and respiratory syndrome virus derived vaccine strain (HuN4-F112) that was used as vector. The recombinant cDNA was in vitro transcribed followed by transfection of BHK-21 cells for 36 h. Then, the supernatant of the cell culture was continuously seeded to monolayer of MARC-145 cells for recovery of the recombinant virus. CPE was obviously visible after a couple of passages in the seeded MARC-145, and the rescued virus (designated as rPRRSV-F112-O/VP1ep) was identified by Mlu I digestion, sequencing and immunofluorescence assay. Meanwhile, expression of inserted FMDV epitopes was also detected by indirect immunofluorescence assay with polyclonal antibodies against VP1 protein of FMDV. The analysis of biological characteristics shows that the titer of the rescued recombinant PRRSV (TCID50 = -log10(-6.75)/0.1 mL) was similar to its direct parental virus rHuN4-F112-delta508-532, but higher than rHuN4-F112.
Animals
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Antigens, Viral
;
immunology
;
Base Sequence
;
Capsid Proteins
;
immunology
;
Cell Line
;
Cysteine Endopeptidases
;
genetics
;
Epitopes
;
genetics
;
Foot-and-Mouth Disease
;
immunology
;
prevention & control
;
Foot-and-Mouth Disease Virus
;
genetics
;
immunology
;
Molecular Sequence Data
;
Mutation
;
Porcine respiratory and reproductive syndrome virus
;
genetics
;
immunology
;
Recombination, Genetic
;
Swine
;
Transfection
;
Vaccines, Attenuated
;
genetics
;
immunology
;
Viral Envelope Proteins
;
genetics
;
immunology
;
Viral Vaccines
;
genetics
;
immunology