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Immunogenicity Evaluation of Porcine Epidemic Diarrhea Virus S1 Protein

CHEN Weicong, LIU Yunchao, ZHOU Chuanjie, YANG Suzhen, WEI Qiang, CHAI Shujun, ZHANG Gaiping

Journal of Henan Agricultural Sciences 2022, 51 (11): 127-134. DOI: 10.15933/j.cnki.1004-3268.2022.11.015

Abstract （111）

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In order to evaluate the immunogenicity of porcine epidemic diarrhea virus（PEDV）S1 protein， Drosophila melanogaster embryo S2 cells were used to express recombinant S1 protein.The 4‑week old Kunming mice were immunized with purified S1 protein，and the serum and spleen lymphocytes were collected.The immunogenicity of the expressed recombinant S1 protein was analyzed by ELISA，virus neutralization test and flow cytometry. The results showed that，compared with the control group（PBS），the level of specific IgG antibody in serum of mice immunized with recombinant PEDV S1 protein was significantly increased. At 14 days after the second immunization，the neutralizing antibody titers of mice immunized with recombinant PEDV S1 protein was 1∶320.The splenic lymphocytes of immunized mice had stronger proliferative ability.The percentage of peripheral blood CD3 ⁺T cells increased significantly，and the ratio of CD4 ⁺/CD8 ⁺T cells was higher than that of the control group. The content of IL‑4 and IFN‑γ in mice serum increased significantly. In summary，the recombinant PEDV S1 protein is successfully expressed，and it can induce mice to produce high titer neutralizing antibody and promote cellular immune response.

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Prokaryotic Expression，Purification and Immunogenicity Study of Outer Membrane Protein AHA1182 of Aeromonas hydrophila

JIAN Sijie, CHAO Jia, SUN Wei, CHEN Rui, DING Rui, CHEN Chen, LIU Xiang

Journal of Henan Agricultural Sciences 2022, 51 (11): 135-144. DOI: 10.15933/j.cnki.1004-3268.2022.11.016

Abstract （103）

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To evaluate the immunogenicity of outer membrane protein AHA1182 of Aeromonas hydrophila，bioinformatics method was firstly performed to analyze the relationship among A.hydrophila， A.salmonicida， A.schubertii， A.diversa， A.jandaei， Providenci alcalifaciens and Edwardsiella ictaluri.Recombinant expression strain of AHA1182 was constructed by molecular cloning and the optimal expression conditions were identified. AHA1182 was purified by inclusion body washing and SDS‑PAGE，and was used to immunize Carassius auratus.Western blot was used to assess the specificity and titer of the C.auratus serum，and enzyme‑linked immunosorbent assay（ELISA） was used to simulate the recognition effect between anti‑AHA1182 serum and A.hydrophila. Nonspecific immunity was assessed by acid phosphatase（ACP），alkaline phosphatase（AKP）and leukocyte phagocytosis.Furthermore，the toxicity of AHA1182 immunization to C.auratus was investigated by histopathological sections of visceral organ.The results showed that AHA1182 was genetically related among different bacteria，especially in Aeromonas，which meant that AHA1182 serum might have cross‑immune protective ability.AHA1182 was cloned，expressed and purified successfully，and the optimal expression conditions for AHA1182 included strain OD ₆₀₀ value of 1.0，isopropyl‑β‑D‑thiogalactoside（IPTG）concentration of 0.5 mmol/L，inducing temperature of 28 ℃ and inducing time of 8 hours.Western blot showed that AHA1182 serum had a high specificity and could recognize A.hydrophila in vitro with the titer of 1∶3 200.The results of AKP，ACP，and leukocyte phagocytosis indicated that AHA1182 stimulated the non‑specific immune of C.auratus. AHA1182 immunization had no toxicity to the visceral structure of C.auratus.The results suggest that A.hydrophila AHA1182 has good immunogenicity.

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Virus Titer Determination and Quality Evaluation of the Commercial Marek’s Disease Vaccines Sold in 2020—2021

ZHENG Luping, TENG Man, LIU Jinling, LUO Qin, CHU Yushu, WANG Weidong, ZHANG Wenkai, LUO Jun

Journal of Henan Agricultural Sciences 2022, 51 (6): 134-143. DOI: 10.15933/j.cnki.1004-3268.2022.06.015

Abstract （298）

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The effective prevention and control of Marek’s disease（MD）mainly depends on vaccine immunization. In order to determine and evaluate the quality of present MD vaccines，virus titers of 11 commercial MD vaccine products provided by 9 distinct companies during 2020—2021 were determined and analyzed by indirect immunofluorescence assay(IFA).Then the potential contamination of avian leukosis virus（ALV）and reticuloendotheliosis virus（REV）was detected using commercial ELISA kits，colloidal gold test strip or RT⁃PCR.The results showed that for all the tested liquid nitrogen vaccines，no significant difference was observed among the three batches of imported or domestic CVI988 vaccines，while significant differences between the two 814 vaccines（ P<0.05） and two bivalent MD vaccines(CVI988+HVT or 814+HVT)( P<0. 01)were found. The virus titers of the tested liquid nitrogen MD vaccine products were all above 5 500 PFU/bird，which were more than two folds of value specified in the national standard.However，the virus titers of four freeze⁃dried HVT vaccine products were only about 83.90—109.50 PFU/bird，much lower than 2 000 PFU/bird as indicated in product introductions.Contamination of ALV or REV was not detected in any of the tested MD vaccines，indicating that these vaccines were clean.

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Construction and Biological Characterization of ssaV Gene Mutant of Salmonella pullorum

XU Guanbing, WANG Liping, CUI Suning, ZHANG Zhen, LIANG Xuerui, ZHAO Jie, DUAN Yanhong, PAN Pengtao, LI Xuehua, YIN Junlei, ZHI Lijuan

Journal of Henan Agricultural Sciences 2021, 50 (11): 139-145. DOI: 10.15933/j.cnki.1004-3268.2021.11.016

Abstract （312）

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The aim of this study was to investigate the effect of ssaV gene on the pathogenicity of Salmonella pullorum.According to the homologous recombination technique，the target DNA with the homologous arms was amplified in the plasmid pKD3.Next the DNA fragment was transformed into Salmonella pullorum C79‑13 which already had the plasmid pKD46，after loss of pKD46，the plasmid
pCP20 was transformed into the recombinant strain C79‑13 ΔssaV∶∶Cm to delete the chloramphenicol resistance gene，then the mutant C79‑13 ΔssaV was confirmed through screening，and its complement strain C79‑13 ΔssaV（pBR322‑ ssaV）was also constructed.The results of basic biological characteristics showed that the growth characteristics and biochemical characteristics of C79‑13 ΔssaV did not change compared to those of C79‑13，and the mutant C79‑13 ΔssaV was stable after lacking of ssaV，the loss of ssaV reduced the colonial ability of Salmonella pullorum in chickens，the LD50 of C79‑13 ΔssaV was at least 100 times that of C79‑13 for chickens after oral challenge.This study demonstrated that loss of ssaV gene could decrease significantly the virulence of Salmonella pullorum，which laid a foundation for further research on the function of ssaV and development of an attenuated vaccine against Salmonella pullorum infection.

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Construction and Biological Characterization of an Attenuated Salmonella typhimurium Type Ⅲ Secretion System Carrying NDV HN Gene

Journal of Henan Agricultural Sciences 2021, 50 (10): 116-123. DOI: 10.15933/j.cnki.1004-3268.2021.10.015

Abstract （180）

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In order to construct a recombinant attenuated Salmonella typhimurium type Ⅲ secretion system which carries the hemagglutinin‑neuraminidase（HN） protein gene of Newcastle disease virus（NDV），the HN gene was amplified from the plasmind pMD18‑T‑ HN and cloned into the vector pYA3493‑ sopE _Nt100.Then the recombinant vector pYA3493‑ sopE _Nt100‑ HN was transformed into attenuated Salmonella typhimurium strain Δ crpΔ asd SL1344 by electroporation，and the growth characteristics，genetic stability，expression characteristics and virulence of the recombinant strain Δ crpΔ asd SL1344（pYA3493‑ sopE _Nt100‑ HN）were analyzed. The identification results of PCR and enzyme digestion showed that the recombinant attenuated Salmonella typhimurium strain Δ crp Δ asd SL1344（pYA3493‑ sopE_Nt100‑ HN）was successfully constructed. The growth curve of the recombinant strain was similar to that of the complementary strain Δ crpΔ asd SL1344（pYA3493‑ sopE _Nt100）and the deletion strain Δ crpΔ asd SL1344，which were significantly different from their parent strain SL1344.The HN gene was stably inherited in the recombinant strain.The HN protein was presented by the attenuated Salmonella typhimurium Ⅲ type secretion system and expressed in chicken embryo fibroblast（CEF）.The LD50 of the recombinant strain was not significantly different from the complementary strain and the deletion strain，but significantly different from the parent strain SL1344. The above results indicated that the recombinant attenuated Salmonella typhimurium Ⅲ secretion system carrying the NDV HN gene was successfully constructed，which had high safety，genetic stability，and expression efficacy of the NDV HN gene.

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Expression and Identification of PCV2b Virus⁃Like Particles with Neutralizing Epitopes in Tandem in Insect Cells

REN Chunxiao, FENG Hua, ZHANG Teng, JIANG Min, LIU Yunchao, JIN Qianyue, ZHANG Gaiping

Journal of Henan Agricultural Sciences 2021, 50 (7): 154-160. DOI: 10.15933/j.cnki.1004-3268.2021.07.018

Abstract （225）

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In order to develop a subunit vaccine that is more efficient to prevent porcine circovirus 2b subtype（PCV2b）infection，the important neutralizing epitope ²²⁶LKDPPLNP ²³³ on the Cap protein was connected to the C⁃terminal of the cap gene（ capE2） in a repeated tandem manner，and the corresponding recombinant proteins were expressed in insect baculovirus expression system.The recombinant proteins CapE2 were identified by means of SDS⁃PAGE，Western blot，dynamic light scattering and transmission electron microscopy observations.Then mice were immunized with purified CapE2 protein，and Cap without the neutralizing epitope，PCV2 subunit vaccine，and PBS were used as controls.Tthe serums post immunization were collected for analyzing the immunogenicity of these target recombinant proteins and the effect of linking neutralizing epitopes on the immunogenicity of Cap protein.The results showed that Cap and CapE2 proteins were successfully expressed in sf21 cells. These purified target proteins could effectively react specifically with the PCV2 monoclonal antibody，and could self⁃assemble to form uniform virus⁃like particles in vitro with a diameter of about 17 nm.Serums antibody test results showed that after the second immunization，the antibody level of CapE2 group increased rapidly，and was significantly higher than other immunization groups after the third immunization. The neutralizing antibody test results showed that the serum antibody titres of the Cap and CapE2 groups were significantly higher than the positive vaccine group，and the neutralization titer of the serum in the CapE2 immunization group could reach up to 1∶2 ⁹. The recombinant Cap proteins that were connected to two repeating neutralizing epitopes were successfully expressed in insect baculovirus expression system and could self⁃assemble to form virus⁃like particles，which had good immunogenicity and provided a new vaccine candidate for the prevention of PCV2 infection.

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Evaluation of Mucosal Immunity of Replication Defective Recombinant Adenovirus Expressing Capsid Protein of PCV2

DENG Zuliying, LIU Yufeng, MA Ningning, CHEN Lu

Journal of Henan Agricultural Sciences 2020, 49 (10): 143-148. DOI: 10.15933/j.cnki.1004-3268.2020.10.020

Abstract （77）

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To develop efficient porcine circovirus type 2(PCV2) vaccine,the mucosal immune effect of PCV2 Cap protein epitope recombinant replication-deficient adenovirus(rAd/Cap/518) was evaluated.The Balb/c mice were immunized by oral,intramuscular and intranasal routes with rAd/Cap/518,and the mice were immunized by intranasal routes with wild rads without exogenous genes as the control group.IgG in serum,IgA antibody in saliva,lung and intestinal lavage fluid,secretion of T lymphocyte subsets and related cytokines IFN-γ and IL-4 in spleen,and PCV2 viral load in lymph nodes,spleen and lung were detected.The results showed that,compared with the control group,the level of serum IgG antibody induced by rAd/Cap/518 via intramuscular route increased significantly.The level of IgA antibody in local mucosa significantly increased by intranasal and oral immunization.The percentage of CD3+T cells induced by rAd/Cap/518 was 46.60%—55.65% by intranasal, intramuscular and oral routes.The percentage of CD3+CD4+T cells was 34.43%—37.29%,the percentage of CD3+CD4+T cells induced by intramuscular and oral immunization was 36.35%—41.58%,the percentage of CD3+CD8+T cells induced by intranasal immunization was 12.39%—18.32%,and the percentage of CD8+T cells induced by oral immunization was 16.29%, which were significantly higher than that of the control group.Intranasal immunization with rAd/Cap/518 could induce IFN-γ production in spleen and mesenteric lymphocytes.The virus load of immunized mice decreased significantly.These results indicate that rAd/Cap/518 mainly induces systemic immune response via intramuscular immunization,and systemic and local mucosal immune response via intranasal and oral immunization. It is a promising mucosal vaccine candidate for PCV2.

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