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Expression and Monoclonal Antibody Preparation of the p22 Protein of African Swine Fever Virus

YAN Shijun, HAO Liying, BAI Lulu, ZHENG Dingding, SONG Huanhuan, LI Shengqiang, WANG Tongyan, TAN Feifei, DENG Junhua, TIAN Kegong

Journal of Henan Agricultural Sciences 2021, 50 (12): 149-154. DOI: 10.15933/j.cnki.1004-3268.2021.12.017

Abstract （342）

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In order to develop an immunodiagnostic reagent for African swine fever virus，the Bac‐to‐Bac baculovirus expression system was used to express the p22 protein of African swine fever virus，the anti‐p22 protein monoclonal antibody was prepared and its reactogenicity with African swine fever virus was identified. The results showed that the recombinant baculovirus AcNPV‐p22 was successfully obtained，and the soluble recombinant protein of p22 with the size of about 22 ku was expressed.Western blot analysis confirmed that the protein had a good reactivity with African swine fever virus positive serum.Positive hybridoma cells（3D2，4A7）stably secreting monoclonal antibodies against African swine fever virus p22 protein were screened. The ascites ELISA titers of the two hybridoma cells were higher than 1∶128 000.Monoclonal antibodies isotype assay showed that the heavy chain was IgG1，and the light chain was κ.The IFA results showed that the prepared monoclonal antibody could specifically react with African swine fever virus.In summary，this study successfully expressed African swine fever virus p22 protein by baculovirus‐insect expression system and the monoclonal antibodies against African swine fever virus p22 protein were successfully prepared.

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Prokaryotic Expression of African Swine Fever Virus K145R Protein and Preparation of Monoclonal Antibody

GENG Xiaolin, SUN Jie, WANG Yanwei, HUANG Tian, LIU Peng, CAO Hongmei, PANG Wenqiang, HAO Liying, DENG Junhua, HUANG Yuxin, TIAN Kegong

Journal of Henan Agricultural Sciences 2021, 50 (8): 154-159. DOI: 10.15933/j.cnki.1004-3268.2021.08.018

Abstract （227）

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In order to develop an immunodiagnostic reagent for African swine fever virus（ASFV），the recombinant ASFV K145R protein was expressed by E. coli system，and the monoclonal antibody against K145R protein was prepared and identified. The results showed that the pET28a‑K145R expression vector was successfully constructed，and the soluble recombinant protein of K145R with the size of about 17 ku was obtained. Western blot analysis confirmed that recombinant K145R protein had a good
reactivity with ASFV positive serum. A monoclonal antibody 1D4 was obtained by cell fusion and screening. The titer was 1∶5 120 000 by ELISA. The heavy chain was identified as IgG2a and the light chain as κ. Western blot analysis showed that it could specifically recognize recombinant K145R protein.IFA test showed that it could react with ASFV. In summary，the soluble expression of K145R protein in E.coli and the preparation of monoclonal antibody were achieved.

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Preparation and Identification of Monoclonal Antibodies against African Swine Fever Virus p17 Protein

BAI Jingjing, SONG Huanhuan, BAI Chenyu, HAO Liying, YAN Shijun, DU Mengmeng, LI Xiangdong, DENG Junhua, TIAN Kegong

Journal of Henan Agricultural Sciences 2020, 49 (12): 137-143. DOI: 10.15933/j.cnki.1004-3268.2020.12.020

Abstract （187）

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In order to develop ASFV immunodiagnostic reagents,the purified recombinant ASFV p17 protein that expressed in baculovirus was utilized for immunization of BALB / c mice,and then the positive hybridoma which produced after fusion of spleen cells of the mice with high serum antibody titers to myeloma cells,was conducted by indirect ELISA. A total of 16 hybridomas that secreted specific MAbs against ASFV p17 were obtained,and the titers of ascites in all strains were between 1∶2.560×10 ⁶and 1∶1.024 ×10 ⁷.MAbs isotype assay showed that heavy chain of 6 MAbs(6H6,4D1,7E8,3D1,2F4 and 2B4) were IgG1,and the rest of 10 MAbs(7H12,10H6,10F3,6E11,4B3,5F7,7H9,6C4,2F1 and 4H7) were IgG2a,while the light chain type of all the MAbs was κ.The results of IFA showed that 8 MAbs could react with ASFV.In summary,MAbs against ASFV p17 protein was successfully prepared.

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Expression and Purification of MGF505-5R Protein of African Swine Fever Virus

ZHANG Suling, WU Peng, YUE Ya’nan, BAI Chenyu, HAO Liying, LI Xiangdong, PANG Wenqiang, TIAN Kegong

Journal of Henan Agricultural Sciences 2020, 49 (10): 130-136. DOI: 10.15933/j.cnki.1004-3268.2020.10.018

Abstract （130）

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In order to obtain African swine fever virus (ASFV) MGF505-5R protein with biological activity and its specific polyclonal antibodies,the His-SUMO,His-TrxA,His-GST,His-MBP and His-NusA gene fragments were recombinanted with MGF505-5R gene respectively,and then the pET28a-SUMO-MGF505- 5R,pET28a-TrxA-MGF505-5R,pET28a-GST-MGF505-5R,pET28a-MBP-MGF505-5R and pET28a- NusAMGF505- 5R were transformed into E.coli BL21(DE3) competent cells,and induced by IPTG,respectively.The optimal expression conditions were explored by optimizing the concentration of IPTG,induction temperature and induction time.The target protein was purified by MBP affinity chromatography and Ni2+affinity chromatography.The BALB/c mice were immunized with purified MGF505-5R protein,and the specificity of the anti-MGF-505-5R serum was identified by Western blot. The results showed that the MBP-MGF505-5R recombinant protein was successfully expressed in E.coli,and the expression quantity of soluble MBP-MGF505-5R recombinant protein was the highest after induced by 0.5 mmol/L IPTG at 20℃ for 12 h.Purified protein was obtained by MBP and Ni ²⁺affinity chromatography,and its purity reached 90%.The polyclonal antibody could specifically recognize the MGF-505-5R protein.In summary,this study successfully expressed ASFV MGF-505-5R protein using E.coli expression system,and the purified recombinant protein had high purity and good biological activity.

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