表达猪附红细胞体ENO基因重组腺病毒的构建及免疫原性分析
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摘要
试验旨在构建表达猪附红细胞体ENO基因的重组腺病毒并分析评价其免疫效果。将重组克隆质粒pMD-19T-ENO与腺病毒穿梭载体AdV4-GFP分别进行双酶切,构建重组腺病毒穿梭质粒AdV4-M/ENO;将经PacⅠ酶线性化后的重组腺病毒穿梭质粒AdV4-M/ENO转染293细胞,获得重组腺病毒Ad4-M/ENO,采用PCR和间接免疫荧光试验(IFTA)鉴定猪附红细胞体ENO基因在293细胞中的表达,再对293细胞进行培养,测定重组腺病毒的滴度;将30只BALB/c小鼠分为3组:重组腺病毒Ad4-M/ENO组、AdV4-GFP空载体对照组和PBS对照组,分别进行免疫接种,采用ELISA方法检测血清中猪附红细胞体IgG、IgG_1、IgG_(2a)抗体水平和IFN-γ、IL-4细胞因子水平,在三免2周后检测小鼠脾脏中CD4~+和CD8~+含量。结果显示,构建的重组腺病毒穿梭质粒AdV4-M/ENO目的基因片段大小为1 182 bp;重组腺病毒Ad4-M/ENO包装成功,能在293细胞中表达,滴度为1×10~9 PFU/mL。经重组腺病毒Ad4-M/ENO免疫后的BALB/c小鼠血清中IgG、IgG_1、IgG_(2a)抗体水平,IFN-γ、IL-4细胞因子水平及淋巴细胞亚群CD4~+、CD8~+含量均显著或极显著高于AdV4-GFP空载体对照组和PBS对照组(P<0.05;P<0.01)。结果表明,本试验成功构建了表达猪附红细胞体ENO基因的重组腺病毒,且该重组腺病毒能诱导小鼠产生特异性的体液免疫和细胞免疫应答反应。
The aim of the experiment was to construct a recombinant adenovirus expressing Mycoplasma suis ENO gene and analyze its immune effect.Recombinant cloning plasmid pMD-19 T-ENO and adenovirus shuttle vector AdV4-GFP were double digested to construct recombinant adenovirus shuttle plasmid AdV4-M/ENO;The recombinant adenovirus shuttle plasmid AdV4-M/ENO,which was linearized by PacⅠ,was transfected into 293 cells to obtain recombinant adenovirus Ad4-M/ENO.Expression of Mycoplasma suis ENO gene in 293 cells was identified by PCR and indirect immunofluorescence assay(IFTA),293 cells were cultured and the titer of recombinant adenovirus was determined.30 BALB/c mice were divided into three groups:Recombinant adenovirus Ad4-M/ENO group,AdV4-GFP empty vector control group and PBS control group,respectively,vaccinated separately.ELISA was used to detect IgG,IgG_1,IgG_(2 a) antibody levels and IFN-γ and IL-4 cytokine levels in serum,and the levels of CD4~+ and CD8~+ in the spleen of mice were detected 2 weeks after the third immunization.The results showed that the recombinant adenoviral shuttle plasmid AdV4-M/ENO gene fragment size was 1 182 bp;The recombinant adenovirus Ad4-M/ENO was successfully packaged and expressed in 293 cells with a titer of 1×10~9 PFU/mL.The serum levels of IgG,IgG_1,IgG_(2 a),IFN-γ,IL-4 and lymphocyte subsets CD4~+ and CD8~+ in BALB/c mice immunized with recombinant adenovirus Ad4-M/ENO were significantly or extremely significantly higher than AdV4-GFP empty vector control group and PBS control group(P<0.05;P<0.01).The results showed that the recombinant adenovirus expressing Mycoplasma suis ENO gene was successfully constructed in this experiment,and the recombinant adenovirus could induce specific humoral and cellular immune responses in mice.
The aim of the experiment was to construct a recombinant adenovirus expressing Mycoplasma suis ENO gene and analyze its immune effect.Recombinant cloning plasmid pMD-19 T-ENO and adenovirus shuttle vector AdV4-GFP were double digested to construct recombinant adenovirus shuttle plasmid AdV4-M/ENO;The recombinant adenovirus shuttle plasmid AdV4-M/ENO,which was linearized by PacⅠ,was transfected into 293 cells to obtain recombinant adenovirus Ad4-M/ENO.Expression of Mycoplasma suis ENO gene in 293 cells was identified by PCR and indirect immunofluorescence assay(IFTA),293 cells were cultured and the titer of recombinant adenovirus was determined.30 BALB/c mice were divided into three groups:Recombinant adenovirus Ad4-M/ENO group,AdV4-GFP empty vector control group and PBS control group,respectively,vaccinated separately.ELISA was used to detect IgG,IgG_1,IgG_(2 a) antibody levels and IFN-γ and IL-4 cytokine levels in serum,and the levels of CD4~+ and CD8~+ in the spleen of mice were detected 2 weeks after the third immunization.The results showed that the recombinant adenoviral shuttle plasmid AdV4-M/ENO gene fragment size was 1 182 bp;The recombinant adenovirus Ad4-M/ENO was successfully packaged and expressed in 293 cells with a titer of 1×10~9 PFU/mL.The serum levels of IgG,IgG_1,IgG_(2 a),IFN-γ,IL-4 and lymphocyte subsets CD4~+ and CD8~+ in BALB/c mice immunized with recombinant adenovirus Ad4-M/ENO were significantly or extremely significantly higher than AdV4-GFP empty vector control group and PBS control group(P<0.05;P<0.01).The results showed that the recombinant adenovirus expressing Mycoplasma suis ENO gene was successfully constructed in this experiment,and the recombinant adenovirus could induce specific humoral and cellular immune responses in mice.
引文
[1] 赵和永,巴彩凤.附红细胞体研究现状分析[J].中国人兽共患病学报,2006,22(7):683-685.ZHAO H Y,BA C F.Research status analysis of Eperythrozoon[J].Chinese Journal of Zoonoses,2006,22(7):683-685.(in Chinese)
[2] 陈启军,尹继刚,刘明远.附红细胞体及附红细胞体病[J].中国兽医学报,2006,26(4):460-464.CHEN Q J,YIN J G,LIU M Y.Eperythrozoon and eperythrozoonosis[J].Chinese Journal of Veterinary Medicine,2006,26(4):460-464.(in Chinese)
[3] 王永,冯昕炜,严光文,等.附红细胞体病原学研究进展[J].中国预防兽医学报,2008,30(6):491-494.WANG Y,FENG X W,YAN G W,et al.Advances in the research of pathogens of Eperythrozoon[J].Chinese Journal of Preventive Veterinary Medicine,2008,30(6):491-494.(in Chinese)
[4] 杨卉新,单思,巴彩凤.猪附红细胞体病研究进展[J].现代畜牧兽医,2013,48(5):48-52.YANG H X,SHAN S,BA C F.Research progress on erythroblastosis in pigs[J].Modern Journal of Animal Husbandry and Veterinary Medicine,2013,48(5):48-52.(in Chinese)
[5] 唐丽,李晓云,贾杏林.猪附红细胞体分子生物学研究进展[J].中国畜牧兽医,2013,40(7):81-83.TANG L,LI X Y,JIA X L.Research progress on the molecular biology of Mycoplasma sui[J].China Animal Husbandry & Veterinary Medicine,2013,40(7):81-83.(in Chinese)
[6] 方剑玉,朱文豪,郭小参,等.表达猪Viperin蛋白重组腺病毒的构建及其对PRRSV复制的抑制作用[J].河南农业科学,2017,46(9):126-131.FANG J Y,ZHU W H,GUO X S,et al.Expression of swine Viperin protein in recombinant adenovirus and study of its effect on PRRSV replication[J].Journal of Henan Agricultural Sciences,2017,46(9):126-131.(in Chinese)
[7] WANG X,JIANG P,LI Y,et al.Protection of pigs against post-weaning multisystemic wasting syndrome by a recombinant adenovirus expressing the capsid protein of porcine circovirus type 2[J].Veterinary Microbiology,2007,121(3-4):215-224.
[8] WESLEY R D,TANG M,LAGER K M.Protection of weaned pigs by vaccination with human adenovirus 5 recombinant viruses expressing the hemagglutinin and the nucleoprotein of H3N2 swine influenza virus[J].Vaccine,2004,22(25-26):3427-3434.
[9] 薛书江,张守发,李海峰,等.猪附红细胞体α-烯醇化酶基因的克隆及生物信息学分析[J].畜牧与兽医,2014,46(5):31-34.XUE S J,ZHANG S F,LI H F,et al.Cloning and bioinformatics analysis of Eperythrozoon suis α-enolase gene[J].Animal Husbandry & Veterinary Medicine,2014,46(5):31-34.(in Chinese)
[10] 张影,钱年超,贾立军,等.猪附红细胞体DnaJ 基因的克隆及真核表达[J].中国兽医科学,2013,43(1):65-69.ZHANG Y,QIAN N C,JIA L J,et al.Cloning and eukaryotic expression of DnaJ gene from Mycoplasma suis [J].Chinese Veterinary Science,2013,43(1):65-69.(in Chinese)
[11] SCHREINER S A,SOKOLI A,FELDER K M,et al.The surface-localised α-enolase of Mycoplasma suis is an adhesion protein[J].Veterinary Microbiology,2012,156(1-2):88-95.
[12] 王淼,倪婷婷,伍生军,等.猪附红细胞体ENO基因重组腺病毒穿梭质粒的构建及真核和表达[J].中国畜牧兽医,2018,45(8):2113-2118.WANG M,NI T T,WU S J,et al.Construction and eukaryotic expression of recombinant adenovirus shuttle plasmid of Mycoplasma suis ENO gene[J].China Animal Husbandry & Veterinary Medicine,2018,45(8):2113-2118.(in Chinese)
[13] 李莉,刘志武,谭榜云.重组腺病毒rAd-mIL-15的构建及表达[J].检验医学与临床,2017,14(22):3300-3302.LI L,LIU Z W,TAN B Y.Construction and expression of recombinant adenovirus rAd-mIL-15[J].Laboratory Medicine and Clinic,2017,14(22):3300-3302.(in Chinese)
[14] 陈娜娜,向冬喜,郑丛龙.腺病毒及其研究进展[J].大连医科大学学报,2010,32(5):586-590.CHEN N N,XIANG D X,ZHENG C L.Adenovirus and its research progress[J].Journal of Dalian Medical University,2010,32(5):586-590.(in Chinese)
[15] 范凌云,谢庆军.腺病毒载体的研究进展[J].中国生物制品学杂志,2008,21(2):153-157.FAN L Y,XIE Q J.Advances in adenoviral vectors[J].Chinese Journal of Biologicals,2008,21(2):153-157.(in Chinese)
[16] GUO Z L,YI Z L,KUI Z C,et al.Immunogenicity of recombinant adenovirus co-expressing the L7/L12 and BCSP31 proteins of Brucella abortus[J].Kafkas Universitesi Veteriner Fakultesi Dergisi,2018,24(2):211-217.
[17] MARTIN V,PASCUAL E,AVIA M,et al.A recombinant adenovirus expressing ovine interferon tau prevents influenza virus-induced lethality in mice[J].Journal of Virology,2016,90(7):3783-3788.
[18] 刘国栋,肖潇,佟颖,等.GFP标记的重组腺病毒在消毒效果评价中的应用研究[J].中国消毒学杂志,2017,34(11):997-1000.LIU G D,XIAO X,TONG Y,et al.Application of GFP-labeled recombinant adenovirus in the evaluation of disinfection effect[J].Chinese Journal of Disinfection,2017,34(11):997-1000.(in Chinese)
[19] 莫武宁,唐安洲,周玲.Ad5F35重组腺病毒载体研究进展[J].病毒学报,2006,22(6):480-483.MO W N,TANG A Z,ZHOU L.Advances in Ad5F35 recombinant adenoviral vectors[J].Chinese Journal of Virology,2006,22(6):480-483.(in Chinese)
[20] 李航,姜利建,刘晋宇,等.新孢子虫与弓形虫交叉抗原AMA1基因重组腺病毒的构建及免疫应答分析[J].中国畜牧兽医,2016,43(12):3336-3342.LI H,JIANG L J,LIU J Y,et al.Construction and analysis of immune response of Neospora caninum and Toxoplasma gondii cross recombinant adenovirus antigen AMA1 gene[J].China Animal Husbandry & Veterinary Medicine,2016,43(12):3336-3342.(in Chinese)
[21] LI L G,GANG C W,PENG J L,et al.Recombinant adenovirus expressing a dendritic cell-targeted melanoma surface antigen for tumor-specific immunotherapy in melanoma mice model[J].Experimental and Therapeutic Medicine,2018,15(6):5394-5402.
[22] JOSE M R,MIGUEL A,ELENA P,et al.Vaccination with recombinant adenovirus expressing peste des petits ruminants virus-F or -H proteins elicits T cell responses to epitopes that arises during PPRV infection[J].Veterinary Research,2017,48:79.
[23] 张金康,孟国林,刘建,等.携带人VEGF165和ANG-1双基因的腺病毒载体的构建及目的基因的表达[J].中国矫形外科杂志,2010,18(4):312-315.ZHANG J K,MENG G L,LIU J,et al.Construction and target gene expression of recombinant adenovirus vectors containing vascular endothelial growth factor 165 and angiopoietin-1[J].Orthopedic Journal of China,2010,18(4):312-315.(in Chinese)
[24] TING W,HUI Q Y,YAN L,et al.Vaccination with recombinant adenovirus expressing multi-stage antigens of Toxoplasma gondii by the mucosal route induces higher systemic cellular and local mucosal immune responses than with other vaccination routes[J].Parasite,2017,24:12.
[2] 陈启军,尹继刚,刘明远.附红细胞体及附红细胞体病[J].中国兽医学报,2006,26(4):460-464.CHEN Q J,YIN J G,LIU M Y.Eperythrozoon and eperythrozoonosis[J].Chinese Journal of Veterinary Medicine,2006,26(4):460-464.(in Chinese)
[3] 王永,冯昕炜,严光文,等.附红细胞体病原学研究进展[J].中国预防兽医学报,2008,30(6):491-494.WANG Y,FENG X W,YAN G W,et al.Advances in the research of pathogens of Eperythrozoon[J].Chinese Journal of Preventive Veterinary Medicine,2008,30(6):491-494.(in Chinese)
[4] 杨卉新,单思,巴彩凤.猪附红细胞体病研究进展[J].现代畜牧兽医,2013,48(5):48-52.YANG H X,SHAN S,BA C F.Research progress on erythroblastosis in pigs[J].Modern Journal of Animal Husbandry and Veterinary Medicine,2013,48(5):48-52.(in Chinese)
[5] 唐丽,李晓云,贾杏林.猪附红细胞体分子生物学研究进展[J].中国畜牧兽医,2013,40(7):81-83.TANG L,LI X Y,JIA X L.Research progress on the molecular biology of Mycoplasma sui[J].China Animal Husbandry & Veterinary Medicine,2013,40(7):81-83.(in Chinese)
[6] 方剑玉,朱文豪,郭小参,等.表达猪Viperin蛋白重组腺病毒的构建及其对PRRSV复制的抑制作用[J].河南农业科学,2017,46(9):126-131.FANG J Y,ZHU W H,GUO X S,et al.Expression of swine Viperin protein in recombinant adenovirus and study of its effect on PRRSV replication[J].Journal of Henan Agricultural Sciences,2017,46(9):126-131.(in Chinese)
[7] WANG X,JIANG P,LI Y,et al.Protection of pigs against post-weaning multisystemic wasting syndrome by a recombinant adenovirus expressing the capsid protein of porcine circovirus type 2[J].Veterinary Microbiology,2007,121(3-4):215-224.
[8] WESLEY R D,TANG M,LAGER K M.Protection of weaned pigs by vaccination with human adenovirus 5 recombinant viruses expressing the hemagglutinin and the nucleoprotein of H3N2 swine influenza virus[J].Vaccine,2004,22(25-26):3427-3434.
[9] 薛书江,张守发,李海峰,等.猪附红细胞体α-烯醇化酶基因的克隆及生物信息学分析[J].畜牧与兽医,2014,46(5):31-34.XUE S J,ZHANG S F,LI H F,et al.Cloning and bioinformatics analysis of Eperythrozoon suis α-enolase gene[J].Animal Husbandry & Veterinary Medicine,2014,46(5):31-34.(in Chinese)
[10] 张影,钱年超,贾立军,等.猪附红细胞体DnaJ 基因的克隆及真核表达[J].中国兽医科学,2013,43(1):65-69.ZHANG Y,QIAN N C,JIA L J,et al.Cloning and eukaryotic expression of DnaJ gene from Mycoplasma suis [J].Chinese Veterinary Science,2013,43(1):65-69.(in Chinese)
[11] SCHREINER S A,SOKOLI A,FELDER K M,et al.The surface-localised α-enolase of Mycoplasma suis is an adhesion protein[J].Veterinary Microbiology,2012,156(1-2):88-95.
[12] 王淼,倪婷婷,伍生军,等.猪附红细胞体ENO基因重组腺病毒穿梭质粒的构建及真核和表达[J].中国畜牧兽医,2018,45(8):2113-2118.WANG M,NI T T,WU S J,et al.Construction and eukaryotic expression of recombinant adenovirus shuttle plasmid of Mycoplasma suis ENO gene[J].China Animal Husbandry & Veterinary Medicine,2018,45(8):2113-2118.(in Chinese)
[13] 李莉,刘志武,谭榜云.重组腺病毒rAd-mIL-15的构建及表达[J].检验医学与临床,2017,14(22):3300-3302.LI L,LIU Z W,TAN B Y.Construction and expression of recombinant adenovirus rAd-mIL-15[J].Laboratory Medicine and Clinic,2017,14(22):3300-3302.(in Chinese)
[14] 陈娜娜,向冬喜,郑丛龙.腺病毒及其研究进展[J].大连医科大学学报,2010,32(5):586-590.CHEN N N,XIANG D X,ZHENG C L.Adenovirus and its research progress[J].Journal of Dalian Medical University,2010,32(5):586-590.(in Chinese)
[15] 范凌云,谢庆军.腺病毒载体的研究进展[J].中国生物制品学杂志,2008,21(2):153-157.FAN L Y,XIE Q J.Advances in adenoviral vectors[J].Chinese Journal of Biologicals,2008,21(2):153-157.(in Chinese)
[16] GUO Z L,YI Z L,KUI Z C,et al.Immunogenicity of recombinant adenovirus co-expressing the L7/L12 and BCSP31 proteins of Brucella abortus[J].Kafkas Universitesi Veteriner Fakultesi Dergisi,2018,24(2):211-217.
[17] MARTIN V,PASCUAL E,AVIA M,et al.A recombinant adenovirus expressing ovine interferon tau prevents influenza virus-induced lethality in mice[J].Journal of Virology,2016,90(7):3783-3788.
[18] 刘国栋,肖潇,佟颖,等.GFP标记的重组腺病毒在消毒效果评价中的应用研究[J].中国消毒学杂志,2017,34(11):997-1000.LIU G D,XIAO X,TONG Y,et al.Application of GFP-labeled recombinant adenovirus in the evaluation of disinfection effect[J].Chinese Journal of Disinfection,2017,34(11):997-1000.(in Chinese)
[19] 莫武宁,唐安洲,周玲.Ad5F35重组腺病毒载体研究进展[J].病毒学报,2006,22(6):480-483.MO W N,TANG A Z,ZHOU L.Advances in Ad5F35 recombinant adenoviral vectors[J].Chinese Journal of Virology,2006,22(6):480-483.(in Chinese)
[20] 李航,姜利建,刘晋宇,等.新孢子虫与弓形虫交叉抗原AMA1基因重组腺病毒的构建及免疫应答分析[J].中国畜牧兽医,2016,43(12):3336-3342.LI H,JIANG L J,LIU J Y,et al.Construction and analysis of immune response of Neospora caninum and Toxoplasma gondii cross recombinant adenovirus antigen AMA1 gene[J].China Animal Husbandry & Veterinary Medicine,2016,43(12):3336-3342.(in Chinese)
[21] LI L G,GANG C W,PENG J L,et al.Recombinant adenovirus expressing a dendritic cell-targeted melanoma surface antigen for tumor-specific immunotherapy in melanoma mice model[J].Experimental and Therapeutic Medicine,2018,15(6):5394-5402.
[22] JOSE M R,MIGUEL A,ELENA P,et al.Vaccination with recombinant adenovirus expressing peste des petits ruminants virus-F or -H proteins elicits T cell responses to epitopes that arises during PPRV infection[J].Veterinary Research,2017,48:79.
[23] 张金康,孟国林,刘建,等.携带人VEGF165和ANG-1双基因的腺病毒载体的构建及目的基因的表达[J].中国矫形外科杂志,2010,18(4):312-315.ZHANG J K,MENG G L,LIU J,et al.Construction and target gene expression of recombinant adenovirus vectors containing vascular endothelial growth factor 165 and angiopoietin-1[J].Orthopedic Journal of China,2010,18(4):312-315.(in Chinese)
[24] TING W,HUI Q Y,YAN L,et al.Vaccination with recombinant adenovirus expressing multi-stage antigens of Toxoplasma gondii by the mucosal route induces higher systemic cellular and local mucosal immune responses than with other vaccination routes[J].Parasite,2017,24:12.