重组蛋白CTB-P97R1对猪支原体肺炎活疫苗滴鼻免疫效果的评价
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摘要
为提高滴鼻免疫条件下猪支原体肺炎活疫苗的免疫效果,本研究设计引物扩增猪肺炎支原体P97R1基因,融合至霍乱毒素B亚单位(CTB)基因下游,构建了重组质粒p ET-CTB-P97R1。重组菌经IPTG诱导表达、分离纯化得到r CTB-P97R1重组蛋白。利用体外神经节苷脂(GM1)结合试验检测r CTB-P97R1的佐剂活性。随后将r CTB-P97R1与活疫苗混合后滴鼻免疫小鼠,免疫后采血和收集小鼠支气管肺泡灌洗液(BALF),分别检测其中的特异性Ig G抗体和s Ig A抗体的水平评价免疫效果。结果显示,r CTB-P97R1可以特异性结合GM1,具有生物学活性。小鼠免疫后的血清Ig G抗体显著高于活疫苗对照组(p<0.01),同时产生了高水平的P97R1特异性Ig G抗体(p<0.01);小鼠BALF中的s Ig A抗体也显著高于活疫苗对照组(p<0.05)。结果表明,r CTB-P97R1能够显著增强经滴鼻免疫的猪支原体肺炎活疫苗的免疫效力,同时可提升P97R1的免疫应答,有望作为经黏膜免疫的猪支原体肺炎活疫苗的新型佐剂。
To enhance the immunostimulating effect of the Mycoplasma hyopneumoniae live vaccine via intranasal injection,the P97 R1 gene of Mycoplasma hyopneumoniae was amplified and fused to the downstream of the CTB gene to construct a recombinant expressing plasmid pETCTB-P97 R1 and transformed to E.coli. The recombinant E.coli was induced by IPTG to express the fusion recombinant protein CTB-P97 R1(rCTB-P97 R1), which was purified by affinity chromatography. The adjuvant bioactivity of rC TB-P97 R1 was detected by the monosialoganglioside(GM1)-ELISA assay. BALB/c mice were immunized with the rC TB-P97 R1 coupled with live vaccine through intranasal route. Serum and bronchoalveolar lavage fluid(BALF) samples were collected and detected for specific IgG and sI gA antibodies against the whole Mycoplasma protein or P97 R1 protein. The results showed that the rC TB-P97 R1 was able to specifically bind to GM1, indicating a well biological activity in vitro. In immunization experiment, high levels of antibodies against the whole Mycoplasma protein and P97 R1 were detected after the immunization inthe serum of the mice immunized with CTB-P97 R1-coupled live vaccine(p<0.01), compared with the mice immunized with live vaccine alone. The sI gA antibody against the whole Mycoplasma protein in BALF of the mice immunized with rC TB-P97 R1-coupled live vaccine was significantly higher than that of the mice in immunized group with live vaccine alone(p<0.05). In conclusion, rC TB-P97 R1 enhanced the immunostimulating effect of the live vaccine through intranasal route, as well as the immune response to the key antigen P97 R1. The data indicated its potential to be used as a new adjuvant for the mucosal immunity live vaccine against M.hyopneumoniae.
To enhance the immunostimulating effect of the Mycoplasma hyopneumoniae live vaccine via intranasal injection,the P97 R1 gene of Mycoplasma hyopneumoniae was amplified and fused to the downstream of the CTB gene to construct a recombinant expressing plasmid pETCTB-P97 R1 and transformed to E.coli. The recombinant E.coli was induced by IPTG to express the fusion recombinant protein CTB-P97 R1(rCTB-P97 R1), which was purified by affinity chromatography. The adjuvant bioactivity of rC TB-P97 R1 was detected by the monosialoganglioside(GM1)-ELISA assay. BALB/c mice were immunized with the rC TB-P97 R1 coupled with live vaccine through intranasal route. Serum and bronchoalveolar lavage fluid(BALF) samples were collected and detected for specific IgG and sI gA antibodies against the whole Mycoplasma protein or P97 R1 protein. The results showed that the rC TB-P97 R1 was able to specifically bind to GM1, indicating a well biological activity in vitro. In immunization experiment, high levels of antibodies against the whole Mycoplasma protein and P97 R1 were detected after the immunization inthe serum of the mice immunized with CTB-P97 R1-coupled live vaccine(p<0.01), compared with the mice immunized with live vaccine alone. The sI gA antibody against the whole Mycoplasma protein in BALF of the mice immunized with rC TB-P97 R1-coupled live vaccine was significantly higher than that of the mice in immunized group with live vaccine alone(p<0.05). In conclusion, rC TB-P97 R1 enhanced the immunostimulating effect of the live vaccine through intranasal route, as well as the immune response to the key antigen P97 R1. The data indicated its potential to be used as a new adjuvant for the mucosal immunity live vaccine against M.hyopneumoniae.
引文
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[13]陈丽,欧阳凤秀,钱炳俊,等.霍乱毒素B亚单位与胰岛素原融合基因的克隆表达及重组蛋白特性分析[J].生物工程学报,2005,21(2):204-210.
[14]Xiong Qi-yan,Wei Yan-na,Xie Hai-dong,et al.Effect of different adjuvant formulations on the immunogenicity and protective effect of a live Mycoplasma hyopneumoniae vaccine after intramuscular inoculation[J].Vaccine,2014,32(27):3445-3451.
[15]Feng Zhi-xin,Shao Guo-qing,Liu Mao-jun,et al.Development and validation of a SIgA-ELISA for the detection of Mycoplasma hyopneumoniae infection[J].Vet Microbiol,2010,143(2-4):410-416.
[16]Sun J,Raghavan S,Sjoling A,et al.Oral tolerance induction with antigen conjugated to cholera toxin B subunit generates both Foxp3+CD25+and Foxp3-CD25-CD4+regulatory T cells[J].J Immunol,2006,177(11):7634-7644.
[17]Dong Hui,Huang Yan-mei,Yao Shu-wen,et al.The recombinant fusion protein of cholera toxin B and neutrophil-activating protein expressed on Bacillus subtilis spore surface suppresses allergic inflammation in mice[J].Appl Microbiol Biotechnol,2017,101(14):5819-5829.
[18]李图帅,陈瑾,乔绪稳,等.利用霍乱毒素B亚单位展示猪O型口蹄疫病毒VP1蛋白主要抗原表位[J].江苏农业学报,2016,32(3):601-607.
[19]Marchioro S B,Sacristan R D,Michiels A,et al.Immune responses of a chimaeric protein vaccine containing Mycoplasma hyopneumoniae antigens and LTB against experimental M.hyopneumoniae infection in pigs[J].Vaccine,2014,32(36):4689-4694.
[20]卢会英,沈青春,宁宜宝.猪肺炎支原体p97R1区基因和大肠杆菌LTB基因的重组和表达[J].中国兽医杂志,2010,46(4):3-6.
[21]Simionatto S,Marchioro S B,Maes D,et al.Mycoplasma hyopneumoniae:From disease to vaccine development veterinary[J].Vet Microbiol,2013,165(3-4):234-242.
[22]马丰英,姚睿玉,邹浩勇,等.猪支原体肺炎亚单位疫苗的免疫应答[J].中国兽医学报,2012,32(4):529-533.
[2]胡桂学,徐浩,郭慧,等.动物黏膜免疫佐剂研究新进展[J].经济动物学报,2014,18(1):1-4.
[3]杨晶晶,杨倩.几种黏膜佐剂口服免疫对鸡空肠、回肠抗体分泌细胞的影响[J].畜牧兽医学报,2016,47(6):1253-1259.
[4]Stratmann T.Cholera toxin subunit B as adjuvant-an accelerator in protective immunity and a break in autoimmunity[J].Vaccines,2015,3(3):579-596.
[5]Hou Jue,Liu Ying,Jenny H,et al.Cholera toxin B subunit acts as a potent systemic adjuvant for HIV-1 DNA vaccination intramuscularly in mice[J].Hum Vaccine Immunother,2014,10(5):1274-1283.
[6]Miyata T,Harakuni T,Taira T,et al.Merozoite surface protein-1 of Plasmodium yoelii fused via an oligosaccharide moiety of cholera toxin B subunit glycoprotein[J].Vaccine,2012,30(5):948-958.
[7]Zhang Qi-jing,Young T F,Ross R F.Identification and characterization of a Mycoplasma hyopneumoniae adhesin[J].Infect Immun,1995,63(3):1013-1019.
[8]Minion F C,Adams C,Hsu T.R1 region of P97 mediates adherence of Mycoplasma hyopneumoniae to swine cilia[J].Infect Immun,2000,68(5):3056-3060.
[9]Okamba F R,Arella M,Music N,et al.Potential use of a recombinant replication-defective adenovirus vector carrying the C-terminal portion of the P97 adhesin protein as a vaccine against Mycoplasma hyopneumoniae in swine[J].Vaccine,2010,28(30):4802-4809.
[10]Lee S H,Lee S,Chae C,et al.A recombinant chimera comprising the R1 and R2 repeat regions of M.hyopneumoniae P97 and the N-terminal region of A.pleuropneumoniae ApxIII elicits immune responses[J].BMC Vet Res,2014,10:43.
[11]李建平,熊祺琰,陈庆梅,等.霍乱毒素B亚单位与p277融合基因在大肠杆菌中表达及免疫分析[J].药物生物技术,2007,14(3):157-163.
[12]刘茂军,邵国青,张映,等.猪肺炎支原体P97基因抗原决定簇R1区的克隆与表达[J].江苏农业学报,2005,21(3):207-211.
[13]陈丽,欧阳凤秀,钱炳俊,等.霍乱毒素B亚单位与胰岛素原融合基因的克隆表达及重组蛋白特性分析[J].生物工程学报,2005,21(2):204-210.
[14]Xiong Qi-yan,Wei Yan-na,Xie Hai-dong,et al.Effect of different adjuvant formulations on the immunogenicity and protective effect of a live Mycoplasma hyopneumoniae vaccine after intramuscular inoculation[J].Vaccine,2014,32(27):3445-3451.
[15]Feng Zhi-xin,Shao Guo-qing,Liu Mao-jun,et al.Development and validation of a SIgA-ELISA for the detection of Mycoplasma hyopneumoniae infection[J].Vet Microbiol,2010,143(2-4):410-416.
[16]Sun J,Raghavan S,Sjoling A,et al.Oral tolerance induction with antigen conjugated to cholera toxin B subunit generates both Foxp3+CD25+and Foxp3-CD25-CD4+regulatory T cells[J].J Immunol,2006,177(11):7634-7644.
[17]Dong Hui,Huang Yan-mei,Yao Shu-wen,et al.The recombinant fusion protein of cholera toxin B and neutrophil-activating protein expressed on Bacillus subtilis spore surface suppresses allergic inflammation in mice[J].Appl Microbiol Biotechnol,2017,101(14):5819-5829.
[18]李图帅,陈瑾,乔绪稳,等.利用霍乱毒素B亚单位展示猪O型口蹄疫病毒VP1蛋白主要抗原表位[J].江苏农业学报,2016,32(3):601-607.
[19]Marchioro S B,Sacristan R D,Michiels A,et al.Immune responses of a chimaeric protein vaccine containing Mycoplasma hyopneumoniae antigens and LTB against experimental M.hyopneumoniae infection in pigs[J].Vaccine,2014,32(36):4689-4694.
[20]卢会英,沈青春,宁宜宝.猪肺炎支原体p97R1区基因和大肠杆菌LTB基因的重组和表达[J].中国兽医杂志,2010,46(4):3-6.
[21]Simionatto S,Marchioro S B,Maes D,et al.Mycoplasma hyopneumoniae:From disease to vaccine development veterinary[J].Vet Microbiol,2013,165(3-4):234-242.
[22]马丰英,姚睿玉,邹浩勇,等.猪支原体肺炎亚单位疫苗的免疫应答[J].中国兽医学报,2012,32(4):529-533.