猪带绦虫TSOL18重组耻垢分枝杆菌疫苗的构建及其表达
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摘要
目的构建猪带绦虫TSOL18重组耻垢分枝杆菌疫苗,分析猪带绦虫TSOL18基因在重组耻垢分枝杆菌中的表达情况。方法采用PCR方法从重组质粒pGEX-TSOL18中扩增TSOL18基因,克隆入pMV361载体,构建pMV361-TSOL18穿梭质粒,经酶切和测序鉴定后电穿孔法转化入耻垢分枝杆菌,进行单克隆菌落筛选及PCR鉴定。构建的猪带绦虫TSOL18重组耻垢分枝杆菌疫苗经热诱导后,以SDS-PAGE分析及Western blot分析TSOL18基因在重组耻垢分枝杆菌中的表达情况。结果成功扩增出TSOL18目的基因,大小约为393 bp,与预期值相符;经酶切验证,pMV361-TSOL18穿梭质粒构建成功;PCR鉴定猪带绦虫TSOL18重组耻垢分枝杆菌疫苗构建成功。SDS-PAGE电泳检测重组菌表达的目的蛋白TSOL18,相对分子质量为15×10~3与预期相符;Western blot分析该蛋白能被TSOL18兔抗血清识别。结论成功构建了猪带绦虫TSOL18重组耻垢分枝杆菌疫苗,猪带绦虫TSOL18基因在耻垢分枝杆菌中成功表达,表达蛋白具有反应原性,其免疫原性有待进一步研究。
Objective To construct a recombinant Mycobacterium smegmatis vaccine containing TSOL18 of Taenia solium in order to ascertain the expression of T. solium TSOL18 in recombinant M. smegmatis. Methods The TSOL18 gene was amplified from the recombinant plasmid pGEX-TSOL18 using PCR and cloned into the vector pMV361 to construct a pMV361-TSOL18 shuttle plasmid. After digestion and identification, the shuttle plasmid was transformed into M. smegmatis via electroporation. After the monoclonal colonies were screened and identified using PCR, a recombinant M. smegmatis vaccine containing T. solium TSOL18 was constructed. After heat induction, SDS-PAGE and Western blotting were used to analyze and identify the expression of the TSOL18 gene in M. smegmatis. Results PCR indicated that the TSOL18 gene(393 bp in length) was successfully amplified. The shuttle plasmid pMV361-TSOL18 was successfully constructed using enzyme digestion. A recombinant M. smegmatis vaccine containing T. solium TSOL18 was successfully constructed according to PCR. After heat induction, the target protein TSOL18 was detected using SDS-PAGE. Its relative molecular mass was 15×10~3, which was consistent with the expected value. Western blotting indicated that the protein was recognized by TSOL18 rabbit antiserum. Conclusion A recombinant M. smegmatis vaccine containing T. solium TSOL18 was successfully constructed. Recombinant TSOL18 protein was successfully expressed in M. smegmatis. The recombinant TSOL18 protein is immunoreactive, though its immunogenicity needs to be studied further. This finding has laid the foundation for further study of M. smegmatis vaccines.
Objective To construct a recombinant Mycobacterium smegmatis vaccine containing TSOL18 of Taenia solium in order to ascertain the expression of T. solium TSOL18 in recombinant M. smegmatis. Methods The TSOL18 gene was amplified from the recombinant plasmid pGEX-TSOL18 using PCR and cloned into the vector pMV361 to construct a pMV361-TSOL18 shuttle plasmid. After digestion and identification, the shuttle plasmid was transformed into M. smegmatis via electroporation. After the monoclonal colonies were screened and identified using PCR, a recombinant M. smegmatis vaccine containing T. solium TSOL18 was constructed. After heat induction, SDS-PAGE and Western blotting were used to analyze and identify the expression of the TSOL18 gene in M. smegmatis. Results PCR indicated that the TSOL18 gene(393 bp in length) was successfully amplified. The shuttle plasmid pMV361-TSOL18 was successfully constructed using enzyme digestion. A recombinant M. smegmatis vaccine containing T. solium TSOL18 was successfully constructed according to PCR. After heat induction, the target protein TSOL18 was detected using SDS-PAGE. Its relative molecular mass was 15×10~3, which was consistent with the expected value. Western blotting indicated that the protein was recognized by TSOL18 rabbit antiserum. Conclusion A recombinant M. smegmatis vaccine containing T. solium TSOL18 was successfully constructed. Recombinant TSOL18 protein was successfully expressed in M. smegmatis. The recombinant TSOL18 protein is immunoreactive, though its immunogenicity needs to be studied further. This finding has laid the foundation for further study of M. smegmatis vaccines.
引文
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[14] 杨凤娇,周必英.猪带绦虫重组BCG-TSOL18疫苗构建及其表达[J].中国人兽共患病学报,2015,31(10):952-6,962.
[15] 江楠,杨凤娇,周必英.猪带绦虫重组BCG-TSOL18疫苗的稳定性研究[J].中国病原生物学杂志,2017,12 (02):148-50.
[16] Sha S,Shi X,Deng G,et al.Mycobacterium tuberculosis Rv 1987 induce Th2 immune responses and enhances Mycobacterium smegmatis surviv in mice[J].Microbiol Res,2017,(197):74-80.
[17] Yan S,Xu M,Wang R,et al.Overexpression of Rv2788 increases Mycobacterium stresses survival[J].Microbiol Res,2017,(195):51-9.
[18] Deshpande V,Krishnan R,Philip S,et al.Oral administration of recombinant Mycobacterium smegmatis expressing a tripeptide construct derived from endogenous and microbial antigens prevents atherosclerosis in ApoE-/-mice[J].Cardiovasc Ther,2016,34(5):314-24.
[19] Fernandez-Cabezon L,Galan B,Garcia JL.Engineering Mycobacterium smegmatis for testosterone production[J].Microbial Biotechnol,2017,10(1):151-61.
[20] 李想,周必英.基因工程重组耻垢分枝杆菌研究进展[J].中国病原生物学杂志,2018,13(1):96-101.
[21] 李俊华,吴少庭,翁亚彪,等.弓形虫GRA4基因体外扩增、克隆及在耻垢分枝杆菌中的表达[J].中国病原生物学杂志,2006,1(6):428-31.
[22] 陈勇,夏惠,陶志勇,等.间日疟原虫pvMSP1-19基因重组耻垢分枝杆菌疫苗的构建与鉴定[J].中国病原生物学杂志,2011,6(7):507-10.
[23] 郑春福,吴少廷,陈雅棠,等.恶性疟原虫FCC-1/HN株环子孢子蛋白基因在耻垢分枝杆菌M.smegmatis mc2155中的表达[J].中国人兽共患病杂志,2001,17(5):27-9.
[2] Molinari JL,Rodriguez D,Tato P,et al.Field trial for reducing porcine Taenia solium cystircercosis in Mexico by systematic vaccination of pigs[J].Vet Parasitol,1997,69(1):55-63.
[3] Verast EM,Gilman RH,Gonzales A,et al.Taenia solium oncosphere antigens induce immunity in pigs against experimental cysticercosis[J].Vet Parasitol,2002,108(1):49-62.
[4] 周必英.猪带绦虫重组抗原研究进展[J].中国人兽共患病学报,2014,30(4):418-22.
[5] Kim BJ,Gong JR,Kim GN,et al.Recombinant Mycobacterium smegmatis with a pMyong2 vector expressing human immunodeficiency virus type I gag can induce enhanced virus-specific immune responses[J].Sci Rep,2017(7):44776.
[6] 李文桂,陈雅棠.载体介导的刚地弓形虫SAG疫苗的研制现状[J].中国病原生物学杂志,2017,12(2):189-95.
[7] 周必英,周泠,刘美辰,等.猪带绦虫TSOL18基因的表达、纯化和兔抗血清的制备[J].中国人兽共患病学报,2013,29(10):977-80,985.
[8] FAO.No authors identifies top 10 foodborne parasites[J].Vet Rec,2014,175(3):58.
[9] 马承旭,王宏伟,杨艺萱.猪带绦虫基因组学及猪囊尾蚴病候选疫苗的研究进展[J].中国寄生虫学与寄生虫病杂志,2016,34,(2):161-5.
[10] 肖红利,申万文,高健安,等.猪带绦虫TSO45W-4B-TSOL18重组蛋白疫苗免疫小鼠诱导的免疫应答[J].中国病原生物学杂志,2015,10(2):159-64.
[11] 李想,孙俊超,周必英.猪带绦虫重组质粒pMG36e-TSOL18和pMG36e-SP-TSOL18在乳球菌中的表达[J].中国病原生物学杂志,2018,13(9):989-93.
[12] 周必英,刘美辰,贺莉芳.猪带绦虫重组Bb(pGEX-TSO45W-4B-TSOL18)疫苗的构建及鉴定[J].中国病原生物学杂志,2014,9(4):289-92,298.
[13] Ding J,Zheng Y,Wang Y,et al.Immune respones to a recombinant attenuated Salmonella typhimurium strain expressing a Taenia solium oncosphere antigen TSOL18[J].Comp Immunol Microbiol Infect Dis,2013,36(1):17-23.
[14] 杨凤娇,周必英.猪带绦虫重组BCG-TSOL18疫苗构建及其表达[J].中国人兽共患病学报,2015,31(10):952-6,962.
[15] 江楠,杨凤娇,周必英.猪带绦虫重组BCG-TSOL18疫苗的稳定性研究[J].中国病原生物学杂志,2017,12 (02):148-50.
[16] Sha S,Shi X,Deng G,et al.Mycobacterium tuberculosis Rv 1987 induce Th2 immune responses and enhances Mycobacterium smegmatis surviv in mice[J].Microbiol Res,2017,(197):74-80.
[17] Yan S,Xu M,Wang R,et al.Overexpression of Rv2788 increases Mycobacterium stresses survival[J].Microbiol Res,2017,(195):51-9.
[18] Deshpande V,Krishnan R,Philip S,et al.Oral administration of recombinant Mycobacterium smegmatis expressing a tripeptide construct derived from endogenous and microbial antigens prevents atherosclerosis in ApoE-/-mice[J].Cardiovasc Ther,2016,34(5):314-24.
[19] Fernandez-Cabezon L,Galan B,Garcia JL.Engineering Mycobacterium smegmatis for testosterone production[J].Microbial Biotechnol,2017,10(1):151-61.
[20] 李想,周必英.基因工程重组耻垢分枝杆菌研究进展[J].中国病原生物学杂志,2018,13(1):96-101.
[21] 李俊华,吴少庭,翁亚彪,等.弓形虫GRA4基因体外扩增、克隆及在耻垢分枝杆菌中的表达[J].中国病原生物学杂志,2006,1(6):428-31.
[22] 陈勇,夏惠,陶志勇,等.间日疟原虫pvMSP1-19基因重组耻垢分枝杆菌疫苗的构建与鉴定[J].中国病原生物学杂志,2011,6(7):507-10.
[23] 郑春福,吴少廷,陈雅棠,等.恶性疟原虫FCC-1/HN株环子孢子蛋白基因在耻垢分枝杆菌M.smegmatis mc2155中的表达[J].中国人兽共患病杂志,2001,17(5):27-9.