幽门螺旋杆菌lpp20-cagA融合基因在乳酸球菌中的表达及免疫原性研究
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摘要
为研究载体疫苗对幽门螺旋杆菌(HP)感染的免疫保护作用,本实验应用无缝克隆技术将HP的两个候选抗原基因lpp20和cagA直接连接后克隆于表达载体pNZ8149中,构建重组表达质粒pNZ8149-lpp20-cagA。将重组质粒电转化乳酸球菌(L.lactis) NZ3900,构建了不含任何抗生素筛选标记的食品级原核表达系统pNZ8149-lpp20-cagA/NZ3900,经nisin诱导表达,得到的重组蛋白分子量约为50.3 ku。将以上得到的重组L.lactis灌胃免疫BALB/c小鼠,利用ELISA方法分别检测各组小鼠血清中IgG、IgG1、IgG2a及细胞因子IL-4、IFN-γ水平。结果显示,与对照组相比,实验组小鼠血清中IgG、IgG1、IL-4水平显著升高,表明重组蛋白Lpp20-CagA可以诱导良好的体液免疫应答;利用HP攻毒经重组菌灌胃免疫的小鼠,检测小鼠胃组织中HP的定植密度及脲酶活性,结果显示重组蛋白Lpp20-CagA在一定程度上可以减少HP在小鼠胃组织中的定植。本实验构建的载体疫苗在预防HP感染方面具有重要的应用价值。
To study the inmmunological protection of vector vaccine against Helicobacter pylori(HP) infection, two candidate antigen genes of HP, lpp20 and cagA were directly ligated into the expression vector PNZ8149 by seamless cloning technique, and the recombinant expression plasmid pNZ8149-lpp20-cagA was constructed. The recombinant plasmid was electroporated into Lactococcus lactis(L.lactis) competent cell NZ3900. A food-grade prokaryotic expression system, NZ3900/PNZ8149-lpp20-cagA was successfully constructed without any antibiotic screening markers. pNZ8149-lpp20-cagA/NZ3900 was induced by nisin, and the molecular weight of the recombinant protein was about 50.3 ku. BALB/c mice were immunized with the recombinant lactic acid bacteria. The levels of serum IgG, IgG1, IgG2 a and cytokine IL-4, IFN-γ were detected by ELISA assay. The results showed that the levels of IgG, IgG1 and IL-4 in serum of mice were significantly increased, which indicated that the recombinant protein could induce a good humoral immune response. The colonization density and urease activity of HP in gastric tissue of mice were determined. The results showed that the recombinant protein could alleviate the colonization of HP in gastric tissue of mice to some extent. The vector vaccine constructed in this study plays an active role in the prevention of HP infection.
To study the inmmunological protection of vector vaccine against Helicobacter pylori(HP) infection, two candidate antigen genes of HP, lpp20 and cagA were directly ligated into the expression vector PNZ8149 by seamless cloning technique, and the recombinant expression plasmid pNZ8149-lpp20-cagA was constructed. The recombinant plasmid was electroporated into Lactococcus lactis(L.lactis) competent cell NZ3900. A food-grade prokaryotic expression system, NZ3900/PNZ8149-lpp20-cagA was successfully constructed without any antibiotic screening markers. pNZ8149-lpp20-cagA/NZ3900 was induced by nisin, and the molecular weight of the recombinant protein was about 50.3 ku. BALB/c mice were immunized with the recombinant lactic acid bacteria. The levels of serum IgG, IgG1, IgG2 a and cytokine IL-4, IFN-γ were detected by ELISA assay. The results showed that the levels of IgG, IgG1 and IL-4 in serum of mice were significantly increased, which indicated that the recombinant protein could induce a good humoral immune response. The colonization density and urease activity of HP in gastric tissue of mice were determined. The results showed that the recombinant protein could alleviate the colonization of HP in gastric tissue of mice to some extent. The vector vaccine constructed in this study plays an active role in the prevention of HP infection.
引文
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[2]Meza B,Ascencio F,Sierra-Beltrán A P,et al.A novel design of a multi-antigenic,multistage and multi-epitope vaccine against Helicobacter pylori:An in silico,approach[J].Infect Genet Evol,2017,49:309-317.
[3]Liu Kai-yun,Shi Yun,Luo Ping,et al.Therapeutic efficacy of oral immunization with attenuated Salmonella typhimurium expressing Helicobacter pylori CagA,VacA and UreB fusion proteins in mice model[J].Vaccine,2011,29(38):6679-6685.
[4]Tobias J,Lebens M,Sun N W,et al.Surface expression of Helicobacter pylori,HpaA adhesion antigen on Vibrio cholerae,enhanced by co-expressed enterotoxigenic Escherichia coli,fimbrial antigens[J].Microb Pathog,2017,105:177-184.
[5]Zhang Rong-guang,Peng Xiao-yan,Duan Guang-cai,et al.An engineered Lactococcus lactis strain exerts significant immune responses through efficient expression and delivery of Helicobacter pylori Lpp20 antigen[J].Biotechnol Lett,2016,38(12):1-7.
[6]Vallese F,Mishra N,Pagliari M,et al.Helicobacter pylori antigenic Lpp20 is a structural homologue of Tipαand promotes epithelial-mesenchimal transition[J].BBA-Gen Subjects,2017,1861(12):3263-3271.
[7]Wang Hong-ping,Zhu Yong-liang,Shao Wei,et al.Role of Helicobacter pylori virulence factor cytotoxin-associated gene A in gastric mucosa-associated lymphoid tissue lymphoma[J].World J Gastroentero,2013,19(45):8219-8226.
[8]Zhang Hong-xin,Qiu Yu-yu,Zhao Ying-hui,et al.Immunogenicity of oral vaccination with Lactococcus lactis derived vaccine candidate antigen(UreB)of Helicobacter pylori fused with the human interleukin 2 as adjuvant[J].Mol Cell Probe,2014,28(1):25-30.
[9]Liu Wei,Tan Zhou-lin,Xue Jin-feng,et al.Therapeutic efficacy of oral immunization with a non-genetically modified Lactococcus lactis-based vaccine CUE-GEM induces local immunity against Helicobacter pylori infection[J].Appl Microbiol Biotechnol,2016,100(14):6219-6229.
[10]Abadi A H,Mahdavi M,Khaledi A,et al.Study of serum bactericidal and splenic activity of Total-OMP-CagA combination from Brucella abortus and Helicobacter pylori in BALB/c mouse model[J].Microb Pathog,2018,121:100-105.