重组轮状病毒疫苗VP8~*蛋白与组织血型抗原的结合性研究
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摘要
目的评价轮状病毒(Rotaviruses,RVs)疫苗株Rotarix和RotaTeq VP8~*蛋白与组织血型抗原(histo-blood group antigens,HBGAs)基因融合的免疫效果。方法从GenBank获取Rotarix和RotaTeq两种疫苗VP8~*蛋白基因序列,根据大肠埃希菌常用密码子进行序列优化后人工合成Rotarix和RotaTeqVP8~*蛋白基因并采用PCR扩增合成的两种基因,引物上、下游分别引入Nde I和Xho I限制性内切酶酶切位点。将PCR产物及载体pET-30a用Nde I和Xho I双酶切,经1.5%琼脂糖凝胶电泳回收目的片段后在T4连接酶作用下连接,连接产物转化至DH5α感受态细胞并进行PCR鉴定,取阳性菌液提取质粒,经Nde I和Xho I双酶切鉴定正确后测序。将合成的基因序列与表达载体pET-30a连接后导入大肠埃希菌BL21(DE3),加入IPTG诱导表达,经Ni~(2+) beads亲和层析纯化得到VP8~*-His融合蛋白,进行SDS-PAGE电泳分析。利用寡糖结合试验检测目的蛋白与lea、lex、leb、ley、A、B、H1、H2 8种寡糖的结合特性,利用唾液结合试验检测VP8~*蛋白与不同血型组织抗原表型唾液的结合特征。结果成功构建重组质粒,SDS-PAGE显示目的蛋白相对分子质量约为26×10~3,与预期的分子质量相符合。寡糖结合试验显示Rotarix和RotaTeq VP8~*蛋白能与H1型HBGAs结合,唾液结合试验显示二者能与分泌型唾液结合而不与非分泌型唾液结合。结论成功克隆表达了轮状病毒疫苗株Rotarix和RotaTeq VP8~*蛋白,两种蛋白均有与H1型HBGAs结合的特性。
Objective To evaluate the immune effects of VP8~* proteins of the rotavirus(RV) vaccine strains Rotarix and RotaTeq and fusion genes of histo-blood group antigens(HBGAs). Methods Gene sequences coding for the VP8~* protein of the Rotarix and RotaTeq vaccine strains were obtained from GenBank. After sequence optimization based on codons commonly used in E. coli, genes coding for the VP8~* protein of Rotarix and RotaTeq were synthesized, and PCR was used to amplify the two genes. Nde I and Xho I restriction endonuclease sites were respectively introduced upstream and downstream of the primers. The PCR product and the vector pET-30a were digested with Nde I and Xho I. The target fragment was recovered using 1.5% agarose gel electrophoresis and ligated under the action of T4 ligase. The ligated product was transformed into DH5α competent cells and identified using PCR. The positive bacterial solution was extracted, and the plasmid was identified using Nde I and Xho I double digestion and sequenced. The synthetic gene sequence was ligated to the expression vector pET-30a, introduced into E. coli BL21(DE3), and IPTG was used to induce its expression. A VP8~* -His fusion protein was purified using affinity chromatography on Ni~(2+) beads and analyzed using SDS-PAGE. The binding properties of the target protein to eight oligosaccharides-lea, lex, leb, ley, A, B, H1, and H2-were detected using an oligosaccharide binding assay. A saliva binding assay was used to analyze the binding characteristics of the VP8~* protein to antigen serotypes in different blood groups. Results A recombinant plasmid was successful constructed, and SDS-PAGE indicated that the molecular weight of the target protein was about 26 ku, which was consistent with the expected molecular weight. Oligosaccharide binding experiments indicated that VP8~* proteins from Rotarix and RotaTeq can bind to H1 HBGAs. Results of a saliva binding assay indicated that the two can bind to secreted saliva and do not bind to non-secreted saliva. Conclusion The VP8~* proteins of RV vaccine strains Rotarix and RotaTeq were successfully cloned and expressed, and both proteins are characterized by binding to type H1 HBGAs.
Objective To evaluate the immune effects of VP8~* proteins of the rotavirus(RV) vaccine strains Rotarix and RotaTeq and fusion genes of histo-blood group antigens(HBGAs). Methods Gene sequences coding for the VP8~* protein of the Rotarix and RotaTeq vaccine strains were obtained from GenBank. After sequence optimization based on codons commonly used in E. coli, genes coding for the VP8~* protein of Rotarix and RotaTeq were synthesized, and PCR was used to amplify the two genes. Nde I and Xho I restriction endonuclease sites were respectively introduced upstream and downstream of the primers. The PCR product and the vector pET-30a were digested with Nde I and Xho I. The target fragment was recovered using 1.5% agarose gel electrophoresis and ligated under the action of T4 ligase. The ligated product was transformed into DH5α competent cells and identified using PCR. The positive bacterial solution was extracted, and the plasmid was identified using Nde I and Xho I double digestion and sequenced. The synthetic gene sequence was ligated to the expression vector pET-30a, introduced into E. coli BL21(DE3), and IPTG was used to induce its expression. A VP8~* -His fusion protein was purified using affinity chromatography on Ni~(2+) beads and analyzed using SDS-PAGE. The binding properties of the target protein to eight oligosaccharides-lea, lex, leb, ley, A, B, H1, and H2-were detected using an oligosaccharide binding assay. A saliva binding assay was used to analyze the binding characteristics of the VP8~* protein to antigen serotypes in different blood groups. Results A recombinant plasmid was successful constructed, and SDS-PAGE indicated that the molecular weight of the target protein was about 26 ku, which was consistent with the expected molecular weight. Oligosaccharide binding experiments indicated that VP8~* proteins from Rotarix and RotaTeq can bind to H1 HBGAs. Results of a saliva binding assay indicated that the two can bind to secreted saliva and do not bind to non-secreted saliva. Conclusion The VP8~* proteins of RV vaccine strains Rotarix and RotaTeq were successfully cloned and expressed, and both proteins are characterized by binding to type H1 HBGAs.
引文
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[16] Jiang X,Liu Y,Tan M.Histo-blood group antigens as receptors for rotavirus,new understanding on rotavirus epidemiology and vaccine strategy[J].Emerg Microbes Infect,2017(6):e22.
[17] Marionneau S,Cailleau-Thomas A,Rocher J,et al.ABH and Lewis histo-blood group antigens,a model for the meaning of oligosaccharide diversity in the face of a changing world[J].Biochimie,2001(83):565-73.
[2] Tate JE,Burton AH,Boschi-Pinto C,et al.Global,regional,and national estimates of rotavirus mortality in children <5 years of age,2000-2013[J].Clin Infect Dis,2016(62):S96-105.
[3] Fleming FE,Graham KL,Takada Y,et al.Determinants of the specificity of rotavirus interactions with the α2β1 integrin[J].J Biol Chem,2011,286(8):6165-74.
[4] Grove J,Marsh M.The cell biology of receptor-mediated virus entry[J].J cell Biol,2011,195(7):1071-82.
[5] Isa P,Arias CF,Lopez S.Role of sialic acids in rotavirus infection[J].Glycoconj J,2006,23(1-2):27-37.
[6] Kuhlenschmidt TB,Hanafin WP,Gelberg HB,et al.Sialic acid dependence and independence of group A rotaviruses [J].Adv Exp Med Biol,1999(473):309-17.
[7] Huang P,Xia M,Tan M,et al.Spike protein VP8* of human rotavirus recognizes histo-blood group antigens in a type-specific manner [J].J Virol,2012,86 (9):4833-43.
[8] Liu Y,Huang P,Tan M,et al.Rotavirus VP8*:phylogeny,host range,and interaction with histo-blood group antigens [J].J Virol,2012,86(18):9899-910.
[9] Liu Y,Huang P,Jiang B,et al.Poly-LacNAc as an age-specific ligand for rotavirus P[11] inneonates and infants [J].PloS One,2013,8(11):e78113.
[10] Bohm R,Fleming FE,Maggioni A,et al.Revisiting the role of histo-blood group antigens in rotavirus host-cell invasion [J].Nat Commun,2015(6):5907.
[11] Lopez S,Arias CF.Multistep entry of rotavirus into cells:a Versaillesque dance[J].Trends Microbiol,2004,12(6):271-8.
[12] Arias CF,Romero P,Alvarez V,et al.Trypsin activation pathway of rotavirus infectivity[J].J Virol,1996,70(9):5832-9.
[13] Clark SM,Roth JR,Clark ML,et al.Trypsin enhancement of rotavirus infectivity:mechanism of enhancement[J].J Virol,1981,39(3):816-22.
[14] Arias CF,Romero P,Alvarez V,et al.Trypsin activation pathway ofrotavirus infectivity[J].J Virol,1996,70(9):5832-9.
[15] Estes MK,Graham DY,Mason BB,et al.Proteolytic enhancement of rotavirusinfectivity:molecular mechanisms[J].J Virol,1981,39(3):879-88.
[16] Jiang X,Liu Y,Tan M.Histo-blood group antigens as receptors for rotavirus,new understanding on rotavirus epidemiology and vaccine strategy[J].Emerg Microbes Infect,2017(6):e22.
[17] Marionneau S,Cailleau-Thomas A,Rocher J,et al.ABH and Lewis histo-blood group antigens,a model for the meaning of oligosaccharide diversity in the face of a changing world[J].Biochimie,2001(83):565-73.