刚地弓形虫重组蛋白表面抗原1与4诱导小鼠免疫保护性的比较研究
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摘要
目的比较刚地弓形虫重组蛋白表面抗原1 (rSAG1)与rSAG4单独及联合诱导昆明小鼠的免疫保护性作用,为研制复合型疫苗分子提供参考。方法以弓形虫RH株基因组DNA为模板, PCR扩增SAG1和SAG4,构建重组质粒pET-28a (+)-SAG1和pET-28a (+)-SAG4;经双酶切和测序鉴定后,将序列正确的重组质粒转入BL21 (DE3)中,挑选阳性菌落,异丙基-β-D-硫代半乳糖苷(IPTG)诱导表达,收集菌体,超声裂菌。在变性条件下经镍离子亲合层析柱纯化目的蛋白,采用透析逐步降低尿素浓度的方法进行目的蛋白复性; 125只昆明小鼠按照随机数字表法分为5组,每组25只,分别为r SAG1免疫组、 rSAG4免疫组、 rSAG1+rSAG4联合免疫组、PBS对照组、 PBS+佐剂对照组,首次免疫为重组蛋白100μg/鼠,与等体积完全弗氏佐剂充分乳化后进行皮下多点注射,间隔2周加强免疫1次,连续2次,加强免疫时重组蛋白为50μg/鼠,与等体积弗氏不完全佐剂乳化,对照组小鼠注射等量PBS。各组小鼠于免疫前(0周)及首次免疫后第2、 4、 6周断尾取血,分离血清,间接ELISA法检测小鼠血清特异性抗体IgG。在首次免疫后第6周,每只小鼠经腹腔注射3 000个弓形虫RH株速殖子进行攻击感染,观察小鼠生存时间,采用SPSS 16.0软件进行统计学分析。结果弓形虫RH株SAG1和SAG4基因经PCR扩增后,目的片段大小分别为780和438 bp,与理论大小一致;重组质粒pET-28a (+)-SAG1和pET-28a (+)-SAG4经双酶切和测序鉴定后,特异片段长度与SAG1和SAG4理论大小780和438 bp一致,测序结果显示,重组基因与目的基因序列完全一致; rSAG1、 rSAG4相对分子质量(Mr)为28 970、 17 720,与理论值一致,经纯化和复性后获得较纯的可溶性蛋白; rSAG1免疫组小鼠血清特异抗体IgG吸光度(A450值)为1.821±0.184,明显高于rSAG4免疫组(0.695±0.089)(P <0.05),但低于rSAG1+rSAG4联合免疫组(1.955±0.097)(P <0.05)。PBS组和PBS+佐剂组分别为0.019±0.002、 0.020±0.004, rSAG1、 rSAG4和rSAG1+r SAG4联合免疫组均高于两对照组(P <0.05);经弓形虫速殖子攻击感染后,对照组小鼠均在224 h内全部死亡, rSAG1、 rSAG4免疫组及联合免疫组分别在296、 288及320 h内全部死亡,各免疫组与各对照组之间、 rSAG4免疫组与联合免疫组之间生存时间差异有统计学意义(P <0.05)。结论 rSAG1、 rSAG4和rSAG1+rSAG4均能诱导小鼠产生抗弓形虫感染的免疫保护作用,明显延长实验小鼠的存活时间,联合免疫保护效果要略优于单抗原免疫。
Objective To determine the protective immunity induced by the immunization of recombinant surface antigen-1(rSAG1) and surface antigen-2(rSAG2) against Toxoplasma gondii infection in mice and compare their potential as vaccine. Methods The DNAs coding for T. gondii SAG1 and SAG4 were amplified by PCR from T. gondii genomic DNA and cloned into expression plasmid pET-28 a(+). The success of cloning was confirmed by DNA sequencing, The recombinant plasmid DNAs were transformed into E. coli BL21(DE3) for expression as recombinant proteins under isopropyl-β-D-thiogalactopyranoside(IPTG) induction. The induced bacteria were lysed and the expressed rSAG1 and rSAG4 were purified by Ni-NTA resin under denatured condition. The denatured recombinant proteins were refolded by dialysis method. The refolded recombinant proteins were formulated with Freund 's adjuvant and used to immunize mice(n = 25 for each group) for 3 times. PBS and PBS + adjuvant were used as controls. Sera were collected after each immunization and specific IgG in the sera were measured by ELISA.Each mouse was challenged with 3 000 T. gondii tachyzoite two weeks after the final immunization. The survival time of the infected mice was observed and the data was analyzed using SPSS 16.0. Results The DNAs coding for full length SAG1(780 bp) and SAG4(438 bp) were successfully amplified from T. gondii genomic DNA and then cloned into pET-28 a(+) to generate recombinant pET-28 a(+)-SAG1 and pET-28 a(+)-SAG4. The correct sequence and reading frame were confirmed by DNA sequencing. After being transformed into E. coli BL21(DE3), both rSAG1 and rSAG4 were expressed as inclusion bodies. After being denatured with urea, the rSAG1 and rSAG4 were purified and refolded as soluble proteins. All mice produced high level of specific IgG in sera after being immunized three times with each recombinant protein compared to pre-immunization sera(P < 0.05). The ELISA A450 value of rSAG1 group(1.821 ± 0.184) was significantly higher than r SAG4 group(0.695 ± 0.089) when sera were diluted at 1 ∶ 400(P <0.05), but it is less than the combined group(rSAG1 + rSAG4)(1.955 ± 0.097)(P < 0.05). And the A450 value of 3 experimental groups was higher than PBS(0.019 ± 0.002) group and PBS + adjuvant group(0.020 ± 0.004). All control mice(without immunization) died 224 h after being infected with 3 000 T. gondii tachyzoite, however, the mice immunized with rSAG1, rSAG4 and rSAG1 + rSAG4 survived till 296 h, 288 h and 320 h, respectively, after being challenged with the same number of T. gondii tachyzoite, with significant difference compared to the control group( P < 0.05).The increased survival time in the rSAG1 + rSAG4 group was significantly longer than each individual antigen group(P < 0.05). Conclusion Mice immunized with rSAG1, rSAG4 and rSAG1 + rSAG4 produced significant protective immunity against T. gondii infection with prolonged survival time than group without immunization. The co-immunization with both rSAG1 and rSAG4 produced synergic protective immunity compared to the individual antigen.
Objective To determine the protective immunity induced by the immunization of recombinant surface antigen-1(rSAG1) and surface antigen-2(rSAG2) against Toxoplasma gondii infection in mice and compare their potential as vaccine. Methods The DNAs coding for T. gondii SAG1 and SAG4 were amplified by PCR from T. gondii genomic DNA and cloned into expression plasmid pET-28 a(+). The success of cloning was confirmed by DNA sequencing, The recombinant plasmid DNAs were transformed into E. coli BL21(DE3) for expression as recombinant proteins under isopropyl-β-D-thiogalactopyranoside(IPTG) induction. The induced bacteria were lysed and the expressed rSAG1 and rSAG4 were purified by Ni-NTA resin under denatured condition. The denatured recombinant proteins were refolded by dialysis method. The refolded recombinant proteins were formulated with Freund 's adjuvant and used to immunize mice(n = 25 for each group) for 3 times. PBS and PBS + adjuvant were used as controls. Sera were collected after each immunization and specific IgG in the sera were measured by ELISA.Each mouse was challenged with 3 000 T. gondii tachyzoite two weeks after the final immunization. The survival time of the infected mice was observed and the data was analyzed using SPSS 16.0. Results The DNAs coding for full length SAG1(780 bp) and SAG4(438 bp) were successfully amplified from T. gondii genomic DNA and then cloned into pET-28 a(+) to generate recombinant pET-28 a(+)-SAG1 and pET-28 a(+)-SAG4. The correct sequence and reading frame were confirmed by DNA sequencing. After being transformed into E. coli BL21(DE3), both rSAG1 and rSAG4 were expressed as inclusion bodies. After being denatured with urea, the rSAG1 and rSAG4 were purified and refolded as soluble proteins. All mice produced high level of specific IgG in sera after being immunized three times with each recombinant protein compared to pre-immunization sera(P < 0.05). The ELISA A450 value of rSAG1 group(1.821 ± 0.184) was significantly higher than r SAG4 group(0.695 ± 0.089) when sera were diluted at 1 ∶ 400(P <0.05), but it is less than the combined group(rSAG1 + rSAG4)(1.955 ± 0.097)(P < 0.05). And the A450 value of 3 experimental groups was higher than PBS(0.019 ± 0.002) group and PBS + adjuvant group(0.020 ± 0.004). All control mice(without immunization) died 224 h after being infected with 3 000 T. gondii tachyzoite, however, the mice immunized with rSAG1, rSAG4 and rSAG1 + rSAG4 survived till 296 h, 288 h and 320 h, respectively, after being challenged with the same number of T. gondii tachyzoite, with significant difference compared to the control group( P < 0.05).The increased survival time in the rSAG1 + rSAG4 group was significantly longer than each individual antigen group(P < 0.05). Conclusion Mice immunized with rSAG1, rSAG4 and rSAG1 + rSAG4 produced significant protective immunity against T. gondii infection with prolonged survival time than group without immunization. The co-immunization with both rSAG1 and rSAG4 produced synergic protective immunity compared to the individual antigen.
引文
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[10]王钊哲,许瑞,洪炀,等.刚地弓形虫表面抗原1、2 B细胞表位基因的融合表达和鉴定[J].中国寄生虫学与寄生虫病杂志,2017,35(6):575-579.
[11]张佃波,周林涛,王海防,等.刚地弓形虫ROP2-P30复合重组蛋白疫苗对BALB/c小鼠免疫保护性的研究[J].中国病原生物学杂志,2008,3(10):763-767.
[12]Wang Y,Yin H.Research progress on surface antigen 1(SAG1)of Toxoplasma gondii[J].Parasit Vectors,2014,7:180.
[13]吴斌,吕芳丽.CD8+T细胞免疫应答在刚地弓形虫感染免疫中的功能研究进展[J].中国寄生虫学与寄生虫病杂志,2014,32(2):143-147.
[14]Zhou J,Wang L.SAG4 DNA and peptide vaccination provides partial protection against T.gondii infection in BALB/c mice[J].Front Microbiol,2017,8:1733.
[15]杨雯,田春林,朱穗京,等.弓形虫表面抗原SAG1基因的克隆表达、纯化与鉴定[J].广西医科大学学报,2010,27(2):205-208.
[16]杨雯,万孝玲,田春林,等.弓形虫表面抗原SAG4基因的克隆、表达和鉴定[J].中国寄生虫学与寄生虫病杂志,2009,27(4):340-343.
[2]Villena I,Ancelle T,Delmas C,et al.Congenital toxoplasmosis in France in 2007:first results from a national surveillance system[J].Euro Surveill,2010,15(25):19600.
[3]Moncada PA,Montoya JG.Toxoplasmosis in the fetus and newborn:an update on prevalence:diagnosis and treatment[J].Expert Rev Anti infect Ther,2012,10(7):815-828.
[4]严晓岚,闻礼永,官亚宜,等.《弓形虫病的诊断》标准解读[J].中国寄生虫学与寄生虫病杂志,2016,34(4):387-389.
[5]Liu Q,Wang Z,Huang S,et al.Diagnosis of toxoplasmosis and typing of Toxoplasma gondii[J].Parasit Vectors,2015,8:292.
[6]蒋子阳,董锴,戴红,等.弓形虫疫苗研究进展[J].热带医学杂志,2018,18(2):269-274.
[7]Di Cristina M,Del Porto P,Buffolano W,et al.The Toxoplasma gondii bradyzoite antigens BAG1 and MAG1 induce early humoral and cell-mediated immune responses upon human infection[J].Microbes Infect,2004,6(2):164-171.
[8]郭凯,陈兴智.弓形虫主要抗原的研究进展[J].蚌埠医学院学报,2012,37(4):486-490.
[9]孙怡,何深一,丛华,等.弓形虫SAG1-SAG2复合DNA疫苗免疫小鼠诱导的免疫保护性[J].国际医学寄生虫病杂志,2006,33(1):2-5.
[10]王钊哲,许瑞,洪炀,等.刚地弓形虫表面抗原1、2 B细胞表位基因的融合表达和鉴定[J].中国寄生虫学与寄生虫病杂志,2017,35(6):575-579.
[11]张佃波,周林涛,王海防,等.刚地弓形虫ROP2-P30复合重组蛋白疫苗对BALB/c小鼠免疫保护性的研究[J].中国病原生物学杂志,2008,3(10):763-767.
[12]Wang Y,Yin H.Research progress on surface antigen 1(SAG1)of Toxoplasma gondii[J].Parasit Vectors,2014,7:180.
[13]吴斌,吕芳丽.CD8+T细胞免疫应答在刚地弓形虫感染免疫中的功能研究进展[J].中国寄生虫学与寄生虫病杂志,2014,32(2):143-147.
[14]Zhou J,Wang L.SAG4 DNA and peptide vaccination provides partial protection against T.gondii infection in BALB/c mice[J].Front Microbiol,2017,8:1733.
[15]杨雯,田春林,朱穗京,等.弓形虫表面抗原SAG1基因的克隆表达、纯化与鉴定[J].广西医科大学学报,2010,27(2):205-208.
[16]杨雯,万孝玲,田春林,等.弓形虫表面抗原SAG4基因的克隆、表达和鉴定[J].中国寄生虫学与寄生虫病杂志,2009,27(4):340-343.